Lecture Slides on Cell Biology

Kedir W.
(M.Sc. Molecular Biology and
Biotechnology)
Department of Biology
Hawassa University

1
 Cell Biology
 Cell is the basic, structural and functional unit of life.
 The science that deals with cells is known as cell
biology(cytology)
How cells were discovered?
 1665- English Scientist, Robert Hooke, discovered cells while
looking at a thin slice of cork using his own microscope.
He described the cells as tiny boxes or a honeycomb
He thought that cells only existed in plants and fungi
 1673- Anton van Leuwenhoek: Used a handmade microscope to
observe pond scum & discovered single-celled organisms
He called them “animalcules”q `

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 Development of Cell Theory
 1838- German Botanist, Matthias Schleiden, concluded that all
plant parts are made of cells
 1839- German physiologist, Theodor Schwann, who was a close
friend of Schleiden, stated that all animal tissues are composed
of cells.

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 Cont.
 1858- Rudolf Virchow, German physician, after
extensive study of cellular pathology, concluded
that cells must arise from preexisting cells.
 The 3 Basic Components of the Cell
Theory
 All organisms are composed of one or
more
cells. (Schleiden & Schwann)(1838-39)
 The cell is the basic unit of life in all living
things. (Schleiden & Schwann)(1838-39)
 All cells are produced by the division of
preexisting cells. (Virchow)(1858)

BUT HOW DID THE FIRST CELL AROSE?

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 Evolution of the cell
 Cells are divided into two main classes, initially defined by
whether they contain a nucleus.
• Prokaryotic cells (e.g. bacteria) lack a nuclear envelope
• Eukaryotic Cells have a nucleus in which the genetic
material is separated from the cytoplasm.
 Prokaryotic cells are
• generally smaller and simpler than eukaryotic cells;
• in addition to the absence of a nucleus, their genomes
are less complex and
• they do not contain cytoplasmic organelles or a
cytoskeleton.
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 Cont.
 In spite of these differences, the same basic molecular
mechanisms govern the lives of both prokaryotes and eukaryotes,
indicating that all present-day cells are descended from a single
primordial ancestor.
How did this first cell develop? And how did the
complexity and diversity exhibited by present-day cells
evolve?

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 1. Evolution
 Living from
cells probably Molecules
arose to the
on earth about 3.5 First
billion Cell
years
ago by spontaneous reactions between molecules in pre-
biotic condition
Pre-biotic conditions
 the earth was a violent place with volcanic eruptions,
lightning, and torrential rains
 the atmosphere contain small molecules such as
ammonia, carbon dioxide, methane.
 There was no free oxygen and no layer of ozone to absorb
the ultraviolet radiation from the sun.
 These conditions may have helped to keep the atmosphere
rich in reactive molecules that produced simple organic
molecules such as amino acids, sugars, purines and
pyrimidines

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 Evidence
 The best evidence came from laboratory
experiments by Stanley Miller.
 If mixtures of gases such as CO2, CH4, NH3,
and H2 are heated with water and energized
by electrical discharge or by ultraviolet
radiation, they react to form small organic
molecules.
 Among these products are compounds, such as
 hydrogen cyanide (HCN) and formaldehyde
(HCHO) that readily undergo further reactions
in aqueous solution.
 Most important, representatives of most of the
major classes of small organic molecules found in
cells are generated, including amino acids sugars
the purines and pyrimidines

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 Cont.
 Simple organic molecules such as amino acids and
nucleotides can associate to form polymers known as
polypeptides and polynucleotides respectively.
 Catalysts for early polymerization
 The earliest polymers may have formed in any of several
ways - for example,
by the heating of dry organic compounds or
by the catalytic activity of high concentrations of
inorganic polyphosphates or
By crude mineral catalysts.

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What are the critical characteristic required of the macromolecule from which life
evolved ?
 Origin of life requires molecules that have the property of:
• autocatalysis (the ability to catalyze reactions that lead to
production of more molecules of the catalyst itself) and
• Self-replicating (Molecules that are Capable of Directing Their
Own Synthesis)
Which molecules do have these properties?
1. Polypeptides: In present-day living cells, the most versatile catalysts
are polypeptides. Although polypeptides are versatile as catalysts,
there is no known way in which one such molecule can reproduce
itself by directing their own synthesis
2. Polynucleotides: have properties that contrast with those of
polypeptides. Both DNA and RNA directly guide the formation of exact
copies of their own sequence. In addition to self replicating RNA
molecules have catalytic property (e.g. Ribozymes).

