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Structure and Properties of Cell Membrane Structure
and Properties of Cell Membranes Volume II First
Edition Benga Digital Instant Download
Author(s): Benga
ISBN(s): 9781351085427, 1351085425
Edition: First edition
File Details: PDF, 24.73 MB
Year: 2017
Language: english
Structure
and
Properties
of
Cell Membranes
Volume II
Molecular Basis
of
Selected Transport Systems
Editor
Gheorghe Benga, M .D., Ph.D.

Head, Department of Cell Biology
Faculty of Medicine
Medical and Pharmaceutical Institute
Cluj-Napoca, Romania
(ci*; CRC Press

Taylor & Francis Group
Boca Raton London N ew York
CRC Press is an im p rin t o f th e
T aylo r & Francis G ro u p , an informa business
First published 1985 by CRC Press
Taylor & Francis Group
6000 Broken Sound Parkway NW, Suite 300
Boca Raton, FL 33487-2742
Reissued 2018 by CRC Press
© 1985 by CRC Press, Inc.

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No claim to original U.S. Government works
This book contains information obtained from authentic and highly regarded sources. Reasonable efforts have been made to publish
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Library of Congress Cataloging in Publication Data

Main entry under title:
Molecular basis of selected transport systems.
(Structure and properties of cell membranes; v. 2)

Includes bibliographies and index.
1. Biological transport. 2. Molecular biology.
I. Benga, Gheorghe. II Series. [DNLM: 1. Cell
Membrane-physiology. 2. Cell Membrane—ultrastructure.
3. Biological Transport. QH 601 S9285]
QH509.M65 1985 574.87’5 84-19942
ISBN 0-8493-5765-9
A Library of Congress record exists under LC control number: 84019943
Publisher’s Note
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CRC Press Web site at http://www.crcpress.com
PREFACE
In recent years it has become apparent that many essential functions of living cells are
performed by membrane-associated events. Membranes are highly selective permeability
barriers that impart their individuality on cells and organelles (Golgi apparatus, mitochondria,
lysosomes, etc.) by forming boundaries around them and compartmentalizing specialized
environments. By receptor movement and responses to external stimuli, membranes play a
central role in biological communication. Owing to various enzymes attached to or embedded
into membranes they are involved in many metabolic processes. The two important energy
conversion processes, photosynthesis in chloroplasts and oxidative phosphorylation in mi
tochondria, are carried out in membranes. To understand all these processes, which are
essential for living organisms, it is necessary to understand the molecular nature of membrane
structure and function.
The main purpose of this book is to provide in-depth presentations of well-defined topics
in membrane biology, focusing on the idea of structure-function relationships at the m o
lecular level. The book consists of three volumes.
Volume 1 covers general aspects of structure-function relationships in biological mem
branes. Attention has been paid both to protein and lipid components of cell membranes
regarding the interactions between these components, mobility of proteins and lipids, as
well as to the physiological significance of membrane fluidity and lipid-dependence of
membrane enzymes. Since some molecular components of the plasma membrane appear to
function in concert with some component macromolecules of basement membranes a review
of this expanding topic has been included.
Volume 2 is devoted to models and techniques which allow molecular insights into cell
membranes. After the first chapter describes quantum chemical studies of proton translo
cation, several chapters present the most extensively used model systems (monomolecular
films, planar lipid bilayers, and liposomes) in relation to biomembranes as well as the
reconstitution of membrane transport systems. The description of some biophysical tech
niques (X-ray, spin labeling ESR, NMR) is focused on their use in studying stucture-function
relationships in cell membranes. The remaining chapters in this volume are devoted to the
physiological significance of surface potential of membranes and of the dietary manipulation
of lipid composition.
Volume 3 covers transport at the molecular level in selected systems. The first chapters
present basic kinetics and pH effects on membrane transport, while subsequent chapters
focus on the effect of membrane lipids on permeability in prokaryotes or on Ca2+ perme
ability. Three chapters describe structure-function relationships in mitochondrial H -
ATPase, cytochrome oxidase, and adenine nucleotide carrier. The last chapter is devoted
to exocytosis, endocytosis, and recycling of membranes, which are distinct, albeit overlap
ping, cellular processes.
From this survey it is obvious that by application of biochemical and biophysical techniques
it is possible to explain membrane phenomena at the molecular level in a meaningful way.
M oreover, it is now clear that the study of cell membranes at the molecular level is important
for understanding the alterations leading to abnormal cells or the understanding of drug and
pesticide action. The multidisciplinary approach of research in this area and the permanent
need for information regarding the recent advances require new books on cell membranes.
The present collection of reviews is by no means a comprehensive treatise on all aspects of
“ m embranology” , rather a sampling of the status of selected topics. The volumes, providing
contributions for reference purposes at the professional level, are broadly aimed at bioche
mists, biologists, biophysicists, physicians, etc., active investigators working on cell mem
branes and hopefully will also be of great help to teachers and students at both the undergraduate
or postgraduate levels.
THE EDITOR
Dr. Gheorghe Benga, M .D ., P h.D ., is the Head of the Department of Cell Biology at
the Medical and Pharmaceutical Institute, Cluj-Napoca, Romania. He is also heading the
Laboratory of Human Genetics of the Cluj County Hospital.
In 1967, Dr. Benga received an M .D. with academic honors from the Medical and
Pharmaceutical Institute. After 3 years of internship (1966 to 1969) in basic medical sciences
(biochemistry, microbiology), he studied for a Ph.D. in medical biochemistry from 1969 to
1972 under Prof. Ion Manta, Department of Biochemistry, at the same Institute. In 1972,
Dr. Benga received a B.Sc. in Chemistry and in 1973, an M .Sc. in Physical Chemistry of
Surfaces from the University of Cluj.
From 1972 to 1978, Dr. Benga was Lecturer and Senior Lecturer in the Department of
Biochemistry at the Medical and Pharmaceutical Institute. In 1974 he was awarded a Well
come Trust European Travelling Fellowship and spent 1 year in England as a postdoctoral
research worker under Prof. Dennis Chapman, Department of Chemistry and Biochemistry,
University of Sheffield and Chelsea College University of Fondon. In 1978, Dr. Benga was
appointed to head the newly formed Department of Cell Biology at the Medical and Phar
maceutical Institute. He is currently teaching cell biology to medical students.
In addition to his other duties, Dr. Benga has spent several 1- to 3-month periods as a
Visiting Scientist at many British and American universities and in 1983 was a Visiting
Professor at the University of Illinois at Urbana-Champaign.
Dr. Benga has attended several international courses on biomembranes and has presented
numerous papers at international and national meetings, as well as guest lectures at various
universities and institutes in Romania, England, the U .S., the Netherlands, and Switzerland.
He has taken an active part in the organization of three international workshops on biological
membranes (1980, Cluj-Napoca — Romanian-British; 1981, Cluj-Napoca — Romanian-
American; 1982, New York City — American-Romanian) and has published over 80 papers
to date.
Dr. Benga is the author of several text books of cell biology for medical students and of
the book, Biologia molecularä a membranelor cu aplicapi medicale, published by Editura
Dacia, Cluj-Napoca, 1979. He is the co-author of Metode biochimice in laboratorul clinic,
Editura Dacia, 1976; co-editor of Biomembranes and Cell Function, New York Academy
of Sciences, 1983; and co-editor of Membrane Processes: Molecular Biology and Medical
Applications, Springer-Verlag, New York, 1984.
His major interests in the field of biological membranes include the characterization of
molecular composition and functional properties of human liver subcellular membranes, the
molecular interactions (lipid-protein, lipid-sterol, and drug effects) in model and natural
biomembranes, and the investigation of water diffusion through red blood cell membranes.
Dr. Benga is President o f the Cluj-Napoca Section of the Romanian National Society of
Cell Biology and Vice-President of this Society. He is on the board of the Subcommission
of Biochemistry of the Romanian Academy and is on the editorial board of Clujul Medical.
C O N TRIBUTO RS
Angelo Azzi Josef Houstëk

Medizinisch-Chemisches Institut Institute of Physiology
Universität Bern Czechoslovak Academy of Sciences
Bern, Switzerland Prague, Czechoslovakia
Kurt Bill
Jan Kopecky
Medizinisch-Chemisches Institut
Institute of Physiology
Universität Bern Czechoslovak Academy of Sciences
Bern, Switzerland
Prague, Czechoslovakia
M arc R. Block
Laboratoire de Biochimie Arnost Kotyk
Departement de Recherche Fondamentale Institute o f Physiology
Centre d ’Etudes Nucléaires Czechoslovak Academy of Sciences
Grenoble, France Prague, Czechoslovakia
Reinhard Bolli
Guy J.-M . Lauquin
M edizinisch-Chemisches Institut
Laboratoire de Biochimie
Universität Bern
Departement de Recherche Fondamentale
Bern, Switzerland
Centre d ’Etudes Nucléaires
Grenoble, France
François Boulay
Laboratoire de Biochimie
Departement de Recherche Fondamentale Ronald N. McElhaney
Centre d ’Etudes Nucléaires Professor
Grenoble, France Deparment of Biochemistry
University of Alberta
Gérard Brandolin Edmonton, Alberta
Laboratoire de Biochimie Canada
Departement de Recherche Fondamentale
Centre d’Etudes Nucléaires
Grenoble, France D. James Morré
Professor
Robert P. Casey Departments of Medicinal Chemistry and
Medizinisch-Chemisches Institut Biological Sciences, and Purdue Cancer
Universität Bern Center
Bern, Switzerland Purdue University
West Lafayette, Indiana
Zdenëk Drahota
Director Katarzyma A. Nafçcz
Institue of Physiology Medizinisch-Chemisches Institut
Czechoslovak Academy of Sciences Universität Bern
Prague, Czechoslovakia Bern, Switzerland
Ross P. Holmes
Senior Food Scientist Maciej J. Nalçcz
Burnsides Research Laboratory Department of Cellular Biochemistry
University of Illinois Nencki Institute of Experimental Biology
Urbana, Illinois Warsaw, Poland
Paul O ’Shea Pierre V. Vignais
Medizinisch-Chemisches Institut Laboratoire de Biochimie
Universität Bern Departement de Recherche Fondamentale
Bern, Switzerland Centre d ’Etudes Nucléaires
Grenoble, France
Lech Wojtczak
Department of Cellular Biochemistry
Nencki Institute of Experimental Biology
Warsaw, Poland
STRUCTURE AND PROPERTIES OF CELL MEMBRANES
Gheorghe Benga
Volume I
The Evolution of Membrane Models

Protein-Protein Interactions in Cell Membranes
Lateral Mobility of Proteins in Membranes
Lateral Diffusion of Lipids
Topological Asymmetry and Flip-Flop of Phospholipids in Biological Membranes
Membrane Fluidity: Molecular Basis and Physiological Significance
Lipid Dependence of Membrane Enzymes
Protein-Lipid Interactions in Biological Membranes
Basement Membrane Structure, Function, and Alteration in Disease
Volume II
Basic Kinetics of Membrane Transport

pH Effects on Membrane Transport
The Effect of Membrane Lipids on Permeability and Transport in Prokaryotes
The Influence of Membrane Lipids on the Permeability of Membranes to Ca2+
Molecular Aspects of Structure-Function Relationship in Mitochondrial H + -ATPase
M olecular Aspects of the Structure-Function Relationship in Cytochrome c Oxidase
Molecular Aspects of the Structure-Function Relationships in Mitochondrial Adenine
Nucleotide Carrier
Exocytosis, Endocytosis, and Recycling of Membranes
The Surface Potential of Membranes: Its Effect on Membrane-Bound Enzymes and
Transport Processes
Volume III
Quantum Chemical Approach to Study the Mechanisms of Proton Translocation Across

M embranes Through Protein Molecules
M onomolecular Films as Biomembrane Models
Planar Lipid Bilayers in Relation to Biomembranes
Relation of Liposomes to Cell Membranes
Reconstitution of Membrane Transport Systems
Structure-Function Relationships in Cell Membranes as Revealed by X-Ray Techniques
Structure-Function Relationships in Cell Membranes as Revealed by Spin Labeling EPR
Structure and Dynamics of Cell Membranes as revealed by NMR Techniques
The Effect of Dietary Lipids on the Composition and Properties of Biological Membranes
TA B LE OF CONTENTS
Volume II
Chapter 1
Basic Kinetics of Membrane T ransport......................................................................................... 1
Arnost Kotyk
Chapter 2
pH Effects on Membrane T ran sp o rt.............................................................................................. 11
Arnost Kotyk
Chapter 3
The Effect of Membrane Lipids on Permeability and Transportin Prokaryotes..................... 19
Ronald N. McElhaney
Chapter 4
The Influence of Membrane Lipids on the Permeability of Membranes to Ca2+ ................. 53
Ross P. Holmes
Chapter 5
M olecular Aspects of Structure-Function Relationship in Mitochondrial H +-A TPase 77
Jan Kopeckÿ, Josef Housték, and Zdenëk Drahota
Chapter 6
M olecular Aspects of the Structure-Function Relationship in Cytochrome c O xidase... 105
Angelo Azzi, Kurt Bill, Reinhard Bolli, Robert P. Casey, Katarzyma A. Nalçcz, and
Paul O ’Shea
Chapter 7
M olecular Aspects of Structure-Function Relationships in Mitochondrial
Adenine Nucleotide C a r r ie r .......................................................................................................... 139
Pierre V. Vignais, Marc R. Block, François Boulay, Gérard Brandolin, and Gùy J.-
M . Lauquin
Chapter 8
Exocytosis, Endocytosis, and Recycling of M em branes.........................................................181
D. James Morré
Chapter 9
The Surface Potential of Membranes: Its Effect on Membrane-Bound
Enzymes andTransport Processes.................................................................................................215
Lech W ojtczak and Maciej J. Nalçcz
Index 243
Volume II 1
Chapter 1
BA SIC K INETICS OF M E M BR A N E TRANSPO R T
Arnost Kotyk
TA B LE OF CONTENTS
I. Introduction................................................................................................................................2
II. Nonspecific Perm eation.......................................................................................................... 2
III. Specific Transport ................................................................................................................... 3