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 Cont.
 An RNA molecule therefore has these two special characteristics:
1. it carries information encoded in its nucleotide sequence that it can
pass on by the process of replication and
2. the catalytic properties.
• There are strong suggestions, therefore, that between 3.5 and 4 billion years
ago, somewhere on earth, self-replicating systems of RNA molecules, mixed
with other organic molecules including simple polypeptides, began the process
of evolution- a period of evolution known as the RNA world

• The first cell is presumed to have arisen by the

enclosure of self-replicating RNA in a membrane
composed of phospholipids
 The enclosure of self replicating RNA and
associated molecules in phospholipids membrane
would thus have maintained them as a unit,
capable of self reproduction and further evolution.
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 Evolution of metabolism
• Cells were originated in a sea of organic molecules, they were able to
obtain food and energy directly from their environment.
• As resources get depleted cells needed to evolve their own mechanism
for generating energy.
 All present cells use ATP as their source of metabolic energy. The
mechanisms used by cells for generation of ATP are thought to have
evolved in three stages corresponding to the evolution of:
Glycolysis
Photosynthesis
Oxidative metabolism
 In the initially anaerobic atmosphere of earth, the first energy generating
reaction involved the breakdown of organic molecules in the absence of
oxygen. These reactions are likely to have been a form of present day
Glycolisis C6H12O6 _____ 2C3H6O3 + 2ATP

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 Cont.
 The development of photosynthesis
 is the next major evolutionary step, which allowed the cell to
harness energy from sunlight
 provided independence from the utilization of preformed organic
molecules.
 The first photosynthetic bacteria probably used
H2S as source of electron and hydrogen to convert CO2 to organic
molecules.
• The use of H2O as a donor of electron and hydrogen for conversion of CO2
to organic molecules evolved later
 had important consequence of changing the earths atmosphere
by producing free O2 as a byproduct
6CO2 + 6H2O­­______C6H12O6 + 6O2

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 Cont.
 The release of O2 changed the environment in which cells
evolved
 have led to the development of oxidative metabolism
 Oxidative metabolism utilizing O2
provided a mechanism for generating energy from organic
molecules that is more efficient than anaerobic glycolisis.

C6H12O6 + 6O2 ______ 6CO2 + 6H2O + 36-38ATP

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 Evolution from prokaryotes to Eukaryotes
 all organisms living now on earth were derived from a single
primordial cell born more than 3.5 billion years ago.
This first cell was prokaryotic, unicellular and anaerobic
 prokaryotes are divided into two groups
1. The archaebacteria and
2. The eubacteria
 Indication:
 Archaebacteria
 live in extreme environments, which are unusual today but
may have been prevalent in primitive Earth.
 For example, thermoacidophiles live in hot sulfur springs
with temperatures as high as 80°C and pH values as low as
2.
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 Cont.
 The eubacteria
Also live in a wide range of environments, including soil, water,
and other organisms (e.g., human pathogens).
 Photosynthesis was first evolved in prokaryotes known as
Cyanobacteria
 Consequences of accumulation of molecular oxygen in the
atmosphere
i. some anaerobic prokaryotes become extinct
ii. others may have evolved the capacity for aerobic respiration
iii.others found niches in which oxygen was largely absent, where
they could continue an anaerobic way of life.
iv.Others became predators or parasites on aerobic cells.

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 Evolution of Eukaryotic Cells
 A critical step in the evolution of eukaryotic cells was the
acquisition of membrane-enclosed sub cellular organelles,
 allowing the development of the complexity characteristic
of these cells.
 The organelles are thought to have been acquired as a result of
the association of prokaryotic cells with the ancestor of
eukaryotes.
 The hypothesis that eukaryotic cells evolved from a symbiotic
association of prokaryotes—endosymbiosis—is particularly well
supported by studies of mitochondria and chloroplasts, which
are thought to have evolved from bacteria living in large cells.

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 Evidence
 Both mitochondria and chloroplasts are similar to bacteria in
size, and like bacteria,
They reproduce by dividing in two(Binary Fission).
 Most important, both mitochondria and chloroplasts contain
their own DNA, which encodes some of their components.
 Like bacteria they contain 70s ribosome
• An endosymbiotic origin for these organelles is now generally
accepted,
 with mitochondria thought to have evolved from aerobic
bacteria and
 chloroplasts from photosynthetic bacteria, such as the
cyanobacteria.

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Features of prokaryotes which make them different from
eukaryotic Cells

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 The Development of Multicellular Organisms
 Multicellular organisms evolved from unicellular eukaryotes at least
1.7 billion years ago.
 Some unicellular eukaryotes form multicellular aggregates that
appear to represent an evolutionary transition from single cells to
multicellular organisms.
For instance, the cells of many algae (e.g., the green alga Volvox)
associate with each other to form multicellular colonies, which are
thought to have been the evolutionary precursors of present-day
plants.
 Increasing cell specialization then led to the transition from colonial
aggregates to truly multicellular organisms.
 Continuing cell specialization and division of labor among the cells of
an organism have led to the complexity and diversity observed in the
many types of cells that make up present-day plants and animals,
including human beings.
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 Cont.

Figure . Four closely related genera of green algae, showing a progression from
unicellular to colonial and multicellular organization.

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Figure Evolution of cells: Present-day cells evolved from a common prokaryotic ancestor
along three lines of descent, giving rise to archaebacteria, eubacteria, and eukaryotes.
Mitochondria and chloroplasts originated from the endosymbiotic association of aerobic
bacteria and cyanobacteria, respectively, with the ancestors of eukaryotes.
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 Cell size
 Cells vary greatly in size which ranges from less than1 micrometer - 200
micrometers.
 The size of a cell is directly related to its level of activity and the rate that
molecules move across its membranes. In order to stay alive, a cell must have a
constant supply of nutrients, oxygen, and other molecules. It must also be able
to get rid of carbon dioxide and other waste products that are harmful to it.
The larger a cell becomes, the more difficult it is to satisfy these requirements;
consequently, most cells are very small.
Advantages of being small: Small cells have large surface to volume ratio, so
things can be moved in and out efficiently
 There is a mathematical relationship between the surface area and volume of a
cell referred to as the surface area-to-volume ratio.
 As cells grow, the amount of surface area increases by the square (X 2) but
volume increases by the cube(X3). The surface area increases at a slower rate
than the volume. Thus, the surface area-to-volume ratio changes as the cell
grows

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 There is a mathematical relationship between the surface area and
volume of a cell referred to as the surface area-to-volume ratio.
 As cells grow, the amount of surface area increases by the square
(X2) but volume increases by the cube(X3). The surface area
increases at a slower rate than the volume. Thus, the surface area-
to-volume ratio changes as the cell grows.
 As a cell gets larger, cells have a problem with transporting
materials across the plasma membrane. For example, diffusion of
molecules is quite rapid over a short distance, but becomes slower
over a longer distance. If a cell were to get too large, the center of
the cell would die because transport mechanisms such as diffusion
would not be rapid enough to allow for the exchange of materials.
When the surface area is not large enough to permit sufficient
exchange between the cell volume and the outside environment,
cell growth stops.

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 Cell shape
 Cells show great variation in their shape .
 In human beings for example, there are about 200 types of cells in that vary greatly in
shape. Examples
 Squamous cells: are thin and flat cells. Such cells line the esophagus and cover the
skin.
 Polygonalcells : have irregularly angular shapes with four, five, or more sides.
 Stellate cells: Some nerve cells have multiple extensions that give them a star like, or
stellate, shape.
 Cuboidal cells: are squarish and approximately as tall as they are wide; liver cells are a
good examples.
 Columnar cells: such as those lining the intestines, are markedly taller than wide.
 Spheroid cells: Egg cells and fat cells are spheroid too void(round to oval).
 Discoid cells: Red blood cells are discoid (disc-shaped).
 Fusiform cells: Smooth muscle cells are fusiform - thick in the middle and tapered
toward the ends.
 Fibrous cells: Skeletal muscle cells are described as fibrous because of their threadlike
shape

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28
Tools of Cell Biology
 As in all experimental sciences, research in cell biology depends on
the laboratory methods that can be used to study cell structure
and function.
 An appreciation of the experimental tools available to the cell
biologist is thus critical to understanding both the current status
and future directions of this rapidly moving area of science.
 Some of the important general methods of cell biology are:
Light Microscopy:
Electron Microscopy:
Sub-cellular Fractionation:
Cell Culture:
Viruses …..etc

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 Light Microscopy
 Because most cells are too small to be seen by the naked eye,
the study of cells has depended heavily on the use of
microscopes.
 Robert Hooke first coined the term "cell" following his
observations of a piece of cork with a simple light microscope
in 1665
 Antony van Leeuwenhoek, in 1673, was able to observe a
variety of different types of cells, including sperm cells, red
blood cells, and bacteria.
 Microscopic studies of plant and animal cell by Matthias
Schleiden and Theodor Schwann respectively led to the
proposal of the cell theory.
 Thus, the cell achieved its current recognition as fundamental
unit of all living organisms because of observations made with
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the light microscope.
 Since most cells are between 1 µm and 100 µm in diameter, they can be
observed by light. However, the light microscope is not sufficiently powerful to
reveal fine details of cell structure, for which resolution—the ability of a
microscope to distinguish objects separated by small distances—is even more
important than magnification.
 The limit of resolution of the light microscope is approximately 0.2 µm
 Two objects separated by less than 0.2 µm appear as a single image,
rather than being distinguished from one another.
• Theoretical limitation of light microscopy is determined by two factors
 The wavelength (λ) of visible light and
 The light-gathering power of the microscope lens (numerical aperture,
NA)—according to the following equation:

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  The wavelength of visible light is 0.4 to 0.7 µm, so the value of A is
fixed at approximately 0.5 µm for the light microscope.
 The numerical aperture can be envisioned as the size of the cone of
light that enters the microscope lens after passing through the
specimen. It is given by the equation