A. Specific Channels or P o re s..................................................................................... 3
B. Specific Carriers.........................................................................................................4
References.............................................................................................................................................. 9
2 Structure and Properties o f Cell Membranes
I. IN T R O D U C T IO N
In this chapter, we shall deal with the rates of movement of substances across membranes
during their transport or translocation. At the outset, let us accept some restrictions and
simplifications for the purposes of this treatment, as they are common throughout transport
literature.
1. The membrane is “ infinitely” thin so that no intramembrane gradients are to be

considered.
2. The solutions separated by the membrane are sufficiently mixed so that there is a
unique concentration of solute at any time in each of them.
3. The transport system resides in the membrane and cannot leave it so that its “ con
centration” in the membrane does not change during the experiment.
Two fundamentally different mechanisms of membrane transport exist: nonspecific per

meation (often called simple diffusion) and specific, saturable transport (often termed carrier
or porter transport).
II. NO NSPECIFIC PERM EATION
In the first type of transmembrane movement, no specific reactions take place between
the permeant solute and the permeable membrane, although nonspecific interactions, such
as stripping of hydration water, attraction by opposite electrical charges, and the like, are
possible and, in fact, likely.
The rate of such transport (in kmol s _ ' m ~ 2) is defined from Fick’s First Law of diffusion
for movement along one of the coordinates
Js = - D ( S „ - S,)/8 = P(S, - S„) (1)
where S, and S„ are solute concentrations (kmol m -3) at the starting and at the target sides
of the membrane, respectively, D is the diffusion coefficient across the membrane in
m2s ~ ‘, 8 the membrane thickness in m, and P the permeability constant in m s -1 .
D itself may be defined as RTU where R is the gas constant (8.314 J m o L 1 K ‘), T the
absolute temperature (in K), and U the mobility (in m2 s -1 J -1 mol). This suggests that
diffusion processes have a poorly expressed temperature dependence, something like 3%
increase in D per 10°C rise in the physiological temperature range. This is, however, not
true for transmembrane crossing. We must consider here a different definition of P, which
must be taken as phenomenological, only as it holds both for passage through membrane
lipid domains as well as for movement through hydrophilic channels. It states that
P = K Dm/8 (2)
where Dn, isthe diffusion coefficient of the solute within the membrane and K the partition
coefficient between the membrane and the exterior aqueous solution. Itis in this quantity
that processes such as stripping of hydration water molecules are included, and these may
possess much steeper temperature dependences, corresponding to apparent activation energies
of 50 to 80 kJ m o L 1. This, by the way, is the range of activation energies commonly found
with the specific types of transport discussed below.
Equation (1) predicts that: (1) the net movement ceases (Js = 0) when S, = S„, so that,
for an uncharged solute not adsorbed within the cell (such as urea in human erythrocytes),
the equilibrium concentrations are the same in and out of the cell; (2) initial transport rate
Volume II 3
(when Sn = 0) as well as unidirectional flux, followed with the aid of labeled substance,
is linearly related to concentration over the entire reasonable concentration range.
It should be noted, however, that if an ion is translocated by the same type of simple
diffusion, it will distribute itself according to the existing membrane potential so that, in
equilibrium,
S„ = s 1e ~ nFAl|'m/RT (3)
where Ai|»m is the membrane potential in V, F is the Faraday constant (96.5 kC mol '), and
n is the number of positive charges of the ion. Thus, for a membrane potential of — 100
mV, passively distributed K + will reach an S„/S[ of 46, while Mg2+ might attain a S„/S,
ratio of 2120 at 30°C.
III. S P E C IF IC T R A N S P O R T
Characteristics of this type of transport are its specificity and its saturability, both indicating
that a finite number of membrane proteins are involved in the process.
Although the molecular mechanisms of this type of membrane translocation are many (cf.
other chapters of this book), they all involve binding of the transported solute to a receptor
(binding) site in the membrane, its movement across the membrane, and dissociation toward
the other membrane face.
There is an important distinction between a situation where the binding site is simulta
neously accessible from both sides of the membrane (we may call this a channel or pore)
and one where it is accessible alternately from the one and from the other side (we may call
this a carrier). The limited accessibility in this last case may be due to isomerization of the
carrier protein during the transport process or to the shifting of a “ gate” which is part of
the surrounding membrane, presumably another associated protein.
A. Specific Channels or Pores

This case may be formally depicted as follows:
b aSj c S jj d
— ES —
where a, b, c, d are rate constants for the association and dissociation of solute with channel
receptor site E. The site is accessible both to S, and to S„, although the rates of association
a and c may be different (say, for steric reasons).
The equation for rate of transport of S is easily derived to be
adS, — bcS,
J = E ---------- ü----- (4)
s ‘ b + d + aS, + cSn
which, for unidirectional flux (as followed by a tracer) becomes
adS[
I = F — (5)
b + d + aS, + cS,,
For initial flux
ad S. d E,S,
T = F -------------- = (6)
s0 ' b + d + aS, (b + d)/a + S,
from which the Jmax is equal to dE, and the half-saturation constant K, is equal to (b + d)/
a. E, represents the total receptor site concentration.
In analogy with enzymological practice. Equation (4) may be converted to the form
(S, ~ S ../K J
J. = (7)
KTi (1 + S„/Kx,) + s,
where Keq, the equilibrium constant (for a simple channel equal to 1), is defined by ad/bc;
Jâx, the maximum rate going from side I to side II, is equal to dE,; KX1, the half-saturation
constant going from I to II, is (b 4- d)/a; and KX2, the half-saturation constant going from
II to I, is (b + d)/c. Finally, J,7ax = Kx, J ^ j K X| Kaq.
The channel described here does not operate in an active manner and it does not show
the phenomenon of countertransport (see below).
B. Specific Carriers
The specific carrier may be formally represented as follows:
EII
aSj cSn
ESj ES„
where the two membrane sides are distinct with respect to the location of the carrier.1
(Phenomenologically equivalent expressions are obtained from a model where ES, and ES„
are identical but E, and EMdistinct.)2
Using a steady-state approach, such that the individual carrier “ concentrations” are con
stant during the measurable reaction time but none of the steps of scheme II is rate-limiting,
one obtains the following equation for the rate of transport of S:
= adfgS, - bcehS,,__________________
5“ 1 (e + f)(bh + bd + dg) + a[d(f + g) + f(g + h)]S, +
c[h(b + e) + e(b + g)]S„ + ac(g + h ^ S ,,
the unidirectional flux from side I to side II is then
JS(,^ M) = E,adgS,[f + bchS„/(bh + bd + dg)]/D (9)
D being the same denominator as in Equation (8). The opposite unidirectional flux may be
obtained by replacing each of the rate constants with its opposite counterpart from the
schematic model above.
The initial rate of reaction from side I to side II is then
j _ E a d f§ S ' ____________
s0 (e + f)(bh + bd + dg) + a[d(f + g) + f(g + h)]S,
so that Jmax = E,dfg/[d(f + g) + f(g + h)]
and Kx = (e + f)(bh + bd + dg)/a[d(f + g) + f(g + h)] (10)

Volume / / 5
Like in the case of channel transport, the equations can be readily converted to forms
containing only measurable kinetic constants, thus. Equation (8) becomes
J = ------------- lS| ~ N2Sh------------ (11a)

' C + D ,S, + D2S„ + D^S.S,,
and
(S, - S„/K )
Js =
KTi (1 + S„/KT2) + S,(l + S../K,)
with Keq = N ,/N 2, Kt1 = C/D,, KT2 = C/D2, J ^ x = N,/D, ( lib )
and, a new expression not present in Equation (7), K, = D ,/D 12.Inversion of this equation
leads to
1/J„ = [KTl (1 + S„/KT2)/J-ax + S, (1 + S . / K . y j ^ K J
x 1/(S, - S„/Kcq) + (1 + SII/K ,)/J^1X (12)
A plot of 1/JS against 1/(S, - SM/Kcq) for different S„ leads to a set of l/Jm;lXappwhich are
related to true Jâx by
= S n /J^ K , + 1 /J - X (13)
A plot of 1/Jmax against S„ will yield 1/Jâx as the intercept with the ordinate and —K, as
the intercept with the abscissa.
The plot of 1/JSagainst 1/(S, — Sn/Kcq) above also yields a family of Kx which is related
to the true Kx’s as follows:
KTapl, = KX| (1 + Sn/K l2)/( 1 + Sn/K,) + Sn/Keq (14)
Now KX| is known from measurement of initial rate of uptake at different S,; K, is known
from the 1/J : S„ plot; Keq is equal to the equilibrium accumulation ratio (unity for
nonenergized transports); then Kx, can be calculated from any Kx at an appropriate
Sn. J~ax is then derived from the condition that Kcq = KX,J^;1X/KXJ ^ . - 1
There are a number of ways to distinguish between the “ channel” and the “ carrier”
mechanism, based on measuring unidirectional fluxes under different conditions.4 In the (1)
zero-trans procedure (zt), we measure initial rates (S„ = 0); in the (2) equilibrium exchange
(ee), we measure flows when S, = S„; in the (3) infinite-trans (it) procedure, we set up a
very high S„ and follow rates of flow of different S,; in the (4) infinite-cis procedure (ic),
we follow the rate of flow from extremely high S, toward various small SH’s. Procedures
(1) and (2) do not qualitatively distinguish between the concepts, but in (3) and (4) finite
values for K r are obtained only for the carrier mechanism so that the channel can be rejected.
If 1/Jmaxe, = . „ the simple channel concept is valid.
Another important diagnostic feature is the occurrence of the so-called countertransport
which is caused by the bilateral “ competition” of S, and SMfor the same carrier. Although
this is predictable for all the complex carrier mechanisms we shall demonstrate it on a
simplified version of Equation (8) for two competing solutes (most conveniently S and R
as the unlabeled and labeled forms of the same compound, respectively), in a mediated
6 Structure and Properties o f CelI Membranes
diffusion system. Equation (8) can be drastically simplified if pseudoequilibria of the

association-dissociation reactions can be assumed so that b/c = d/c = K and e = f and
g = h (and the same for S and R). Let S/K = S' and R/K = R '. The appropriate equation
for the rate of flow of R in the presence of S is
____________R,(gS,< + gR„ + e) - R„(gS; + gR, + e)____________

Res) g t + gR^ + e^ j + + R^ ^g<^ + gR^ + e^ j + s i + R^
(15)
In an experimental arrangement where we preincubate cells with R until R, = R„ = R and

then add S,, at that moment (when S„ = 0)
. = T ______________________ ~ g R ' s l_____________________ (16)

R<S, max ( g S - + g R , + e ) ( 1 + R () + ( g R , + e ) ( j + S J + R ()
Since, by convention, movement into cells is taken as positive solute R will move out of
cells against its concentration gradient at the expense of a flow of S into cells down its
concentration gradient.5
In a channel mechanism the countertransport phenomenon does not occur. If Equation
(4) is expanded to include movement of S so that
JR(S) = E,(adR,/S, - bcR n/SJ/Ib + d + aR,/S, + cR^S,,)
under the above condition that S„ = 0 and R, = R„ = R, JR(S) = —b has no meaning.

Coming back to Equation (8) we shall see that net flow will cease when
Sn/S, = adfg/bceh (17)
If no energy flows into the system through a coupled reaction S„ will reach the value of S,
in equilibrium and hence adfg = bceh. This applies to the so-called mediated or facilitated
diffusion. If, however, energy is used to drive the transport of S uphill, then in equilibrium
(for nonmetabolized molecules or ions!) SM> S,.
Then one or more of the constants in the numerator will be increased by this energy
coupling, say, with the ATP-splitting reaction (in one type of primary transport) or with the
dissipation of the gradient of H + or N a+ (in the so-called secondary active transport).
The primary active transport systems so far known concern the transport of ions and,
again, mainly the transport of cations, and most of them are rather complicated molecules
or molecular complexes. It has not been possible to ascribe a molecular-level meaning to
the rate constants in such transports so as to satisfy Equation (17), although one may envisage
a “ phosphorylation potential” , an “ oxidative pressure” , “ photon pressure” , and the like,
to affect the rate constants driving the process uphill.
The situation is somewhat clearer with the so-called secondary active transport which is
“ energized” by mediating a downhill (both concentration- and potential-wise) transport of
a driving ion, either H + or N a + .
However, to derive the rate equations one must consider the various possibilities of
association of solute S and ion A with the carrier (E + S + A, E -I- A + S) and of its
dissociation (ESA - S - A, ESA - A - S), as well as the fact that some of the complexes
may not move across the membrane.
The schematic model, in rapid equilibrium
Volume 11 1
es
^ ES, V ES„ £
I
Ei vs
ke
lb
Ell
V
EAt ^
êa s lb
EAu
K4
11 V
ESA[ x-
b-
*esa \ 1h
ESAn -
or in steady state
f 4f
ESAj ESA II ESA ESA ESA] ; ESA II
4g
A
EAj
/
EAj
V ' -I
ESj ESn
‘11-EAj ESn
b
, 4' -II
4' En
4b Ei ^ ; En
4‘
El El ^
yields rate equations of considerable complexity. The initial rate equations are somewhat
simpler and the kinetic parameters Kx and Jmax can be derived from them (Table 1).
Table 1 of the section on pH effects lists the influences that increasing A,, A„, and Ai|i
will have on the parameters of these and similar models. Other predictions are made if a
negatively charged carrier is assumed to exist.6
A somewhat more complicated situation arises if the steady-state approach is applied to
the so-called random-sequence of binding, i.e., E + A + S as well as E + S + A. The
general expression for the initial rate here is of the form
Js0 = (a S f + P S ,) / ( 7 S f + 8S, + e) (18)
and it is known about such rate expressions that, in a Lineweaver-Burk arrangement, a