 Where I is the refractive index of the medium through which light

travels between the specimen and the lens. The value of for air is
1.0, but it can be increased to a maximum of approximately 1.4 by
using an oil-immersion lens to view the specimen through a drop of oil.
 The angle α corresponds to half the width of the cone of light collected
by the lens. The maximum value of α is 90°, at which sin α = 1, so the
highest possible value for the numerical aperture is 1.4.
 The theoretical limit of resolution of the light microscope can therefore
be calculated as follows:

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33
 Types of light Microscope
 Bright-field microscopy
 Light passes directly through the cell and the ability to distinguish
different parts of the cell depends on contrast resulting from the
absorption of visible light by cell components.
 cells are stained with dyes that react with proteins or nucleic acids in
order to enhance the contrast between different parts of the cell.
 Staining procedures kill the cells
 not suitable for many experiments in which the observation of living
cells is desired.
 Phase-contrast microscopy
 Use optical systems that convert variations in density or thickness
between different parts of the cell.
 Enhances contrast in unstained cells by amplifying variations in
density within specimen
 useful for examining living, unsyatined cells.

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 Fluorescence microscopy
 A fluorescent dye is used to label the molecule of interest within either
fixed or living cells.
 The fluorescent dye is a molecule that absorbs light at one wavelength
and emits light at a second wavelength.
 used to study a variety of molecules within cells
 Differential-interference-contrast (Nomarski).
 Like phase-contrast microscopy, it uses optical modifications to
exaggerate differences in density, making the image appear almost 3D.
 Confocal Microscopy
 Uses lasers and special optics for “optical sectioning” of fluorescently-
stained specimens.
 Uses computer.

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 Electron Microscopy
 Because of the limited resolution of the light microscope, analysis of
the details of cell structure has required the use of more powerful
microscopic techniques—namely electron microscopy,
 The electron microscope can achieve a much greater resolution and
magnification than that obtained with the light microscope because
the wavelength of electrons is shorter than that of light.
 The wavelength of electrons in an electron microscope can be as short
as 0.004 nm: about 100,000 times shorter than the wavelength of
visible light. Theoretically, this wavelength could yield a resolution of
0.002 nm, but such a resolution cannot be obtained in practice,
because resolution is determined not only by wavelength, but also by
the numerical aperture of the microscope lens.
 Modern electron microscopes have a practical resolving power of
about 2 nm.
 Enhanced resolution and magnification allowed researchers to clearly
identify sub-cellular organelles and to study cell ultra-structure.
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 Types of electron microscopes
 The transmission electron microscopy (TEM)
 Thin section of specimen is stained with metals to absorb electrons and
enhance contrast.
 Electrons transmitted through the specimen are focused and the image
is magnified by using electromagnetic lenses (rather than glass lenses)
 Image is focused onto a viewing screen or film.
 It is used to study internal cellular ultra-structure.
 Scanning electron microscopy
 used to provide a three-dimensional image of cells.
 In scanning electron microscopy the electron beam does not pass
through the specimen. Instead, the surface of the cell is coated with a
heavy metal, and a beam of electrons is used to scan across the
specimen.
 Electrons that are scattered or emitted from the sample surface are
collected to generate a three-dimensional image as the electron beam
moves across the cell.
 Can usually only view dead cells because of the elaborate preparation
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required. May also introduce structural artifacts.
 Sub cellular Fractionation
 Although the electron microscope has allowed detailed visualization of cell
structure, microscopy alone is not sufficient to define the functions of the
various components of eukaryotic cells.
 Used to isolate the organelles of eukaryotic cells in a form that can be used
for biochemical studies.
 accomplished by differential centrifugation—a method used to separate the
components of cells on the basis of their size and density.
 The process of cell fractionation involves
• Homogenization of tissue: sonication, grinding in a mechanical
homogenizer, or treatment with a high-speed blender. All these
procedures break the plasma membrane and the endoplasmic reticulum
into small fragments while leaving other components of the cell (such as
nuclei, lysosomes, peroxisomes, mitochondria, and chloroplasts) intact.
• Centrifugation of the resulting homogenate at a slow speed. Nuclei and
other larger particles settle at the bottom of the tube, forming a pellet.
The unpelleted fluid or supernatant is decanted into another tube and
centrifuged at a faster speed, separating out smaller organelles.
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 Homogenization
Tissue
cells
Homogenate
1000 g
(1000 times the
force of gravity)
10 min
Differential centrifugation

Supernatant poured
into next tube

20,000 g
20 min

80,000 g
60 min
Pellet rich in
nuclei and
cellular debris
150,000 g
3 hr

Pellet rich in
mitochondria
(and chloro-
plasts if cells
are from a
plant)
Pellet rich in
“microsomes”
(pieces of
plasma mem-
branes and
cells’ internal Pellet rich in
membranes) ribosomes
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Organelles and Protein Sorting
 Each eukaryotic cell is subdivided into functionally distinct, membrane-
bound compartments called organelles
 Each of the various organelles within cells is specialized for one or more
tasks and therefore needs a specialized set of proteins to carry out its
function.
 These proteins (with the exception of those produced in mitochondria and
chloroplast) are synthesized on ribosomes in the cytosol and have to be
destined to their proper locations
 For example
 Receptors – functions on plasma membrane
 DNA polymerase – functions in the nucleus
 Catalase – functions in the peroxisomes
 Insulin – functions outside the cell
 The delivery of specialized set of proteins to their proper cellular
destinations is referred to as protein targeting or protein sorting or protein
translocation
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 Protein targeting can follow two distinct pathways:
1. Post-translational targeting
 Involve protein targeting after polypeptide synthesis is completed
on cytosolic ribosome
 Proteins that are going to nucleus, mitochondria, chloroplasts and
peroxisomes are targeted post-translationally
2. Co-translational targeting
 The synthesis of proteins begin on cytosolic ribosomes. The
ribosome with the nascent peptide is targeted to the endoplasmic
reticulum(ER) and sorting is done during translation (co-
translationally)
 This pathway is utilized by proteins destined for the ER, Golgi,
lysosome, the plasma membrane and protein that is going to be
secreted from the cell
How do proteins know where to go?

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 Targeting Sequences
 Targeting of proteins to their proper destination is done by short amino acid
sequences called targeting sequences or signal sequence or signal peptide or
sorting signals
 Targeting sequences function like cellular Postal Codes targeting proteins
towards their destination in the cell.
 Upon the delivery of the protein, the signal sequence may be removed by the
resident protein called SIGNAL PEPTIDASE or may remain as permanent part of
protein
Retention signal
 Another class of sorting signal that give a signal to the cell that the protein has
reached its final destination and should not be moved
Other requirements for protein targeting
 Specific receptor that recognize the signal sequence
 A channel (translocon) or two (for double membrane organelles) to
translocate (cross the organelle’s membrane)
 ATP to provide energy
 Chaperones proteins for proper folding of proteins
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43
The Nucleus
 found in all the eukaryotic cells. certain eukaryotic cells such as the mature
sieve tubes of higher plants and mammalian erythrocytes contain no
nucleus
 the number of the nucleus may vary from cell to cell. According to the
number of the nuclei following types of cells have been recognized :
Mononucleate cells. E.g. Most plant and animal cells
Binucleate cells. E.g. Paramecium and cells of cartilage and liver.
Polynucleate cells. The cells which contain many (from 3 to 100) nuclei .
The polynucleate cells of the animals are termed as syncytial cells, while
the polynucleate cells of the plants are known as coenocytes. The most
common example of the syncytial cells are the osteoblast
(polykaryocytes of the bone morrow) which contain about 100 nuclei per
cell and striated muscle fibers each of which contains many hundred
nuclei.
Structure of the nucleus
is the largest organelle surrounded by two concentric membranes called
the inner and outer nuclear membranes separated by an intermembrane
44 space
• The outer nuclear membrane is continuous
with the endoplasmic reticulum
• The nuclear envelop contains small channels
or holes that allow regulated exchange of
molecules between nucleus and cytoplasm.
These pores are called nuclear pore
complexes(NPC).
• An NPC is made up of multiple copies of some
50 different proteins called nucleoporins.
 The spherical inner nuclear membrane
contains specific proteins that act as binding
sites for the supporting fibrous sheath of
intermediate filaments (IF), called nuclear
lamina.
 Nuclear lamina maintains the shape of the
nucleus. These lamins also serve as sites of
chromatin attachment and organize other
proteins into functional nuclear bodies.
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Internal Organization of the Nucleus
Nucleoplasm
 The cell nucleus contains the fluid filled space known as nucleoplasm or
karyolymph. The majority of the cell's genetic material (DNA) is located in
nucleoplasm
Nucleolus
 The nucleolus is a discrete densely stained structure found in the nucleus
 Main roles of the nucleolus are to synthesize rRNA and assemble rRNA and
proteins to ribosome
Protein targeting to the nucleus
 All proteins found in the nucleus are synthesized in the cytoplasm and imported
into the nucleus through nuclear pore complexes.
 Such proteins contain a targeting sequences called nuclear-localization signal
(NLS)
 NLS directs the selective transport of proteins into the nucleus.
 Examples: Histones, Ribosomal proteins, DNA and RNA polymerases, and
Transcription factors are guided into nucleus with the help of NLS.
 NLS consist of four to eight amino acid residues and include several
consecutive basic aminoacids (Arg or Lys).
 In addition to NLS, Nuclear import requires
 Nuclear import receptor: Importin α and β
 GTP as source of energy
 Ran GTPase
 Fully folded cargo protein: protein to be transported into nucleus