biphasic convex plot is obtained if a 8 > (38 and ae < [38, while various types of concave
plots are obtained for other combinations of these restrictive conditions.7 It seems to hold
for this situation that the convex plot is obtained for relatively low concentrations of the
activator ion, while a concave one is obtained as A is increased.
The maximum accumulation ratio predicted by the above models generally relates S„/S|
to A,/An and the membrane potential as exponent of e. In the simplest cases, where only
one loop of the carrier scheme is present, such as where kes and kea are equal to zero, the
expression is
S„/S, = (ion/ion,,)" e nFA,1,/RT = acegik/bdfhjl (19)
where n is the number of cations H + or N a + bound to the carrier for efficient solute transport.
For all other models the S„/S, ratio is less than shown by Equation (19).8
It should be noted that even if the system is tightly coupled (kinetically, this means that
kea and kes are zero) the ratio predicted by Equation (19) is not reached at higher concentrations
<
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Volume II 9
of S. In fact, Sn/S, may be less than zero at very high concentrations. This situation is
predictable on the basis of the following assumption. If the source of energy for the active
transport, Q — e .g ., the pH difference — is seen by the transport system only as it is limited
within the membrane, its local “ concentration” will be Q = Q, — ES, Q, being the total
concentration and ES the concentration of the carrier complex to which Q can be bound. If
the carrier concentration is high enough S may be bound to form a large concentration of
ES so that Q will decrease substantially. If Q is part of one of the numerator constants of
Equation (19) it can under these conditions decrease so that 8,,/S, < l . 9
In all the previous derivations it was assumed that one solute molecule is bound per carrier.
If two molecules or ions are bound per carrier the binding of the second one or movement
across the membrane of the doubly bound carrier will be either supported by the first molecule
on the carrier (positive cooperativity) or it will be impaired (negative cooperativity). The
corresponding reciprocal plots will then be concave or convex, respectively, corresponding
to the general rapid-equilibrium equation for such a case which will have the form of Equation
(18) and a 8 > (3~y, a e > (38 for positive cooperativity and a 8 > (3y, a e < (38 for negative
cooperativity.
From positive cooperativity in transport one can predict an interesting kinetic phenomenon
that has been termed cotransport.10 In a situation like that used for the study of counter
transport, if equilibrium is reached with a low concentration of solute, addition of another
low concentration of the same or related solute will evoke a transient increase of the pre
equilibrated intracellular concentration, due to the parabolic dependence of rate on concen
tration in the low concentration range. If we assume a rapid-equilibrium system in which
Kes is the dissociation constant of the first molecule, Kess that of the second molecule, then
preequilibrated labeled R will move inward (cotransport) if 1 > S(S + R)/KesKess.
REFERENCES
1. Kotyk, A. and Janacek, K ., M embrane Transport — An Interdisciplinary Approach, Plenum Press, New
York, 1977. 207.
2. Stein, W . D. and Lieb, W . R ., A necessary simplification of the kinetics of carrier transport, Isr. J.
C hem ., 11, 325. 1973.
3. Cuppoletti, J. and Segel, I. H ., Kinetic analysis of active membrane transport systems: equations for net
velocity and isotope exchange, J. Theor. B iol., 53, 125, 1975.
4. Stein, W . D ., Concepts of mediated transport, in M embrane Transport, Bonting, S. L. and de Pont, J.,
E ds., Elsevier/North-Holland, Am sterdam . 1981, chap. 5.
5. R osenberg, T . and YVilbrandt, YV., Uphill transport induced by counterflow, J. Gen. Physio!., 41, 289,
1957.
6. K otyk, A ., Coupling of secondary active transport with |i H- , J. Bioenerg. B iom em br., 15, 307, 1983.
7. Segel, I. H ., E nzym e Kinetics, W iley-Interscience, New York, 1975.
8. Kotyk, A ., Critique of coupled vs. noncoupled transport of nonelectrolytes, in 5th Winter School on
B iophysics o f M em brane Transport, Vol. 2, Agricultural University Press, W roclaw, 1979, 49.
9. K otyk, A and Struzinskÿ, R ., Effect of high substrate concentrations on active transport param eters,
Biochim. Biophys. Acta, 470, 484, 1977.
10. K otyk, A. and Janacek, K ., C ell M embrane Transport — Principles and Techniques, 2nd ed ., Plenum
Press, New York, 1975, 89.
Volume II 11
Chapter 2
pH EFFECTS O N M E M BR A N E TRANSPO R T
Arnost Kotyk
T A B LE OF CONTENTS
I. Kinetic E ffects........................................................................................................................ 12
II. Thermodynamic E ffects........................................................................................................ 13
III. Other pH-Dependent Translocations ............................................................................... 16

A. Dissociable M olecules.......................................................................................... 16
B. Indirect E ffects.........................................................................................................17
VI. C onclusion................................................................................................................................17
References............................................................................................................................................ 17
I. KINETIC EFFECTS
The majority of transport mechanisms and practically all those that have to do with the
physiologically important solutes (both nonelectrolytes and ions) include a specific interaction
of the transported solute with a membrane protein. Such membrane proteins resemble en
zymes known from the aqueous “ soluble” phase both in their molecular structure1 and in
their kinetic behavior,2 the “ product” of the reaction being identical with the substrate but
translocated to the other membrane side.
One of the characteristic properties of enzymes, as well as membrane carrier proteins, is
the dependence of their catalyzed reaction rate on pH which, in the vast majority of cases,
is bell-shaped, there being an optimum at intermediate pH values. This has been analyzed
for soluble enzym es’ 4 and shown convincingly to be due to protonation of the enzyme-
active site which is functional only if it is half protonated, i.e., it does not function when
no protons are bound, as well as when two protons are bound, as shown in Scheme I.
E
K2 \ l K,
EHH EH
k* 4 ks k a)
EHHS 5= ^ EHS product
ES
(E is the enzyme, S the substrate, and H hydrogen ion.)

The expression for initial rate based on the rapid-equilibrium assumption (i.e., all the
forms above may be considered to be in a virtual equilibrium with one another) states that
Et ‘ ks
v0
1 + [H + ]/Ks2 + KS|/[H +] (1)
____________________________ S____________________________
’ k s(1 + [H + ]/K2 + K,/[H + ])/(l + [H + ]/Ks2 + Ksl/[H + ]) + S
From this, the apparent maximum rate V is easily derived as the first fraction of the
expression, the apparent half-saturation constant Km as the fraction appearing in the denom
inator of the right-hand-side compound expression.
Plotting the logarithm of V, Km and their ratio against pH yields the curves shown in
Figure 1.
In a transport reaction, the pH dependence is similar but somewhat more complicated, as
one must take into account the fact that the membrane protein is exposed alternately to the
extracellular and to the intracellular pH.
Assuming again that it is only the properly protonated carrier form which is productive,
i.e., which moves across the membrane, and introducing the simplification that the system
is intrinsically symmetrical, i.e., all the various dissociation constants are identical with
their counterparts on the other membrane side, we may write the kinetic scheme as follows:
Volume II 13
El EII
EHHj
K2
7
K
11
EHj
a I* K2
|K , (II)
k s2 Ksf b t Ks k s2
EHHS! ** — ^ EHSj EH Sn EH H Sn
K4 ESj
| k„
ESn
The parameters of the initial rate equation (for V = Jmax; Km = K r) are as follows:
j = ________________________________ (2a)
max 1 + [H + ],/Ks2 + Ksl/[H + ]I + (b/a)(l + [H + ]„/Ks2 + K,/[H +]„) v ’
Ks(2 + K ,/[H H [H + ],/K2 + K,/[H +] m + [H + ]„/K2)

Kt = (2b)
1 + [H +],/Ks2 + Ksl/[H + ], + (b/a)(l + [H + ]„/Ks2 + K,/[H +]„)
A plot analogous to that in Figure 1A, but taking into account intracellular pH, is shown
in Figure 2. Spreading of the intersections of the straight lines with respect to Figure 1
indicates that in assigning the apparent pH values to the amino acid residues within the
carrier-active site, one must be aware of the intracellular pH exerting its effect.5
II. T H ER M O D Y N A M IC EFFECTS
In the so-called secondary active transport, a cation (H + or N a+ , but only very excep
tionally interchangeably) is cotransported with another solute, usually an organic molecule,
such as sugar or amino acid. In fact, the concentration difference of this ion across the
membrane, plus the membrane potential, constitutes the driving force for transporting such
solutes uphill. If the cotransported ion is H + we can describe effects of pH at each of the
membrane sides on kinetic parameters, as well as of ApH across the membrane on the
equilibrium distribution of the transported solute. The kinetic equations involved derive from
a transport scheme, such as
E es
! ES¡ S’ ESij
s ke
11
El EII
u
EHj
> k eh
7 EH II
(HI)
Jl
-ESI •
k esh
/ ESHn
which includes all sequences of binding of solute S and activating ion H to the carrier enzyme
E. Depending on whether the system is in rapid equilibrium (like above in the effect of pH
-1 '
Km
log Km
-3
-5 ■
2
log V
0 ■
P *$ 2 P *2 P *l P *s,
-2 ■
3-
log V /K'm
m
] -
-] -
2 3 4 5 6 7 8 9 10
pH„
FIGURE 1. Plot to determ ine intrinsic dissociation con

stants o f an enzym e-catalyzed reaction. Calculated on the
basis of K, = 1 0 - 7 Ai, K , = 10 5 M , K, = 10 1 M , Ksl
= 1 0 1' M , Ks2 = 10 3 M , k, = 1.
on rates), whether the sequence of binding is E + S + H o r E + H + S, and on whether

all the complexes involved are mobile in the membrane, one may derive more or less complex
equations such as shown in the section on transport kinetics. It should be noted that all the
complexes that move across the membrane, including the free carrier, are in fact half-
protonated as discussed in the earlier section. However, the protons shown in the present
scheme are ligands that are transported across and released at the other side of the membrane,
whereas the protons involved in the catalytic effects on rates remain bound to the carrier
unless ambient pH changes considerably during the transport process.
Since the binding of a proton to the carrier increases its positive charge, the movement
of the complex will be favored in the direction into cells by the existing transmembrane
potential, which is under practically all conditions oriented with its negative side inward. It
will be seen in Table 1 how changes of pHout, pHin, and of the membrane potential affect
the kinetic parameters of proton-driven transports, for a variety of models, both in rapid
equilibrium and in steady state.
It should be observed that these effects may obscure or enhance the catalytic pH influences
discussed above.
Now as the ApH is part of the driving force it will be reflected in the accumulation ratio
of a H +-symported solute as may be derived either from the appropriate rate equation or
from the thermodynamic expression for the electrochemical potential gradient of protons
across the membrane6
Volume II 15
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A p.H- = Ali-n;,', - (J.Ho«u, - RT ln([H + ]in/[H +]out) - FA v)j (3)
where the standard zeroed potentials are concentration- and potential-independent and taken
to be equal; R is the gas constant (8.314 J mol T is the absolute temperature (in
K), F is the Faraday constant (96.5 kC m o l" 1), and Ai|» the membrane potential (in V). It
is often assumed that in Scheme III only kcsh and ke are nonzero, the argument being that
if kch were appreciable it would short-circuit the H + concentration difference at the two
membrane sides uselessly, and if kes were mobile this would represent an intrinsic leak such
that the system would operate less efficiently. Be it as it may, a system thus defined (termed
“ tightly coupled” ) is much easier to describe kinetically and it is the only variant that can
be handled by a thermodynamic approach, proceeding from Equation (3). The maximum
accumulation ratio of a FI+-driven solute is then derived by assuming that the Gibbs free
energy available from the electrochemical potential gradient of H + is
AG = nA |i,H+ and —AG = RT In ([solute]in/[solute](H„) (4)
where n is the number of protons accompanying the solute during transport. Hence, in
equilibrium
[solute]in/[solute]OUI = ([H + ]out/[H +]in)n e nFAiJj/RT (5)
It is often difficult to determine the true ApH just across the membrane, not to mention
uncertainties of estimating the actual membrane potential,6 so that more often than not the
accumulation of solute calculated from macroscopic assays of ApH and Ai|) does not tally
with experimentally determined A|xM)hll(!. M oreover, the maximum attainable value of A|xM)|Ute
is observed only at low concentrations of solute, as if the saturation of the carrier with solute
at its high concentration were not adequately met by the supply of H + .7
III. OTHER pH -D E PEN D EN T T R A N S LOCATIONS
A. Dissociable Molecules
It should be observed that some molecules capable of dissociating or associating a proton
but existing also in the undissociated form, such as weak organic acids or weak bases, will
distribute themselves unevenly across biological membranes, according to the following
simple principle. If the neutral form is the only one that can permeate freely — and this is
most often the case in the above-mentioned examples — it will reach identical concentrations
in the two membrane-bathing solutions, but then will dissociate or associate the proton
according to the ambient pH. From measuring the analytical concentrations of the substances
(neutral plus charged) at both membrane sides, one may in fact deduce the intracellular pH,
using the following formulas:
[solute],,,
pHm = pHolil + log (J _|_ J 0 P Ka “ PHout^ -)- lQ P K i*-pHout (6)
[solute]oul
(for weak acids, used if pHm > pHout), and
[solute], „ (1 + 1 0 pH out + pKfc 14^ _|_ J Q p H out + p K b 14

pH m = pHout - log (7)
[solute]oul
(for weak bases, used if pH in < pHnllt). Here pKa and pKb are the negative logarithms of
the dissociation constants of the acid and the base, respectively.
Volume II 17
B. Indirect Effects
There are various indirect effects of pH on membrane transport parameters that have to
do with the local interactions of membrane constituents, especially charged phospholipids.
Among the more important ones one should mention the surface potential tjis, generally
believed to be caused by the negative charges predominating on membrane surfaces, due
especially to phosphate anionic residues. It causes the concentration of a charged solute at
the membrane (Sm) to be different from the “ analytical” bulk concentration (Sb) as follows:
Sm = S b e - n* /kT (8)
where n is the number of positive charges on the solute, q is the charge of the electron
(0.1602 fC), and k is the Boltzmann constant (1.38 • 10“ 23 J K _1). From this expression it
is useful to extract the quantity e ~q,K/kl and set it equal to, say, y. Then Sm = Sby".
This potential exerts a variety of effects on the transport of ions and ion-accompanied
transports of other solutes.8 Thus, in a symport of a cation with an anion the Kx of anion
uptake (in the simplest case of random binding) will be Ka-y, the Kx of cation uptake will
be Kc/y. The Jmax of the anion will be given by Jmax-0,Scy/(Kc + Scy), that of the cation by
Jmax.o‘Sa/(K ay + Sa). (Here subscripts a refer to anion, subscripts c to cation concentrations
or constants.)
Because cations decrease the surface potential low pH will do the same and thus affect
the Kx and Jmax of a variety of transports.
IV. C O N C LUSIO N
It will have become apparent that ambient pH can affect various transports in different
ways.
1. Every saturable transport will be less effective (lower Jmax and higher Kx) at the ends
of the pH scale because of improper protonation of the carrier-active site.
2. Most transports of cations will have both K, and Jmax increased and most transports
of anions will have the two parameters decreased at low pH because of decreased
surface potential.
3. Transports of solutes in symport with H + will be affected in a variety of ways by low
pH, invariably by a decrease of K, and, in most cases, by an increase of Jmax.
REFERENCES
1. Carafoli, E. and Scarpa, A ., Transport ATPases, Ann. N.Y. Acad. Sci., 402, 207, 1982.
2. K otyk, A. and Janacek, K ., Cell M embrane Transport — Principles and Techniques, 2nd ed ., Plenum
Press, New York, 1975.
3. Dixon, M . and W ebb, E. C ., Enzymes, Longmans, Green, London, 1958, 116.
4. M ichaelis, L ., D ie W asserstoffionenkonzentration, Springer-Verlag, Berlin, 1922, 48.
5. K otyk, A. and H orak, J ., Effects of pH and tem perature on saturable transport processes, in Water and
Ions in Biological Systems, V asilescu, V ., Pullm an, B ., Packer, L ., and Leahu, L ., E ds., Plenum Press,
New York, 1985.
6. K otyk, A ., Coupling of secondary active transport with p.H- , J. Bioenerg. B iom em br., 15, 307, 1983.
7. K otyk, A. and Struzinsky, R ., Effect of high substrate concentrations on active transport param eters,
Biochim. Biophys. Acta, 470, 484, 1977.
8. Room ans, G . M . and B orst-Pauw els, G. W . F. H ., Co-transport o f anions and neutral solutes with
cations across charged biological membranes. Effects o f surface potential on uptake kinetics, J. The or.
B iol., 73, 453, 1978.
Volume II 19
Chapter 3
THE EFFECT OF M E M B R A N E LIPIDS O N PERM EABILITY A N D