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Mechanism of Nuclear Import
 Importin α and β bind to the cargo protein at its NLS, forming cargo-
importin complex
 The complex is translocated through the nuclear pore complex(NPC)
into the nucleus
 In the nucleoplasm, interaction of Ran·GTP with the importin causes a
conformational change that decreases its affinity for the NLS, releasing
the cargo in the nucleoplasm.
 To support another cycle of import the importin-Ran·GTP complex
diffuses back through the NPC.
Ribosomes
 Ribosomes are non membrane bounded cytoplasmic organelle found in all
organisms.
 are complexes of rRNA and protein constructed in the nucleolus in eukaryotic
cells
 Cells with high rates of protein synthesis have prominent nucleoli and many
ribosomes (e.g., human liver cell has a few million).
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 It is the site for protein synthesis
 Ribosomes function either free in the cytosol or bound to endoplasmic
reticulum.
 Most proteins made by free ribosomes will function in the cytosol. Bound
ribosomes generally make proteins that are destined for membrane
inclusion or export.
Types of ribosomes
 According to the size and the sedimentation coefficient (S) two types of
ribosomes have been recognized
 70S Ribosomes.
 comparatively smaller in size and have sedimentation coefficient 70S
and the molecular weight 2.7× 106 daltons.
 occurs in the prokaryotic cells of the blue green algae and bacteria and
also in mitochondria and chloroplasts of eukaryotic cells.
 consists of two subunits, viz., 50S and 30S. The 50S ribosomal subunit is
larger in size and the 30S ribosomal subunit is smaller in size
 80S Ribosomes.
 have the sedimentation coefficient of 80S and the molecular weight 40 ×
49 106 daltons.
 The 80S ribosomes occur in eukaryotic cells. The 80S ribosome also
consists of two subunits, viz., 60S and 40S.

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Mitochondria
 Surrounded by a double membrane, the inner and outer mitochondrial
membranes are separated by intermembrane space.
 The inner membrane forms numerous folds called cristae that surround
the inner space, matrix
 Found in almost all eukaryotic cells
 Mitochondria plays a critical role in generation of metabolic energy
(ATP): power house of cells
 The matrix contains the enzymes for TCA cycle, the inner membrane
contains protein complexes involved in ETS and oxidative metabolism
 Mitochondria have their own DNA and manufacture a small number of
their own proteins.

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 Import of proteins to Mitochondria
 The majority of mitochondrial proteins are coded for by nuclear genes. These are
synthesized on free ribosomes and only imported into the mitochondrion post-
translationally.
 Only unfolded proteins can be imported into the mitochondria in order for the proteins
to be pulled through small pores of mitochondrial translocons.
 The unfolded state of such proteins is maintained by proteins called cytosolic chaperones

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 Proteins targeting to mitochondria requires
 Mitochondrial targeting sequences called pre-sequences
 One TOM complex (translocase of outer mitochondrial membrane)-
for targeting to outer mitochondrial membrane and
 Two TIM complexes (Translocase of inner mitochondrial membrane).
One TIM complex for targeting to mitochondrial matrix and one TIM
complex for inner mitochondrial membrane
• After protein import, the uptake-targeting sequence is removed by
proteases called signal peptidase within the matrix and the proteins are
folded into their correct shape
Plastids:
 Plastids are groups of plant and algal membrane-bound organelles that
include Leucoplasts, chromoplasts and chloroplasts.
Leucoplasts:
 The leucoplasts are the colorless plastids which are found in
embryonic and germ cells.
 They store the food materials as carbohydrates, lipids and proteins

53
Chromoplasts
 are the colored plastids containing carotenoids and other pigments.
 They impart color (e.g., yellow, orange and red) to certain portions of
plants such as flower petals, fruits and some roots.
Chloroplast
 are chlorophyll-containing plastids which are the sites of
photosynthesis
 Found in eukaryotic algae, leaves and other green plant organs.
 Like the mitochondrion, the chloroplast is surrounded by an outer and
an inner membrane and posses their own genetic material
 Chloroplasts contain an extensive internal system of interconnected
membrane called thylakoids which serve as the site of electron
transport system for ATP synthesis

54
 Thylakoids are arranged in stacks called grana embedded in a matrix, the
stroma.

 The thylakoid membranes contain green pigments (chlorophylls) and other

pigments that absorb light and generate ATP during photosynthesis

55
Protein targeting to Chlorolast
 Most chloroplast proteins are synthesized on free ribosomes in the cytosol and
targeted into chloroplast in unfolded state
 Protein import into the chloroplast requires
 Targeting sequences called transit sequences that target chloroplast proteins
to the import machinery in the chloroplast membranes
 TOC complex (Translocase at outer membrane of chloroplast) and
 TIC complexes (Translocase at inner membrane of chloroplast.
 After targeting, the transit sequence is cleaved by stromal processing
peptidase (SPP), and the proteins are folded into their correct shape by
chaperon proteins
Peroxisomes
 Peroxisomes are small single membrane bounded organelles that contain
enzymes involved in variety of metabolic reactions
 Functions include:
 Decomposing harmful chemicals such as H2O2.
 Fatty acid oxidation: into acetyl group that provide source of enery