TR A N SPO R T IN PROKARYOTES
Ronald N. McElhaney
T A B L E OF CONTENTS
I. Introduction............................................................................................................................20
II. Definition of Membrane Lipid F lu id ity ...........................................................................22
III. Arrhenius Plots of Permeability and Transport P rocesses........................................... 23
IV. Membrane Lipids and Passive D iffusion......................................................................... 26

A. N onelectrolytes..........................................................................................................26
B. Ions ............................................................................................................................29
V. Membrane Lipids and Facilitated D iffusion....................................................................30
VI. Membrane Lipids and Active Transport........................................................................... 31

A. Sugar T ra n sp o rt........................................................................................................31
B. Amino Acid Transport ........................................................................................... 38
C. Ion T ransport............................................................................................................. 43
D. Other Transport S y ste m s......................................................................................... 46
VII. C onclusions.............................................................................................................................46
A cknow ledgm ent............................................................................................................................... 47
References 47
I. INTRO DUCTION
The cells of prokaryotic microorganisms, because of their relative genetic, morphological,

and biochemical simplicity, possess a number of inherent advantages over the more complex
eukaryotic cells for studies of the structure and function of biological membranes generally,
and for the investigation of membrane permeability and transport processes in particular.
In addition to their relatively rapid generation times and ease of cultivation, most prokaryotes
contain only a single membrane, the limiting or plasma membrane, as well as an extracellular
wall structure. In contrast, most eukaryotic cells contain a variety of cytoplasmic and or
ganelle membrane systems in addition to their plasma and nuclear membranes. Thus, the
isolation of substantial quantities of pure plasma membrane for biochemical or biophysical
study is a fairly straightforward process for most prokaryotic m icroorganisms,1 2 particularly
for the mycoplasmas, which lack a cell w all.3 On the other hand, the isolation of a particular
membrane from eukaryotic cells in relatively pure form and in usable amounts can be a
formidable task, especially in the case of plasma membranes.4 Finally, the lipid polar head
group and fatty acid compositions of prokaryotic membranes are generally considerably
simpler than those of eukaryotic membranes. In prokaryotes, as few as three major phospho-
and/or glycolipid types may be present, and each of these typically possesses only a relatively
small number of saturated, monounsaturated, branched-chain, or alicyclic fatty acids. In
addition, sterols are generally absent in prokaryotes, although pentacyclic triterpenes of the
hopanoid family, which are structurally and functionally analogous to sterols, may be present.5-6
In eukaryotes, a larger number of phospholipids are present, as well as sphingolipids and
sterols. In addition, a number of polyunsaturated as well as saturated and monounsaturated
fatty acids are found. Thus, a prokaryotic plasma membrane may contain as few as ten
major lipid molecular species (chemically unique combinations of polar head groups and
fatty acyl chains), while a typical eukaryotic plasma membrane possesses at least ten times
as m any.7* It is thus not surprising that our knowledge of the structure and function of
prokaryotic plasma membranes generally greatly exceeds that of eukaryotic plasma mem
branes. 13 9 The human erythrocyte membrane may be a partial exception to this generalization,
since red blood cells, which are highly differentiated and simplified eukaryotic cells, possess
many of the advantages offered by prokaryotes.
In addition to possessing relatively simple membrane lipid polar head group and hydro
carbon chain compositions, in many cases considerable alterations in membrane lipid struc
ture can be induced in prokaryotic plasma membranes through genetic and/or environmental
manipulation. In a number of Escherichia coli fatty acid auxotrophs,1-2 for example, or in
several mycoplasma species,3 marked changes in membrane lipid fatty acid composition can
be produced by culturing cells in the presence of appropriate exogenous fatty acids, which
are readily incorporated to high levels. In several mycoplasmas, notably Mycoplasma
m ycoides10 and Acholeplasma laidlawii B ," “ fatty acid-homogeneous” membranes (i.e.,
membranes whose lipids contain essentially only a single species of fatty acyl group) can
even be obtained. M oreover, substantial amounts of cholesterol and other sterols can be
incorporated into the plasma membranes of prokaryotes such as E. coli12 and A. laidlawii,9
even though these organisms normally completely lack these constituents. Conversely, certain
sterol-requiring mycoplasmas, which normally possess high levels of cholesterol in their
plasma membranes, can be adapted to grow with very low levels of cholesterol.1314 The
* The protein com position of prokaryotic plasma mem branes, however, may be quite complex. Thus, the bac
terium , E scherichia coli, has been estim ated to contain at least 300 different polypeptides in its plasma
m em brane, while the sim pler m ycoplasm a, Acholeplasm a laidlawii B, has at least 140.8 This situation doubt
lessly arises from the fact that in most prokaryotes all mem brane-associated processes must be localized on a
single m em brane system , whereas a num ber o f specialized membranes exist in most eukaryotic cells.
Volume II 21
fluidity and phase state of the membrane lipids of these and other microorganisms can thus
be varied in a marked yet controlled manner through variations in the chemical composition
of the hydrophobic core of their lipid bilayers. O f course, changes in the fatty acid com
position and sterol content of the plasma membranes of some eukaryotic cells are also
possible, but the degree of compositional alteration obtainable is quite limited in comparison
to prokaryotic systems. Moreover, the presence of sterols and of biochemical compensatory
mechanisms in most eukaryotic cells further restricts one’s ability to significantly alter the
physical properties of the membrane lipids.
The membrane lipid polar head group composition of prokaryotic organisms can also be
markedly altered by environmental manipulations or by the utilization of biochemically
defined mutants conditionally defective in various portions of the phospho- or glycolipid de
novo biosynthetic pathways. In E. coli mutants, for example, not only can the proportions
of the “ normal” membrane lipids (phosphatidylethanolamine [PE], phosphatidylglycerol
[PG], and cardiolipin) be considerably varied, but membranes containing substantial quan
tities of metabolic intermediates (phosphatidic acid [PA], phosphatidylserine [PS], or di
glyceride), which are normally present in the membrane only in trace quantities, can be
produced.7 Moreover, the membrane lipid/protein ratio can be altered using conditional
mutants defective at early steps in the lipid biosynthetic pathway. These mutants should
prove very useful in studies of the relationship between lipid bilayer surface properties
(chemical composition, degree of hydration, charge, etc.) and various membrane-associated
processes, including membrane permeability and transport. Moreover, since the fluidity and
phase state of the membrane lipids depend on the structure of their polar head group as well
as on the structure and length of their hydrocarbon chains,15 the physical properties of the
E. coli membrane lipids can be altered without changing fatty acid or sterol composition,
a possibly advantageous feature in certain types of studies. Again, although a small number
of biochemically defined choline, inositol, and sterol auxotrophs have been isolated from
eukaryotic microorganisms and from a few mammalian cell lines, these have proven less
useful for membrane studies, since the increased biochemical complexity and the presence
of compensatory metabolic pathways limit the polar head group variability which can be
obtained with fully viable cells.7
Prokaryotic microorganisms also possess several important advantages over eukaryotic
cells, specifically for investigations of membrane permeability and transport processes.
Because prokaryotic cells normally lack cytoplasmic and organelle membranes, they usually
behave essentially as one-compartment systems bounded by a single permeability barrier,
the plasma membrane. In contrast, eukaryotic cells typically contain a number of intracellular
compartments, each of which is bounded by its own permeability barrier. Moreover, in
prokaryotic cells all protein-mediated transport systems are, of course, associated exclusively
with the plasma membrane, whereas in eukaryotic cells a given molecule or ion may be a
substrate for several different transport systems associated with different cytoplasmic or
organelle membranes in addition to the plasma membrane. Finally, the ability to construct
and isolate transport mutants makes it possible to selectively alter, delete, or enrich pro
karyotic plasma membranes with the various protein components of active transport systems
and, thereby, to gain additional insight into the function of these components, whereas this
is more difficult with eukaryotic cells. For these and related reasons, the actual molecular
mechanisms of facilitated diffusional and active transport processes and the effect of mem
brane lipid physical properties on these processes are better understood in bacteria than in
other cell ty p es.1617
In this chapter I will attempt to summarize and critically evaluate recent studies from this
and other laboratories on the effect of membrane lipids on the movement of molecules across
the membranes of prokaryotic microorganisms. More specifically, this contribution will
focus primarily on the relationship between membrane lipid fluidity and phase state and
passive permeability and various protein-mediated transport processes. Before embarking

on this analysis, however, brief discussions of the definition and measurement of membrane
lipid fluidity and phase state and of the interpretation of diffusional and transport experiments
are in order.
II. D EFINITION OF M E M BR A N E LIPID FLUIDITY
The terms “ viscosity” and “ fluidity” were originally developed by physical chemists to
describe the flow properties of macroscopic fluid systems. Viscosity is a measure of the
frictional resistance that a fluid offers to an applied shearing force, that resistance resulting
from a transfer of momentum from one layer of the moving liquid to the next. The fluidity
of a fluid is simply the reciprocal of its viscosity and is thus a measure of the tendency of
that fluid to flow in response to an applied force. The viscosity (or fluidity) of a system can
be determined by measuring the rate of settling of a sphere in a fluid, as the frictional force
retarding the movement of the sphere is directly proportional to the viscosity of that fluid.
For most fluids, the viscosity decreases with increasing temperature and increases with
increasing pressure. However, under a given set of environmental conditions, a single
coefficient of viscosity is sufficient to fully describe a particular fluid system, at least
macroscopically.
The terms fluidity and viscosity are often used in membrane research, usually in a rather
qualitative way, to indicate something about the tightness of packing and/or about the relative
mobilities of the lipid and protein components in the membrane. Although these terms are
useful, they are inherently ambiguous when applied in a general sense to a highly anisotropic,
asymmetric, two-dimensional structure such as a biological membrane, which, in addition,
is only a few molecules thick .18 19 Clearly, the organization and mobilities of lipids and
proteins will be quite different when measured from within the plane of the membrane than
when measured across the membrane plane. Not only does the effective or “ average” fluidity
of a biological membrane differ within and across the membrane plane, but also within the
membrane structure itself. This is a consequence of the existence of gradients of motion
and orientational order within the lipid22 26 and protein27 28 molecules themselves. It is clear
from these and other considerations that no single “ coefficient of viscosity” can adequately
describe the fluid properties of a biological membrane.
The local or “ m icroviscosity” of the lipid and protein components of model and biological
membranes are usually determined by one or more of a variety of spectroscopic techniques.
In considering the results of such studies, one should remember that these techniques often
measure primarily either average orientational order or average rates o f motion (again only
within certain time scales); often single techniques do not provide reliable measures of both
of these components of fluidity simultaneously. Although orientational order (usually ex
pressed as an order parameter, S) and rates of motion (usually expressed as a relaxation
time, T, or correlation time, t ) seem usually to be inversely related, as in the case of the
phospholipid bilayer gel to liquid-crystalline phase transition, a simple inverse relationship
between order and motion may not always obtain. For example, the presence of integral
membrane proteins in membranes has been reported by several techniques to decrease both
the motional rates and the orientational order of phospholipid hydrocarbon chains9 29 30 (but
see References 23, 24, and 31). Still, it seems most likely that changes in temperature, fatty
acid composition, and cholesterol content do generally affect the order and motional rates
of various portions o f the phospholipid molecule in opposite ways, although this has by no
means been rigorously established, even for simple model systems. In this chapter, the term
“ fluidity” will be used to describe both the relative degree of orientational order and relative
rates of motion, although in most studies both of these parameters may not have been
rigorously determined. M oreover, the term “ fluidity” will be used only to describe the
Volume II 23
biologically relevant liquid-crystalline state of the lipid bilayer. I will thus differentiate
between changes in membrane lipid fluidity (within the liquid-crystalline state) and changes
in membrane lipid phase state, although, of course, orientational order and rates of motion
are profoundly affected by lipid phase transitions.
III. A R R H E N IU S PLOTS OF PERM EABILITY A N D

T R A N SPO R T PROCESSES
Studies of the temperature dependence of chemical, enzymatic, or transport processes can

often provide information valuable for an understanding of the molecular mechanisms of
these processes. The effects of temperature on the rate constants characterizing a chemical
or a biological process are often analyzed in terms of an empirical activation energy (E J
according to the Arrhenius equation, which may be written as
E, 1
log k
2.3R T
where k is the rate constant of interest, R the gas constant, and T the absolute temperature.
In practice the numerical value of E is determined from the slope of a plot of log k vs. 1/
T (the Arrhenius plot). For most chemical reactions, and for chemical reactions catalyzed
by most soluble enzymes, Arrhenius plots are linear over the accessible temperature range,
although in both cases exceptions are know n.32-33 In these cases, the enthalpies of activation
of these processes, which differ from their apparent activation energies only by a quantity
equal to RT (about 0.6 kcal/mol), are essentially temperature invariant. For membrane-
associated transport systems and enzymes, however, nonlinear Arrhenius plots are often
obtained.17-34 In most publications Arrhenius plots consisting of a relatively sharp break
between two (or sometimes more) straight line segments are reported, while in a few cases
Arrhenius plots of membrane-associated functions are depicted as smooth curves. Normally,
even abrupt changes in the apparent activation energies of membrane transport or enzymic
processes are not accompanied by a significant change in the reaction rate, although in
several cases actual jum p discontinuities in Arrhenius plots of membranous enzymes have
been reported.35 It has been proposed that true jump discontinuities can only arise as a
thermodynamic consequence of a phase change.36 However, a break or change in slope in
an Arrhenius plot, which is not accompanied by a marked change in reaction rate, can arise
from a number of causes.32-33
One should be aware that the use of the simplified form of the empirical Arrhenius equation
given above involves a number of assumptions about the process of interest. Specifically,
it assumes that the reaction geometry and activation entropy, as well as the activation
enthalpy, are independent of temperature! These assumptions are not always met, even for
relatively simple chemical reactions.33 O f course, if a simple linear relationship between the
logarithm of k and 1/T is indeed obtained for a particular process, then these assumptions
are probably valid, at least to a first approximation. However, if a validly constructed
Arrhenius plot departs substantially from a simple linear relationship, then at least one (and
perhaps more) of these factors must be temperature dependent. If this is so, then it is no
longer valid to employ a simplified form of the Arrhenius equation and the slope of the
Arrhenius plot is no longer a reliable indicator of the reaction enthalpy. Since it is generally
very difficult experimentally to evaluate activation enthalpies, activation entropies, and
reaction geometries and their temperature dependencies, particularly for complex biological
processes, in practice a valid mechanistic interpretation of an Arrhenius plot of a biological
process that deviates from a simple linear relationship is usually not possible. These points
have recently been stressed by Bagnall and Wolfe37 in their useful critique of the use of
Arrhenius plots in plant research.
It should also be pointed out that an Arrhenius plot of an enzymic reaction or transport
process will normally be linear only if a single species of catalyst is responsible for the
chemical process under study, and only if one particular step in the overall reaction is rate-
limiting over the entire temperature range examined. In biological membranes, several
enzymes or transport systems may simultaneously participate in a given process. Moreover,
membrane enzymic reactions and transport processes are generally complex, multistep pro
cesses, and each partial reaction in the overall process may have a different temperature
dependence (and a different lipid dependence).14 Dixon and W ebb11 and Han12 have discussed
the effects of temperature on the rate of enzymic reactions generally and have formulated
experimental approaches to recognize and correct for some of the more trivial effects of
temperature. These effects can include such things as unrecognized temperature-induced
changes in the pH of aqueous buffers, in solution viscosity, and in substrate-binding affinity
(Km). In addition, Han12 has analyzed other factors that may produce nonlinearity in Arrhenius
plots of enzymic reactions, and classified them into two categories: (1) thermodynamic
factors, including all secondary equilibrium reactions that modify the elementary process
being catalyzed, and (2) kinetic factors, attributed to changes in the rate-limiting step oc
curring within the experimental temperature range. Although the above treatments were
developed for soluble enzymes, they apply to membrane enzymes and transport systems as
well. In much of the transport work reviewed in subsequent sections, it appears that the
factors noted above have usually not been explicitly considered, with the result that at least
some of the conclusions reached must be regarded as tentative.
Several attempts have been made to develop a quantitative and systematic analysis of the
mechanistic basis for nonlinear Arrhenius plots in membranous systems, taking into account
the unique properties of these systems due to their existence in a lipid environment, the
fluidity and phase state of which can also vary with temperature. W ynn-Williams19 has
proposed that the sudden change in the apparent activation energy of membrane enzymes
could be due to the simultaneous presence of pure lipid (in the gel state) and of enzyme
lipid phases (in a fluid state) in the membrane. If enzyme activity depends on the composition
of the enzyme-lipid phase, the temperature dependence of lipid solubility in the enzyme
lipid phase can lead to a sudden change in the apparent enzyme activation energy within
the lipid phase transition temperature range, without the activity of the functional enzyme
molecules undergoing an actual discontinuity. This is because the actual enthalpy of the
“ activated state” of the enzymic reaction is no longer equal to the slope of the Arrhenius
plot of enzyme activity within the phase transition range. If this treatment proves generally
valid for membrane enzymes and transport systems, it removes a theoretical difficulty, since
it is no longer necessary to assume that a marked change in the activation enthalpy is exactly
compensated for by a change in the activation entropy at the break temperature. However,
the existence of an apparent inherent energy-entropy compensation in the passive permeation
of simple lipid bilayers at their phase transition temperatures has recently been presented.40
Thilo et al.41 have provided evidence for membrane lipid phase transition-induced changes
in the activities of several transport systems that are compatible with the proposal of Wynn-
W illiams, in that a differential partitioning of the transport system into gel and liquid-
crystalline domains is postulated (see Section VI.A). However, the recent work of Silvius
and M cElhaney42 on the Mg2+ + N a+-ATPase of the A. laidlawii B plasma membrane is
not supportive of this proposal, since the physical state of the boundary lipid surrounding
this enzyme seems to determine its activity, rather than the composition of the annular lipid.
Finally, Silvius and McElhaney41 have systematically derived the rate-temperature relation
ships for a variety of physical models of membrane rate processes in order to predict the
Arrhenius plot shape appropriate to each. Interestingly, only a few models predict Arrhenius
plots with the “ biphasic linear” form most commonly reported in studies of membrane
enzymes and transport systems. Instead, most models predict Arrhenius plots consisting of
Volume II 25
3 (a) (b)
Tc Te
2-
I-
T0
O)
O
c
3
T¿T0 ) (c) (d)
i T¿T0 )
«_________ I__________l____
32 34 36 32 34 36
io3/r
FIGURE 1. Representative theoretical Arrhenius plots corresponding to

some o f the equations derived for several classes of physically realistic
m odels of lipid-protein interactions (for details, see Silvius and Mc-
Elhaney4'. In all cases the lipid phase transition midpoint (denoted Tc or
T [T „ |) has been arbitrarily set at 25°C (298.2 K). The small arrows along
the x-axis indicate the usual limits of temperature variation (5 to 40°C or
278.2 to 313.2 K) used in most biological studies.
smooth curves (see Figure 1). However, many of the models yield plots which can be fit
to two intersecting straight lines with a quite modest experimental error, particularly if the
slope change around the “ break” temperature corresponds to a change in apparent activation
energy of less than 15 to 20 kcal/mol, and the temperature range examined is not large
(Figure 2). These findings indicate a need for rigorous analysis of Arrhenius plot data in
terms of graph shapes other than sets of intersecting straight lines and for a cautious inter
pretation of the physical basis of Arrhenius plot “ breaks” . The need for accurate deter
minations of the true maximum rates of the membrane-associated process of interest, and
at a large number of experimental temperatures, for the valid interpretation of Arrhenius
plots has been stressed recently by several groups,44-45 and Silvius et al.4h have demonstrated
how unrecognized temperature-dependent variations in Km can produce Arrhenius plot ar
tifacts. Also, Keleti47 has recently called attention to numerical errors common in the im
proper use of Arrhenius and van’t Hoff plots. Moreover, the fitting of Arrhenius plot data
points byeye, as is usually done, may lead to controversies over whether the author’s
subjective representation is the most correct one. In particular, a tendency to draw two
straight lines through data points which actually fall on a single, continuously curving line
is often evident. Recently, Sprague et al.45 have developed statistical methods of assessing
the goodness of fit of Arrhenius plot data points by various types of curves, as well as by
straight-line segments. This is an important development, since of course two straight lines
will always fit a set of curvilinear points better than a single straight line. To utilize these
approaches effectively, however, a number of determinations of the rate of the process of
interest at each experimental temperature must be available, and the variance between
replicate measurements must be determined. Unfortunately, in little of the present literature
-I
0>
O
c -2
-3
-4
■ I» I —
32 33 34 35 36
\ / T x I03
FIGURE 2. The fit of a curved Arrhenius plot with two intersecting

straight lines. The data points plotted were com puter generated from an
equation giving activation enthalpies of 30 kcal/mol at 10°C and 10 kcal/
mol at 35°C and producing a sm ooth, curved plot over the entire tem per
ature range. The vertical line segments represent 59c error bars centered
on the computed curve. This curved Arrhenius plot can actually be quite
well fit, within experim ental error, by two straight-line segments having
apparent activation enthalpies of 11.6 kcal/mol above and 28.1 kcal/mol
below the “ break” temperature. However, no discrete break in this curve
actually exists. (W ith perm ission from Silvius, J. R. and M cElhaney, R.
N ., J. Theor. B iol., 88, 135, 1980. Copyright: Academic Press Inc. (Lon
don) Ltd.)
is this information provided. Thus, in many published studies, evidence for the existence
of a discrete Arrhenius plot “ break” is equivocal, and this has important consequences for
the interpretation of the experimental results.
IV. M E M B R A N E LIPIDS A N D PA SSIV E DIFFUSIO N
A. Nonelectrolytes
The relationship between membrane passive permeability to nonelectrolytes and membrane
lipid composition has been most systematically investigated with the simple prokaryote
A. laidlawii B. In a series of studies by McElhaney and co-workers,48'53 the fatty acid
composition and cholesterol content of the A. laidlawii B plasma membrane were varied
and the rates at which a number of nonelectrolytes passively diffuse into or out of intact
cells, and into and out of liposomes prepared from the total membrane lipid, were studied
as a function of temperature. Since alterations in membrane fatty acid composition or
cholesterol content do not alter the qualitative or quantitative distribution of membrane
Volume II 27
' .8 r 1.8
(o ) (b )
16=0 ’6 ' 16:0
-3 * M 18:2 ^ » r 14 - ‘ 1 8 :l c
• 12 16=0 12 - 16:0 16:0
• -
o io - ' 1 8 :1c o 10- 18:2 18:0
16=0
oc 16:0
e» 0.8 ‘ 18:0
a 0.8- " 18:1,
c
= 06 16:0 J 06 - 16:0
• 18:1, ' 18:0,
* 04 * 0.4 -
LO LO
0.2 : 16=0
18:0,
0.2 -
0o i 1 1 > *----- —1
i 1. 0^ > i_____ i_____ i-------- 1--------1
0 10 20 30 40 50 IO 70 IO 40 50 60
Temperature Temperature
18 1.8
12
(c )
14 :Q
1 8 :1C
iiv::: 1.2 -
(d )
14:0
18: lc
• 16=0
• 160
o
oc
10 18: 1c
a
oc
10 - 18: lc
» 08 18:0 a °8 18=0
0.6 18: lc J• 06 - 18 : lc
J 04
02
16=0
18: l c
*
LO
0.4 -
02 -
16:0
18 : lc
0 -1 0-
IO 20 30 40 30 0 10 20 30 40 30 60
FIGURE 3. (a and c) Initial swelling rates in isotonic glycerol of intact cells of A. laidlawii B grown in the
presence o f different com binations of fatty acids, with or without cholesterol, as a function of temperature; (b and
d) initial swelling rates in isotonic glycerol of liposom es, prepared from the total membrane lipids of A. laidlawii
B. as a function of tem perature. Under the experimental conditions employed here, the relative initial swelling
rates, m easured optically, are proportional to the relative rate of passive glycerol entry into cells and liposomes.
(From M cElhaney. R. N .. De Gier, J., and van der Neut-Kok, E. C. M ., Biochim. Biophys. Acta, 298, 500,
1973. With perm ission.)
proteins, and have only modest effects on lipid polar head group distribution,9 it was possible
to selectively study the effects of variations on the nature of the hydrophobic core of the
plasma membrane of this organism on its passive permeability properties. The nonelectrolyte
permeabilities of intact cells and derived liposomes were found to be markedly dependent
on the chemical structure and chain length of the membrane lipid fatty acids. The biosynthetic
incorporation of branched-chain or unsaturated fatty acids, or fatty acids of reduced chain
length, increased nonelectrolyte permeability to a similar extent in both cells and liposomes
(see Figures 3 and 4, respectively). The nonelectrolyte permeability of both plasma and
liposomal membranes above their phase transition temperatures was also reduced by the
incorporation of cholesterol. The mean activation energy values calculated for the permeation
o f several nonelectrolytes into intact cells and into liposomes were the same, within exper
imental error, and did not depend on the fatty acid composition or cholesterol content of
the membrane (see Table 1). Mean activation energy values did, however, depend on
permanent structure in such a way as to suggest that most nonelectrolytes permeate both
natural and artificial membranes as single, fully dehydrated molecules. The passive perme
abilities of a series of membranes of different fatty acid compositions were inversely related
to their gel to liquid-crystalline membrane lipid phase transition temperatures, suggesting
that permeation rates increase with increases in membrane lipid fluidity. Generally, similar
18 1.8
16:0 16 :Q
<1 ___ 16 io) 18 =2 (b ) 182
^ o l ° 14 ^ 16
16:0 ^ ' 4 16:0
18 : lc 12 18:1c
• 12 «
16:0 16:0
0 1.0 o 10
18:0 oc 18:0
a 0.8 16:0 » 0.8 16:0
c
=
•
06 18:1, J 0.6 18:1,
. I6:Q * 04
l1
o 04 LO
18:0, 16:0
0.2 02 18:0,
0 —t i-------- 1-------- 1-------- 1
10 20 30 40 50 10 20 30 40 50 60
Temperature Tftmn«rntitr«
1.8 18 ■
14:0 _ 14:0 _
(c ) (d )
S I-“
•
u
1.2
18:1c
16:0 _
18:1 c
ih •
12
18: l c
16:0
18:1 c
o 10 16:0 18:0
a 10
oc 18: Ic oc 18: lc
» 08 16:0 a, °8 16:0
J 06 18: lc J• 06 I8:lc
♦
*
LO
04 I 04 hoUttcri
LO
02 02
0
10 20 30 40 50D °0 10 20 30 40 50 60
FIG URE 4. (a and c) Initial swelling rates in isotonic erythritol of intact cells of A. laidlawii B, grown in the
presence o f different com binations of fatty acids, with and without cholesterol, as a function of tem perature: (b
and d) initial swelling rates in isotonic erythritol of liposomes, prepared from the total membrane lipid o f A.
laidlawii B, as a function o f tem perature. Initial swelling rates are m easures of the rate of passive erythritol entry
into cell and liposomes. (From M cElhaney, R. N ., De Gier, J., and van der Neut-Kok, E. C. M ., Biochim.
Biophys. Acta, 298, 500. 1973. With perm ission.)
results have been reported for the lipid dependence of glycerol passive permeation in an E.
coli unsaturated fatty acid auxotroph by Eze and M cElhaney.54
It has also been established that small changes in the chemical structure of the sterol
molecule present in the A. laidlawii B membrane can significantly alter the passive perme
ability of intact cells by altering the physical state of the membrane lipid. De Kruijff et al.51
have demonstrated that the presence of cholesterol itself decreased the fluidity of the hy
drocarbon chains of the membrane lipids and increased the tightness of packing in the lipid
bilayer, thus reducing the nonelectrolyte permeability of both intact cells and liposomes.
The incorporation of epicholesterol, the 3a-hydroxy epimer of cholesterol, had no detectable
effect on membrane lipid fluidity or packing and also did not alter the passive permeability
of model or biological membranes. On the other hand, the presence of choIest-3-one, a keto
analog of cholesterol which actually increased the fluidity of the membrane lipids and resulted
in a more loosely packed bilayer structure, increased the nonelectrolyte permeability of both
cells and liposomes. These studies confirmed that the physical properties of the membrane
lipids, as determined primarily by their fatty acid and sterol compositions, determine the
passive nonelectrolyte permeability properties of the A. laidlawii plasma membrane.
The intrinsic passive nonelectrolyte permeability of A. laidlawii B and E. coli plasma
membranes appears to be very low when their membrane lipids exist entirely in the gel
state.51-55-56 However, at temperatures near or just below the membrane lipid gel to liquid-
Volume II 29
Table 1
ACTIVATION ENERGIES CALCULATED FOR THE
PERM EATION OF GLYCEROL AND ERYTHRITOL
INTO A . L A lD L A W ll B CELLS AND LIPOSOMES
Activation energy (kcal/mol)
Glycerol Erythritol
Fatty acids added Cells Liposomes Cells Liposomes
16:0 + 18:2 18.2 20.2 20.5 20.7