56  Biosynthesis of lipids such as cholestrol etc

Protein import to Peroxisomes
 All proteins destined for peroxisomes are also translated on free cytosolic
ribosomes and then transported as completed polypeptide chain fully folded in
cytosol
 The Import requires
 Two peroxisome targeting signals: PTS1 and PTS2
 Peroxins - peroxisome transport receptors that recognize PTS1 and PTS2
 Translocation channel
 During the transport:
 Peroxins bind to cargo proteins with PTS1 or PTS2 and dock to the
translocation channel
 The complex is transported through the membrane into peroxisome, protein
is released and Peroxin is recycled
 Unlike transport to mitochondria and chloroplast ,the targeting signals are not
usually cleaved during the import of proteins into peroxisomes

57
Endoplasmic Reticulum
 The endoplasmic reticulum is a network of membrane enclosed channels
that run throughout the cell
 It forms a continuous network whose lumen (inside) is separated from the
cytosol by a single membrane.
 The membrane of the endoplasmic reticulum is continuous with the outer
nuclear membrane
 There are two regions of ER
1. Smooth endoplasmic reticulum
2. Rough endoplasmic reticulum
 The basic difference is that, unlike the smooth endoplasmic reticulum,
rough endoplasmic reticulum is covered in ribosomes, which give it its
rough appearance in the electron microscope.
Smooth endoplasmic reticulum
 Used for the synthesis of fatty acids and phospholipids
 in the liver it is the site of detoxication of foreign chemicals including drugs.
It is also the storage site of calcium ions.
Rough endoplasmic reticulum
58  Ribosome bound to the rough ER synthesize proteins
Protein import to ER
 Synthesis of proteins destined for import into the endoplasmic reticulum
starts on free ribosome in cytosol.

60
  Requirements for the import to ER
 Signal peptide of about 20 amino acids long
 Signal recognition particle (SRP): recognize the signal sequence of protein to
be targeted to endoplasmic reticulum
 is made up of a small RNA molecule and proteins
 The signal recognition particle receptor(SRPR): Receptor for SRP
 Protein translocator
Mechanism of targeting
 After synthesis of a protein begins on free ribosomes in the cytosol, the
endoplasmic reticulum signal sequence is recognized by a signal
recognition particle
 The signal recognition particle brings the ribosome to the endoplasmic
reticulum membrane where it interacts with a signal recognition particle
receptor
 The SRP and SRP receptor then mediate insertion of the nascent protein
into the translocon (channel within the membrane)
 As the ribosome attached to the translocon continues translation, the
unfolded protein chain is extruded into the ER lumen
61
 Once the polypeptide
chain has entered the
lumen of the
endoplasmic reticulum,
the signal sequences
may be cleaved off by an
enzyme called signal
peptidase.

62
Post translational modification of proteins
 Membrane proteins and soluble proteins synthesized on the rough ER
undergo a number of modifications before they reach their final
destinations:
 Glycosylation: Most polypeptides synthesized on the rough endoplasmic
reticulum are glycosylated, that is, they have sugar residues added to
them
 Formation of disulfide bonds: disulfide bonds (–S–S–) help stabilize the
tertiary and quaternary structure of many proteins
 Folding: Only properly folded proteins and assembled subunits are
transported from the rough ER to the Golgi complex in vesicles.
Unassembled or misfolded proteins in the ER often are transported back
through the translocon to the cytosol, where they are degraded.

63
Golgi Apparatus
 Golgi, is a distinctive stack of flattened sacks called cisternae.
 The Golgi apparatus is the distribution point of the cell where proteins made
within the rough endoplasmic reticulum are further processed and then
directed to their final destination.
 The stack of flattened Golgi sacs has three defined regions — the cis, the
medial, and the trans.

64
 Transport from the ER through the Golgi Apparatus
(VESICULAR TRAFFICKING)
 In vesicular transport, newly synthesized proteins are transported
from the ER to the Golgi apparatus and from the Golgi apparatus to the
cell surface and elsewhere,

65
Vesicular transport
 Transport mediated by membrane bound vesicles
 It involve formation and budding of a vesicle from one organelle, followed by
fusion with a second membrane to transport proteins to the new
compartment.
Vesicle Formation
 Vesicle formation is the process during which cargo is captured and the lipid
membrane is shaped with the help of cytosolic proteins into a bud, which is
then pinched off in a process called fission.
 There are two types of coats that serve this function:
1. Coatomer coats (COP)
2. Clathrin coats.
Coatomer-Coated Vesicles
 used in trafficking between the endoplasmic reticulum and Golgi
i. COPII-coated vesicles: bud from the ER and transport proteins to Golgi
complex (Anterograde Transport)