16:0 + 18:1,,, 18.5 18.9 21.8 21.0
16:0 + 18: l„,ln, 19.4 17.3 21.5 23.8
16:0 + 18:0,,„ 19.0 16.7 20.3 21.9
16:0 + 18:0 16.8 17.7 21.1 21.6
18:1,„ + 14:0 18.6 18.6 21.7 21.2
18:1,,, + 16:0 18.5 18.9 21.8 21.0
18:1,„ + 18:0 17.3 17.6 22.6 20.3
18:1„, + 16:0 + 17.8 17.6 21.2 21.3
cholesterol
Mean ± S.D. 18.2 ± 0.9 18.1 ± 1.1 21.3 ± 0.7 21.5 ± 1.1
crystalline phase transition midpoint temperature, these membranes can become quite leaky
to nonelectrolytes.51•53-55-56 Similar local increases in passive permeability near the phase
transition temperature have also been reported for a number of model membranes composed
of single or binary mixtures of phospholipids, and it thus appears to be an intrinsic property
of lipid bilayers. It has been proposed that structural defects or mismatches in molecular
packing at the fluid and solid lipid phase domain boundaries, an increased lateral compress
ibility, or an increased magnitude of structural fluctuations near the lipid phase transition
temperature are responsible for this behavior. W hatever its exact molecular basis, this
phenomenon must be considered carefully in the interpretation of protein-mediated transport
experiments as well (see Section VI). Moreover, A. laidlawii and E. coli cells containing
predominantly gel state lipid become very susceptible to cell lysis if subjected to mechanical
or osmotic stress.51 5156
B. Ions
The relationship between the ionic permeability of prokaryotic plasma membranes and
their lipid compositions and physical states has received relatively little systematic study.
In fatty acid-homogeneous A. laidlawii B cells, passive sodium ion permeability was found
by Silvius et a l.53 to be influenced by fatty acid structure and chain length in much the same
way as passive nonelectrolyte permeability. In M . mycoides cells depleted of cholesterol,
passive sodium and potassium permeabilities were reported by Le Grimellec and Leblanc57
to increase relative to cholesterol-rich cells of the same fatty acid composition. Moreover,
in both A. laidlawii B53 and in an unsaturated fatty acid auxotroph of E. coli,55 cells become
leaky to sodium and potassium ions, respectively, near their phase transition temperatures.
It thus appears that the ionic permeability of prokaryotic plasma membranes can be markedly
influenced by the fluidity and phase state of the membrane lipids in a manner similar to that
observed for nonelectrolyte permeability.
No studies of the lipid dependence of the proton and hydroxide ion passive permeabilities
of prokaryotic plasma membranes appear to have been carried out. This is unfortunate, as
these ions play crucial roles in energizing various active transport processes (see Section
VI).
T e m perature ( ° C )
40 30 20 10
-1-0 ■ (b )
(a)
-2 0 ■
0-3 ■
ivi
-3 0 ■
t/5
V-
0-2 ■ _ç
-4 0 ■
01 ■
-5 0 -
I______ 1______ I______ I__
20 30 40 50 3-2 3-3 3-4 3-5
T em perature ( ° C ) IO3 x 1/T em pe ratu re ( K ')
FIGURE 5. (a) Tem perature dependence of the rate of passive glycerol permeation into E. coli K I060 grown
with xylose plus various unsaturated fatty acids. The rates of passive glycerol entry were measured as the reciprocal
relaxation times ( 1 / t of cell swelling in hypertonic glycerol). Unsaturated fatty acids: • , linoleic acid (18:2, ,); ■,
palm itoleic acid (16:1, ); o , oleic acid (18:1,); □, elaidic acid (18:1,). (b) Arrhenius plots of the data in (a). (From
Eze. M. O. and M cElhaney, R. N ., J. Gen. M icrobiol., 124. 299. 1981. With permission.)
V. M E M B R A N E L IP ID S A N D F A C IL IT A T E D D IF F U S IO N
Eze and McElhaney54 studied the influence of membrane lipid fatty acid composition and
temperature on the passive permeation and facilitated diffusion of glycerol in an E. coll
unsaturated fatty acid auxotroph. Cells grown on glycerol as the sole carbon and energy
source contained the components of the g ip regulon, including a membrane-associated
glycerol permease, a protein capable of catalyzing the nonconcentrati ve passage of glycerol
across the plasma membrane. In contrast, the g ip regulon was repressed in cells grown on
glucose or xylose, so that the glycerol permease was absent and glycerol entry occurred
exclusively by passive diffusion. It was found that the relative rates of protein-mediated
glycerol entry varied fairly markedly with membrane lipid fatty acid composition, just as
observed for the passive entry of glycerol; the relative rates of glycerol entry decreased in
the order linoleic > palmitoleic > oleic > elaidic acid-enriched cells (see Figures 5a and
6a). An inverse relationship was observed between the rate of glycerol-facilitated diffusion
and the gel to liquid-crystalline phase transition temperature of the membrane lipids, sug
gesting that the rate of protein-mediated glycerol permeation increases with increasing mem
brane lipid fluidity, again as observed for the passive entry of glycerol into E. coli cells of
similar fatty acid compositions. Finally, Arrhenius plots of the rates of glycerol-facilitated
diffusion were roughly linear and of similar slopes, irrespective of membrane lipid fatty
acid composition (see Figure 6b). The apparent activation energies for glycerol facilitated
entry varied from 9.5 to 11.3 kcal/mol. In particular, no abrupt breaks were observed near
the lipid phase transition temperatures of oleate- or elaidate-enriched cells. In contrast,
Arrhenius plots of passive glycerol permeation exhibited breaks near the membrane lipid
phase transition temperature in cells enriched in oleic and elaidic acids, and the apparent
Volume II 31
T em perature (°C )
40 30 20 10
-2-2 (6)
-2-6
1/5
-3 -0
0-12 (a)
_c -3 -4
008
<S>
- 3 -8
004
4-2
10 20 30 40 50 3-2 3-3 3-4 3-5

T em perature (° C ) IO3 x 1/T em perature ( K “ ')
FIGURE 6. (a) Tem perature dependence of the rate of entry of glycerol by facilitated diffusion into E. coli K1060
grown in the presence of glycerol plus various unsaturated fatty acids. The rates of glycerol facilitated diffusion
were m easured as the difference in reciprocal relaxation times ( 1 / t ) of cell swelling between glycerol- and xylose-
grown cells. Unsaturated fatty acids: • . linoleie acid (18:2, ,): ■, palmitoleic acid (16:1,); o. oleic acid (18:1,); ■,
elaidic acid (18:1,). (b) A rrhenius plot o f the data in (a). (From E/.e. M. O. and McElhaney, R. N ., J. Gen.
M icrobiol., 124, 299, 1981. With perm ission.)
activation energy for glycerol passive diffusion across fluid membranes was higher than for
glycerol-facilitated diffusion, being 15 to 16 kcal/mol (see Figure 5b). The apparent lack of
an effect of the lipid phase transition on the function of the glycerol permease may indicate
that this protein functions as a membrane channel. Flowever, the apparent influence of
membrane fluidity on the rates of mediated glycerol permeation would be difficult to explain
if this were the case, unless the membrane lipid fatty acid composition influences the number
of function carriers in the E. coli membrane. Alternatively, the boundary lipid immediately
adjacent to the glycerol carrier protein may remain in a fluid or semifluid state at temperatures
where the bulk membrane lipid is solid. The ability of intrinsic membrane proteins to disorder
gel state lipid,58 and of membrane enzymes and transport systems to retain activity below
the bulk lipid phase transition temperature42 58 has recently been demonstrated in several
systems.
This study is the only one the author is aware of in which the relationship between
membrane lipid fluidity and phase state and the function of a prokaryotic facilitated diffusion
system has been investigated. Although the results presented above are probably valid, it
would be desirable if these experiments could be repeated using a more direct assay of
glycerol entry. The stopped-flow spectroscopic swelling rate assay utilized by Eze and
M cElhaney59 is necessarily indirect and may be subject to some technical uncertainties.
VI. M E M B R A N E LIPIDS A N D ACTIVE TR ANSPO RT
A. Sugar Transport
The relationship between phospholipid biosynthesis and the induction of a functional
lactose transport system was originally investigated by Fox60 and Hsu and Fox;61 in the first
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To the sound of the drums the five battalions of Grenadiers, in
their long black gaiters, marched to the front. Arrived within forty
paces of the English, they halted, and the two armies, face to face
with each other, waited in solemn, silent hesitation. Old enemies on
a new soil, on how many a European battle-field had their forefathers
fought for dominion! And now they waited, awed, on this virgin soil,
who should begin this mortal duel.
In a clear voice the word of command flew along the English line.
A sound as of thunder broke forth, rolling along, to be repeated in
continuous roar; and as the smoke cleared off, in the French ranks
there were deep gaps, as if a scythe had passed through cutting
them down. The battle was begun.
Another volley, and yet another. The militia, which was
interspersed with the regular French troops, unable any longer to
stand the fire, hesitated. Montcalm saw it.
“Forward, forward!” he cried, showing with the point of his sword
the English ranks still unmoved. At the same moment a ball struck
him.
“You are wounded, General,” said an officer beside him.

“It is of no account, sir. Ride forward and rally the Canadians; they
are retreating.” Himself he sprang forward into their midst.
“Courage, my children, courage!” he cried; but another ball struck
him, and his white uniform was stained with blood.
“Support me; do not let them see me fall,” he murmured, striving
with a superhuman will to keep himself erect.
At that moment Wolfe gave the order to charge, and the wild yell
of the Highlanders, mingled with the British cheer, rose loud and
fierce.
A shot shattered Wolfe’s wrist; he wrapped his handkerchief round
it and went on. A second shot struck him; he still advanced. A third
pierced his breast; he staggered and fell. Then the officers
surrounding him carried him to the rear.
“Send for a surgeon,” said Lord St. Vincent.
“There is no need; it is all over with me,” he answered.
“They run; see how they run!” cried some one.
“Who run?” asked Wolfe, with a sudden return to life.
“The enemy, sir; they give way everywhere.”
“Tell Colonel Burton to cut off their retreat from the bridge,” he
said; and turning on his side, he added, “Now, God be praised, I will
die in peace.”
A few minutes later, for him the battle of life was over.
But to his country he left a rich heritage, with which his name is
ever linked in high honour. Canada became then and is now one of
the brightest jewels of the British Empire. She was bought with the
price of many a young and noble life, but, ever loyal and true to
England and her sovereigns, she has proved herself worthy of the
sacrifice.
Canada has, moreover, taught the world the lesson that two
peoples, supposed to be antagonistic, can live together in perfect
peace and harmony, side by side in the same cities, each speaking
their own language and retaining their own customs. The wisdom
and conciliatory policy of the British Government effected this union,
which has been pacifically maintained ever since. The French
population, which far outnumbered the English, finding themselves
treated with justice, and, instead of being driven forth, encouraged to
remain in the land, assured of religious freedom and the equity of the
laws, willingly submitted to the new rule, and have proved as faithful
subjects as their English brethren.
CHAPTER XXX
THE VANQUISHED
The Indians had fought bravely. Charles Langlade and their chief
Ominipeg had kept them steady. Long after the Canadians were in
full retreat they lay behind a mound firing without ceasing on the
English, who were advancing upon them. Then a strange thing
happened.
Ominipeg stood on a grassy knoll, on the left side of which were
high bushes, and looked around upon the battle-field. He knew that
Montcalm was wounded; he saw the Canadians flying before the
English: the cause was lost; he and his tribe would ere long be
prisoners.
The Black Eagle could not brook defeat. Charles Langlade, lying
on the ground at a little distance firing on the English, saw him
suddenly stoop behind the bushes and gather something in his arms.
A cry, a child’s cry, even through the din of battle reached his ears,
and a terrified baby face, round which the soft fair curls clustered,
appeared before his agonised gaze. To spring forward to seize him
would have been the work of a second, but Ominipeg was too quick
for him. Clasping the child tightly in his arms, with horrible cries,
brandishing his enormous battle-axe, the Indian chief, followed by
his whole tribe, dashed into the midst of the enemy.
The yells and war-whoops of the savages gradually died out as
the English bayonets pierced their naked bodies, and they lay upon
the ground a bleeding mass of humanity. They had fulfilled their code
of honour; they had died for the cause they could not save!
And the Black Eagle, with his daughter’s child, the little “White
Chief,” as he had been surnamed, lay foremost among the slain. A
shot had struck Charles Langlade to the ground before he could
advance a step to save the child.
That morning, at early dawn, when the first alarm had reached
Quebec, a young Indian had passed rapidly through the streets,
gained the house inhabited by Mercèdes, and knocked loudly at the
door of her apartment.
“Who is there?” asked Marthe.
“Langlade; open quickly,” was the answer. She hastened to obey;
the Indian glided into the room, looked round, and saw the child
sleeping in its little bed. To snatch it in his arms, smothering its cries,
and disappear with it, was the work of a second.
Roused by the noise, Mercèdes came running in, but the child
was gone. Marthe was wringing her hands, and in short, incoherent
phrases told Mercèdes what had happened.
But events were to succeed each other so rapidly that they had
hardly time to breathe, much more think. So accustomed had they
become to the bombardment of the city that, though it sounded more
continuous and louder than usual that morning, they attached no
especial importance to it; but a nun with a white, terrified face came
to them from the Superior, bidding them repair at once to the
General Hospital, that the English were on the Heights of Abraham,
and that a great battle was being fought. Wrapping their black cloaks
around them, and drawing their hoods over their heads in such a
way as to conceal their faces, they hastened to obey, passing quickly
through the streets, in some parts crowded by frightened citizens
driven forth from their half-ruined houses, in others swept clean by
the bombs which came whizzing down from the English batteries.
Very white and fixed was the young novice’s face as she glided
along. She suddenly came to a standstill, almost in front of the
Church of the Ursulines, where a crowd was gathered, which opened
to let a party of soldiers, carrying a litter which had been hastily
constructed out of guns crossed one over the other, pass on their
way.
The brilliant rays of the sun fell full upon the livid face of the man
who lay thereon. The waxen features were thrown into relief by the
black military cloak around him.
Not a cry escaped Mercèdes’ lips, though in that second she had
had time enough to recognise her father; but like an arrow she flew
to his side. One of the officers knew her, and gently and pityingly
made way for her, and she entered the church with the litter; then the
heavy doors were closed to keep back the surging crowd. Slowly,
with measured steps, surrounded by his officers, they bore him up
the nave; in front of the high altar the soldiers laid down their
precious burden, and Mercèdes, kneeling beside him, raised his
hand to her lips. He made no sign of being even aware of her
presence; his eyes were fixed, his features immovable; his soul was
still on the battle-field in the agony of that first moment of defeat. A
surgeon had been hastily summoned, who examined the patient and
probed the wound; but not a muscle of Montcalm’s face moved even
under that agony. When it was over, and a temporary dressing had
been applied, he said, “Well, sir, how long have I to live?”
“General,” answered the surgeon, in a low, pained voice, “a few
hours only.”
“All the better,” he said. “I shall not see the English enter Quebec,”
and he closed his eyes. Notwithstanding the wounds received on the
battle-field, borne by the tide of the fugitives the General had ridden
into Quebec at the head of the army, crossed the bridge under the
northern rampart, and entered the palace gate. At that moment
another shot reached him, which, passing through his body, proved
fatal, and he was half lifted, half fell from his horse; and so it came to
pass that his soldiers bore him into the Church of the Ursulines.
Mercèdes and Marthe tended him. Quiet and loving were the
words which from time to time he spoke to them. A few only of those
who surrounded them knew that the pale-faced novice was his
daughter. Michel, the gardener of the Ursuline Convent, fetched and
carried for them, and so that fatal day drew to an end.
Towards evening, Ramsay, the new Governor, came and asked
Montcalm’s advice as to how he might best defend Quebec.
“Have you any orders to give me, General?” he asked.
“Sir,” answered Montcalm, “I deliver into your hands the honour of
France. I shall spend my night with God preparing to die.”
Then he asked for pen and paper, and desired one of his officers
to write at his dictation:—
“General,—The humanity of the English sets my mind at

peace concerning the fate of the French prisoners and the
Canadians.
“May you feel towards them as they have caused me to feel
for them. Do not let them feel that they have changed masters.
Be their protector, as I have been their father.”
“Let this letter be sent without delay to General Wolfe,” he said,

when with difficulty he had succeeded in signing it.
“It is rumoured that James Wolfe is either dead or dying,” replied
one of his officers.
“He also!” said Montcalm. “At least he is happier than I am,” he
added; “he dies in the midst of his country’s triumph.”
Shortly after this his face became livid. His sufferings were
intense; he could only from time to time give utterance to a few
words in a low voice to Mercèdes, tender remembrances for the
loved ones at home! About midnight the Bishop Pont Briand
administered the last Sacraments of the Church in which he had
lived and was now dying.
Gently, almost painlessly, he lingered until the dawn of a new day,
and as the light began to creep into the sacred building his eyes
closed. When the surgeon, who had never left him, saw the eyelids
droop, he shook his head sadly, slipped his hand under the white
uniform so deeply stained with blood, and waited a few minutes, then
he rose.
“Gentlemen,” he said, turning to the group of officers who stood
watching, “that great heart has ceased to beat.”
Mercèdes never moved, her head was bowed low on her father’s
bier; Marthe alone wept, kneeling there beside her master.
Then suddenly the doors of the church were thrown open, and the
crowd which had been waiting patiently outside came flocking up the
nave. Soldiers of that poor defeated army, inhabitants of Quebec,
Canadians, savages, pressed around to take a last look at the brave
General who had so gallantly defended them. In the dim morning
light the torches flared, showing the half-ruined church, the roof laid
open, through which the sky looked down, shattered pillars, the
pavement torn up by bombs which in bursting had made deep holes;
and in the centre of all this ruin, surrounded by his officers, lay that
still figure wrapped in his black mantle, looking grander in death than
he had done in life.
In the afternoon of the same day they carried him into the
forsaken garden of the Convent of the Ursulines. The bursting of a
shell dug his grave, and there they laid him, all who had known and
loved him grieving, not for the hero so much as for the man.
Throughout that night two women knelt and prayed beside that
lonely grave.
CHAPTER XXXI
WEARY WAITING
“No news of the lads yet, Martha! Will they never come home?”
said Nathaniel impatiently, as he sat in the wide porch of Alpha
Marsh one bright autumn day.
“No, there be no news,” answered Martha sadly; “and yet they say
the fighting’s over for the present. I’m minded, if they’ve not both
been killed, they’ll be here before long.”
“Both killed! Our bonnie lads, Martha? Nay, I cannot think God
would have spared my life and taken them. I’m not of much account
now,” and he looked at his arm, which hung helpless in his
coatsleeve.
“You’ve no need to fret; you’re wonderfully better,” said Martha.
“And as for the lads, it isn’t likely they’re together; they’ll be dropping
in when we least expect them, one after the other.”
“God grant it,” said Nat; “but somehow I always see them
together;” and he rose from his chair, and went and stood by the
wicket gate, looking down the road which skirted the forest and led to
the village.
During the year which had elapsed since the Indians invaded
Marshwood, it had gradually resumed its former appearance of
happy prosperity. Most of the houses destroyed by the fire had been
rebuilt; a fresh harvest had been gathered in; and if some hearts still
ached for those who had fallen, time was gradually softening the
horrors of that terrible night, and casting a halo over the memory of
the lost.
Early the previous spring Martha Langlade had returned to Alpha
Marsh, bringing little Susie with her, though in truth she was “little
Susie” no longer, but a tall fine girl, very proud of her knowledge of
city life, and only desirous of returning to Boston, where they had left
Marie, the happy bride of young William Parkmann.
Nathaniel Boscowen had to a great extent recovered his health;
his arm alone was still powerless; but as time went on his restless
longing for the return of the “lads,” as he called them, grew painfully
intense. The news of the fall of Quebec, and of both Montcalm’s and
Wolfe’s death, had reached him in due time, and from that hour he
had, so to speak, waited by night and by day.
“They’ll be here to-morrow,” he would say, with a sigh, when Loïs
bade him “Good-night”; and she would answer with a smile which
grew every day fainter,—
“Yes, Father Nat; they’ll be here to-morrow.”
Several companies of Rangers had returned to their homes,
bringing the assurance that Roger was alive, that they had seen him
after the battle; but of Charles there was no news, and Loïs, like
Nathaniel, waited, going patiently about her daily work, with that look
of hungry longing which grows in women’s eyes from “hope
deferred.”
Between her and Roger there had been no words of
reconciliation, but, beside Nadjii’s grave, when they laid her to rest
under the shadow of the great oak tree in the home meadow, and in
the long night watches by Father Nat’s bedside, the hardness had
melted out of Roger’s face; their hands had touched, their eyes had
looked into each other’s; once more it was “Loïs” and “Roger.” And
so, through all the months of sadness and loneliness after he left
them, Loïs bore up bravely, for hope, blessed hope, was hers.
She worked as she had never done before, comforting the widows
and clothing and feeding the orphan children. Love gave her strength
as only love can. Through the bright short spring and long summer
days she waited, with the never-ceasing prayer upon her lips for
“Peace, blessed peace.” But now for many weeks she had had no
news, save what the stray home-comers had brought; and yet the
war was over—the English were masters of Quebec. Why then did
Roger linger?
Of late the habit had come to her of going to the upper windows
and looking out over the country. Vague rumours of Charles’ death
had reached both her and Marcus, but by common consent they hid
it from Martha and Father Nat, who always repeated, “The two will
come together. Many things may have happened to detain them on
the road,” and both she and Marcus were thankful he should think
thus. But the winter was fast approaching, and then the land would
be icebound, and long dreary months must elapse before they could
hope to see the wanderers. Oh, how earnestly Loïs prayed for news,
only for news, of them, and it came to pass that her prayer was
granted. But alas, how?
Loïs was always up betimes. All the dairy work fell to her lot, and
Martha had been ailing lately, fretting for Charles, they all knew. As
she stood in the dairy, pouring the new milk, which the maids had
just brought in, from the pails into the earthen pans for setting, the
old Indian woman Nokomis crept up to her with a mysterious look on
her face.
“Well, Nokomis, what has happened? Have you burnt the cakes
for breakfast?” asked Loïs.
She shook her grisly head and answered slowly, “Alas, alas,
mistress! there be those who will never eat of my cakes again, and
yet he loved them! Old Nokomis’ cakes—he’d take them half-baked
out of the oven, for the smell of them!”
“Who are you speaking of?” said Loïs, hastily putting down the
half-empty pail.
“Who should I speak of if not of the young master? Ah, it was an
evil day when Boscowen and Langlade parted; they’ll never come
together again.”
“What have you heard?” said Loïs, turning deadly pale.
“The boy’s there; he can speak,” said Nokomis.
“What boy?” asked Loïs. “Oh, Nokomis, if there be news of Roger
and Charles, do not keep me waiting.”
Thus adjured, the Indian woman went to the door, made a sign to
some one, and in another minute an Indian youth entered and stood
before Loïs.
“What have you to tell me?” she asked tremulously.
The boy answered,—
“I am Nadjii’s brother. I carried the boy away, but the White Chief,
his father, found him, and would have hidden him from Ominipeg, but
he could not; the ‘Black Eagle’ took him, and carried him into the
battle, and they were killed together. And last of all the White Chief
was killed; I saw him fall. They are all gone into the land of the Great
Spirit.”
“Do you mean to say my brother is dead?” said Loïs, leaning
against the wall to keep herself from falling.
“Yes, I mean it; they are all dead, and I will stay here and serve
you. I loved the White Chief, and I served him. He told me many
things. I will live with the white man, and pray to the Great Spirit
Jesus”; and suiting the action to the word, he sat down upon the
floor, in token that he meant to abide there.
Silence, a dead silence, fell upon them. The early morning light
came creeping in through the windows, a pale autumn light with no
warmth or brightness in it. A chill feeling of despair overpowered
Loïs; she looked at the dark messenger. Could he be speaking the
truth? Might he not be mistaken? But she knew the Indian lad; he
had often brought her messages from Charles, even when he was a
mere child; now he was about fifteen, and there was no reason why
he should deceive her. What should she do with him? If she took him
into the kitchen the rest of the family would see him, and the news
he brought would spread from mouth to mouth, until it reached the
ears of her mother and Father Nat. At present this must be avoided.
“How have you travelled?” she asked. “And how long have you
been on the road?”
“I travelled the same way as the hunters, through the forests. I
have come often before; I know the way,” said the boy. “The moon
was new when I started; it is full now.”
“You must be tired; you had better rest. Nokomis, take him to the
attic next yours in Omega Marsh, and be careful that neither my
mother nor Father Nat sees him, until I tell you. Give him bread and
meat, and all he needs. You will keep quiet for a day or two, until I
know what to do,” she said to the boy.
Her eyes were full of tears, her lips trembled; she never for one
moment doubted the truth of the story he told. Her brother was dead,
the child was dead, and Roger—where was he?
Nokomis signed to the Indian to follow her, and skirting the
outhouses, they reached the back entrance to Omega Marsh, which
was at present only inhabited by herself and one or two men, Father
Nat having remained since his illness at Alpha Marsh.
“You lie quiet here. Nokomis bring you food: you sleep; no work.”
And to this pleasant prospect the Indian readily acquiesced.
Nevertheless Nokomis, when she left him, took the precaution of
turning the key and putting it in her pocket.
Two days later, when she went in the early morning to take him
his food, he was gone; the dormer window was open, and, looking
out, she knew he had escaped by the roof. Here and there a creeper
had been loosened, and in the grass and on the ground below she
saw traces of feet—not the Indian’s naked feet only, but the print of a
woman’s shoe; and she stood and looked, then went across to Alpha
Marsh, her eyes fixed on the ground, like a dog on the scent. As she
passed Bob’s kennel she saw it was empty.
“Bob, Bob!” she called. There was no answer. “He gone too,” she
muttered between her teeth. Taking the key of the back kitchen from
the hiding place where she put it every night, she entered, looked
round, went into the pantry, examined the safe in which cold meats
and other provisions were kept, lifted the cover of the bread-bin, and
counted the loaves. While she was thus occupied Marcus entered.
“What are you doing, Nokomis?” he asked, watching her curiously
for a few seconds.
“Where’s Loïs?” she asked, looking up at him.
“Not yet up, I suppose,” he answered. “She’s overslept herself—
an unusual thing for her.”
“You go and look in her room. I tell you she’s gone.”
“Gone! Where should she be gone?” said Marcus.
“To bring the lads home,” said Nokomis; and then for the first time
Marcus heard of the arrival of the Indian lad, the story he related,
and how he had disappeared.
“Why did she not tell me?” he thought bitterly; and yet his faith in
Loïs was so great that he checked the angry feeling, and went
straight up to her room. There he found the confirmation of Nokomis’
words. The bed had not been slept in; Loïs was gone! But surely not
without a word! No, there on the table was a letter addressed to
himself.
“Dear Marcus,—Forgive me,” she wrote. “For the last two
days and nights I have prayed unceasingly for God to guide me,
and it has been borne in upon me that, notwithstanding all the
Indian lad tells me, Charles and the child are still living. At first I
did not think so; but now I do. I know where Charles put the
child—in the Convent of the Ursulines at Quebec; I am going
there. Tell Father Nat and the mother that I have had news of
Charles; that he needs me, therefore I am gone to him. They
shall hear soon; but do not let them know the rumour of his
death. Why should they grieve, perhaps without a cause? I have
taken money, my Indian guide, and Bob. Have no fear for me;
God and His angels will guide my steps. I am going forth in His
strength, without fear, to bring our dear ones home. Pray for me,
and tell John Cleveland to pray for me in the congregation on
the Sabbath Day, until I come back to you all, and we settle
down in peace. I go without warning you; not from mistrust, but
because I know you would wish to go in my stead, and that must
not be. You are all that is left to us. If harm befell you, the
Marshes would indeed be without a master and desolate. I am
only a woman!
“Your loving sister,
“Loïs.”
“And truly a brave one!” said John Cleveland, when he had read
the letter, which Marcus took straight down to the minister’s house.
“You can but do as she says; tell Father Nat she has been sent for,
and is gone on the road to meet Charles. You may be sure she’ll
manage to send us news before many days are over; we’ll just live
from day to day in hope and prayer. If any one can bring the lads
home, Loïs can. Go about your work as usual, Marcus; tell Nokomis
to keep a silent tongue in her head. I’ll come up and see your mother
and Father Nat. No need to say she’s gone to Quebec: we don’t
know whether she’ll ever get there; maybe she’ll meet them on the
road.”
Marcus shook his head.
“I do not think there is much chance of that,” he said.
“How dare you say so?” said John Cleveland sharply; “and you
who would be a minister and teach others. With God nothing is
impossible. Have faith, lad—faith which can remove mountains,” and
he clapped him on the shoulder, adding, “And now I’ll just let my
missis know I’m going to breakfast up at the Marshes. I won’t leave
you to face Father Nat alone. How he’ll live the day through without
Loïs, his right hand, is more than I can tell. She thinks she’s of no
account because she’s a woman, but we men should be badly off
without our womankind, even though there are not many like our
Loïs. I only want to live long enough to give her and Roger my
blessing on their wedding-day, and I believe I shall, and that before
long.”
It was no easy matter to hoodwink Father Nat. But she was gone;
there was no remedy: they could not go after her, not knowing which
way she had taken; and so, when Martha wept and wailed “that all
her children were going from her,” Nathaniel said quietly,—
“She’s a wise and a good lass, and the Lord is with her. No harm
will come to her, and maybe she’ll bring both the lads back.”
And so they watched and waited at the Marshes, and the snow fell
covering the earth, and the rivers were icebound, and still there was
no news of the wanderers.
CHAPTER XXXII
ON THE BATTLE-FIELD
The silver light of the moon was shining down on the battle-field,
where the dead and dying lay in hideous confusion, the night after
the fray. Dark figures moved stealthily to and fro, lanterns flashed on
ghastly upturned faces, piteous voices called for help, hands were
stretched out praying for mercy, too often only to meet death and
spoliation. Birds of prey hovered overhead. Alas for poor human
nature! there were those abroad who reverenced neither heroism nor
death, but laid rude hands on their fellow-men, robbing and
mutilating the prostrate forms as they lay writhing in death’s agony.
A group of half a dozen men in the well-known dress of the Royal
Rangers had found their way to that part of the battle-field where the
Indians had made their last fierce onslaught. The near approach of
death had not extinguished the passionate instincts of hatred and
revenge; more than once the treacherous knife gleamed in a dying
hand seeking still to slay. Every precaution had to be taken by the
searchers, as they picked their way over the ground strewn so thickly
with the dead and dying, to avoid the murderous thrusts.
“Look here, Captain!” and the speaker, a young man, pointed to
where a red chief lay, with a little child clasped in his arms. A shot
had pierced the baby heart, in kindly mercy quieting for ever its wild
fluttering; but the blue eyes were wide open still, and retained that
look of terror mirrored in them which gleamed there when death
came, and the long fair curls were dabbled in blood.
The man who had been addressed as Captain stood looking down
upon the group. Pain, bitter pain, was visible in every line of his face.
“It is Ominipeg,” he said, and stooping, he lifted the dead child in his
arms and wrapped it in his bearskin. He and his companions knew
enough of Indian customs to understand how that infant came by his
death—a chief’s son in the foremost ranks of the slain!
They renewed their search; and, at last, amidst those dark naked
figures, with their wild headgear and strange fantastic war-paint, they
found him they sought. He was lying propped up against a tree;
evidently, when the battle was over, he had dragged himself thither.
Was he dead? Roger bent eagerly over him, and took the hand
which hung listlessly by his side.
“Charles,” he said; and the strong man’s voice trembled.
“Roger, am I dreaming, or have you come to take me home?”
The drooping head is raised, and the cold fingers close over
Roger’s.
“We will go home together,” he said. “Are you much hurt,
Charles?”
“I do not know,” he answered dreamily. “Is the battle over? Are we
beaten?”
“The battle is ended,” said Roger; “and God grant it may be our
last,” and he signed to his men that the search was finished, that
their help was needed. They lifted the wounded man in their arms
and slowly bore him off the battle-field to where in the moonlight
clustered the white tents of the Rangers, and there they laid him
down.
Quebec had capitulated, notwithstanding Levis’ rapid march to its
relief. Ramsay paid but little attention to Montcalm’s last words, and,
encouraged by Vaudreuil, on the 18th surrendered to the English.
Honourable terms were granted. The garrison was to march out with
the honours of war, and the troops be carried back to France on
English ships; the inhabitants to have protection in person and
property, free exercise of their religion, and all other privileges of
British subjects. These conditions having been formally agreed to
and signed, the British flag was raised on the heights near Mount
Street, and General Murray was named Governor of Quebec.
As soon as he could do so, Roger had brought Charles into the
city. He was unconscious at the time, and the military surgeon gave
but faint hope of his recovery. It was a battle between life and death,
but youth and a strong constitution aiding, Roger was at last
rewarded by seeing Charles enter upon what might be called
convalescence; but by that time winter had set in, and there was no
possibility of communicating with Marshwood. “I ought to have
thought of sending a messenger immediately after the battle,” Roger
said; “but I didn’t know quite what you meant to do, so I waited, and
now it is too late.” So time passed on.
One evening, a lady, deeply veiled, came to the house where the
two friends lodged, and, asking to see Mr. Langlade, was admitted.
Charles was seated in an armchair near the large open fireplace;
he turned as the stranger entered, and, when she raised her veil,
exclaimed, “Madame Péan!”
“Yes,” she said, coming forward; “I heard you were in Quebec,
where I myself have been detained by severe illness, and I have
come to you with a message from Mercèdes Montcalm.”
“She is well, I trust?” said Charles, in a low voice.
“Yes, she is,” answered Madame Péan, “and the day after to-
morrow she takes the veil. I have done the best I could to dissuade
her, offering to take her back with me to France in the spring, but she
will not listen to me; her place, she says, is by her father’s grave, in
the convent garden, and the Bishop and Mother Superior have
consented to shorten her novitiate. One thing troubles her, the loss
of the child committed to her care by you. When I heard you were in
Quebec I told her, and she entreated me to come to you without
delay, to hear what had become of the child.”
“He is dead,” said Charles; “his mother’s tribe stole him, lest he
should be made a prisoner, and he was killed. Tell her this, or not, as
you deem best.”
“If you will, you can tell her yourself,” said Madame. “She bids
farewell to her friends to-night; if you come to the convent, you can
have speech with her for the last time.”
“I will come,” said Charles, his pale face flushing.
“She thought you would,” said Madame; “she has not many
friends to whom to bid farewell, and the General loved you.”
“Not better than I loved him,” said Charles, rousing himself. “Tell
Mademoiselle Mercèdes I will be at the convent to-night after
vespers; and thank you a thousand times for coming to me. I would
not have missed seeing her once more, for all the world,” and he
held out his hand to Madame Péan.
“I guessed as much,” she answered. Their eyes met, and she
slowly shook her head. “It is too late,” she said; “all that was earthly
in her heart and soul has dropped away from her and lies buried in
her father’s grave. She has no thoughts left which are not of heaven.
And now I will leave you. As soon as to-morrow’s ceremony is over I
go to Montreal. Is there any service I can render you? any request
you have to make to Chevalier Levis? He is well aware how you
have behaved throughout the war, and would be only too glad if you
would join his poor remnant of an army, with which he still hopes to
wrest Canada from the English.”
Charles shook his head.
“He will never do that,” he said. “The cause is lost; he will only
uselessly sacrifice fresh lives. Is it not so, Roger?”
“Most certainly it is. But, Madame,” said Roger, “if you would do
my friend a real service, it would be to obtain from the Chevalier for
him and for me a free pass through all the country still occupied by
the French troops. We are anxious to return to our people, but
without this it would be almost impossible during the winter; we
should have to take such a circuitous route, and my friend’s health is
not sufficiently recovered to resist the cold and fatigue; if we can
pass through Montreal, it will shorten the journey greatly.”
“I will do my best,” said Madame Péan. “And now farewell; we are
none of us likely to meet again in this world. When the last French
ship leaves the shores of Canada, I shall sail in her, and go back to
old France.” She dropped her veil and rose. Charles also rose, and
silently they shook hands; then Roger re-conducted her to her
carriage, and they took leave of each other.
She had said truly they were never to meet again.
That evening, as he had promised, Charles went alone to the
convent. He waited what seemed to him an eternity in the parlour,
watching anxiously a grated window in the wall, across which was a
dark curtain; at last he saw it slowly drawn back, and on the opposite
side, with a face almost as white as her veil, stood Mercèdes.
“Thank you for coming,” she said, in a low, calm voice. “Before
bidding my last farewell to the world, I desired greatly to see you, to
tell you how I have grieved for the child you committed to my care. I
loved him very dearly. I would not have parted from him if I could
possibly have done otherwise; but we were taken by surprise. Before
even Marthe, who was in the room with him, was aware of it, he was
gone; we had no time to prevent it; he was truly spirited away. I pray
you forgive me: it has been a bitter grief to me.”
“Forgive you!” exclaimed Charles. “Surely you never for one
moment thought I blamed either you or Marthe? Knowing the Indians
would use every means in their power to get hold of my poor little
son, I placed him with you, believing he must be safe in the convent.
How could either of us imagine you would be driven out into the
world again? How can I harbour one thought of blame against you!
Indeed, I almost think it best for him to be at rest. Had he lived, his
would have been a very divided life. He must have suffered, and I for
him. I am content. It is well with the child.”
“I am thankful to hear you speak thus,” answered Mercèdes.
“Truly all God does is well done. And now, Monsieur Langlade, I will
bid you farewell. You will go back to the world to which, to-morrow, I
shall for ever bid adieu; but I wish to thank you for many pleasant
hours and for much kindness, but, above all things, for your
faithfulness to my dear father. I beg you to cherish his memory, and
be assured I shall ever remember you in my prayers.”
“No one who has ever lived with General Montcalm as I have can
possibly forget him. I shall cherish his memory as long as I live,” said
Charles, with deep emotion.
“Thanks, I am glad to think it will be so,” and a faint smile lighted
up her pale face. “Adieu!” and she passed her hand between the iron
bars. “Wear this in remembrance of him,” she added, slipping a ring
of great price on his finger.
“I will never part with it. Adieu,” repeated Charles, and stooping,
he touched the tips of her fingers with his lips. When he raised his
head she had disappeared.
The following morning he was amongst the spectators who
witnessed the ceremony of Mercèdes Montcalm taking the veil, and
as he left the chapel his heart was very sad within him.
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Keratin Structure Properties and Applications Structure

Structure and Properties of Atomic Nanoclusters Julio A.

Structure and Functional Properties of Colloidal Systems

Solid State Physics Structure and Properties of Materials

High strain zones structure and physical properties 1st

Polymeric Liquids Networks Structure and Properties 1st

The Myocardial Cell Structure Function and Modification by

Metallic Films for Electronic Optical and Magnetic

Gheorghe Benga, M .D., Ph.D.

(ci*; CRC Press

Reissued 2018 by CRC Press

© 1985 by CRC Press, Inc.

No claim to original U.S. Government works

Library of Congress Cataloging in Publication Data

Molecular basis of selected transport systems.

(Structure and properties of cell membranes; v. 2)

A Library of Congress record exists under LC control number: 84019943

ISBN 13: 978-1-315-89787-5 (hbk)

Angelo Azzi Josef Houstëk

The Evolution of Membrane Models

Basic Kinetics of Membrane Transport

Quantum Chemical Approach to Study the Mechanisms of Proton Translocation Across

BA SIC K INETICS OF M E M BR A N E TRANSPO R T

II. Nonspecific Perm eation.......................................................................................................... 2

III. Specific Transport ................................................................................................................... 3

1. The membrane is “ infinitely” thin so that no intramembrane gradients are to be

Two fundamentally different mechanisms of membrane transport exist: nonspecific per­

II. NO NSPECIFIC PERM EATION

Js = - D ( S „ - S,)/8 = P(S, - S„) (1)

A. Specific Channels or Pores

which, for unidirectional flux (as followed by a tracer) becomes

For initial flux

the unidirectional flux from side I to side II is then

JS(,^ M) = E,adgS,[f + bchS„/(bh + bd + dg)]/D (9)

so that Jmax = E,dfg/[d(f + g) + f(g + h)]

and Kx = (e + f)(bh + bd + dg)/a[d(f + g) + f(g + h)] (10)

J = ------------- lS| ~ N2Sh------------ (11a)

with Keq = N ,/N 2, Kt1 = C/D,, KT2 = C/D2, J ^ x = N,/D, ( lib )

1/J„ = [KTl (1 + S„/KT2)/J-ax + S, (1 + S . / K . y j ^ K J

x 1/(S, - S„/Kcq) + (1 + SII/K ,)/J^1X (12)

KTapl, = KX| (1 + Sn/K l2)/( 1 + Sn/K,) + Sn/Keq (14)

diffusion system. Equation (8) can be drastically simplified if pseudoequilibria of the

____________R,(gS,< + gR„ + e) - R„(gS; + gR, + e)____________

In an experimental arrangement where we preincubate cells with R until R, = R„ = R and

. = T ______________________ ~ g R ' s l_____________________ (16)

JR(S) = E,(adR,/S, - bcR n/SJ/Ib + d + aR,/S, + cR^S,,)

under the above condition that S„ = 0 and R, = R„ = R, JR(S) = —b has no meaning.

Sn/S, = adfg/bceh (17)

Js0 = (a S f + P S ,) / ( 7 S f + 8S, + e) (18)

and it is known about such rate expressions that, in a Lineweaver-Burk arrangement, a

S„/S, = (ion/ion,,)" e nFA,1,/RT = acegik/bdfhjl (19)

< < w> + ’SB

II. Thermodynamic E ffects........................................................................................................ 13

III. Other pH-Dependent Translocations ............................................................................... 16

(E is the enzyme, S the substrate, and H hydrogen ion.)

Ks(2 + K ,/[H H [H + ],/K2 + K,/[H +] m + [H + ]„/K2)

FIGURE 1. Plot to determ ine intrinsic dissociation con­

Two fundamentally different mechanisms of membrane transport exist: nonspecific per

R,(gS,< + gR„ + e) - R„(gS; + gR, + e)

. = T __ ~ g R ' s l_ (16)

FIGURE 1. Plot to determ ine intrinsic dissociation con