66
 ii. COPI-coated vesicles: bud from pre-Golgi compartments and transport mis-
targeted proteins back to ER(Retrograde transport).
 if resident proteins of ER lumen containing a retention signal KDEL (Lys-Asp-
Glu-Leu) are mis-targeted to Golgi, they targeted back to ER, coated by COPI
coated vesicle
Clathrin-Coated Vesicles
 Clathrin-coated vesicles mediate selective transport from
 the Golgi to lysosome, plasmamembrane and secretary proteins to the surface
of plasma membrane
 proteins and lipids from the plasma membrane to the endosome, and operate
in other places where selective transport is required.
Vesicle fusion with target organelle
 vesicle fusion is the process by which a vesicle membrane incorporates its
components into the target membrane and releases its cargo into the lumen
of the organelle or, in the case of secretion, into the extracellular medium
 Vesicle fusion is highly specific. vesicle must only dock with and fuse with
the correct target membrane in order to avoid mixing of proteins

67
 Specificity in targeting is ensured because all transport vesicles and target
membranes display unique surface markers known as SNAREs
 SNARE proteins have a central role both in providing specificity and in
catalyzing the fusion of vesicles with the target membrane
 SNARES located on the vesicles are known as v-SNARES and
 SNAREs on the target membranes are known as t-SNARES
 Each v-SNARE in a vesicular membrane specifically binds to a t-SNARE proteins
in the target membrane, inducing fusion of the two membranes and release
cargo protein.
 After fusion is completed, the SNARE complex is disassembled in an energy
dependent reaction
 GTP hydrolysis by proteins Rabs is thought to provide energy for membrane
fusion.

68
 Lysosome
 Lysosomes are a single membrane bounded acidic organelles that contain a
battery of degradative enzymes
 All the lysosomal enzymes work most efficiently at acid pH values and
collectively are termed acid hydrolases
Import to lysosomes
 Proteins that are destined for the lysosome are synthesized on the rough
endoplasmic reticulum. Because they do not have an endoplasmic reticulum
retention signal such as KDEL, they are transported to the Golgi apparatus
 In the Golgi apparatus the proteins are modified by addition of Mannose
containing oligosaccharides and mannose is phosphorylated at carbon number
6 ( form Mannose-6-phosphate)
 Mannose-6-phosphate is used as a surface marker distinguishing proteins
destined to lysosome from others
 Proteins containing mannose-6-phosphate are coated with a clathrin coat at
transgolgi network and targeted to lysosome

69
70
 The plasma membrane
o The plasma membrane also called plasmalemma or cell membrane is
the structure that encloses the cell & defines cell boundaries
Functions
1. The plasma membrane encloses the cell & defines cell boundaries
2. Maintains the essential differences between the cytosol and the extra
cellular environment.
3. Inside eukaryotic cells, maintain the characteristic differences
between the contents of each organelle and the cytosol.
4. Ion gradients across membranes is established by the activities of
specialized membrane proteins
5. Can be used to synthesize ATP
6. Selective transport of solutes
7. in nerve and muscle cells, to produce and transmit electrical signals.
8. In all cells, the plasma membrane also contains proteins that act as
sensors of external signals

71
 Membrane structure
 Despite their differing functions, all biological membranes have a
common general structure:
 each is a very thin film of lipid bilayer and protein molecules,
held together mainly by non-covalent interactions.
Early observations:
 The first glimpse of the cell membrane's structure came in 1925 when
Gorter & Grendell found a way to release the contents of red blood
cells leaving only the membranes, called erythrocyte ghosts.
 Analysis of these ghosts revealed the presence of lipids.
 Thus, Gorter & Grendell demonstrated that the plasma
membrane consisted of two layers of lipid --it was a bimolecular
layer.
 A little later in the mid-1930s, Schmidt based on observation of the
rotation of polarized light by myelin sheaths (membranes around
neurons) suggested the presence of protein.
72
 Membrane Models
1. The Davson- Danielli Sandwich Model of Membranes
 In part, the work just cited lead Davson and Danielli to develop a model of
membrane structure known as sandwich model
 They defined membrane as a bimolecular lipid layer capped on the inside and
outside by protein.
 also postulated the presence of protein-lined pores for movement small
molecules through membranes
•The Davson-Danielli model
can be thought of as a
sandwich where the bread
represents the outer and
inner layers of protein and
the sandwich filling the lipid
component (however, this
sandwich model is inadequate
since it has no protein pores

73
2. Robertson's Unit Membrane Model
 Electron micrographs of tissue sections fixed in osmium tetroxide (a
fixative) revealed membranes as solid dark lines about 75 Å thick.
 In 1959 Robertson used a different fixative and stained his specimens with
potassium permanganate. This gave much better resolution. He found that
membranes consisted of a light area about 35 Å thick ( lipid) surrounded by
dark lines each 20 Å in thickness (proteins)..
 The membranes in Robertson's electron
micrograph were interpreted as consisting of a
protein-lipid-protein sandwich, consistent with
the Davson-Danielli model.
 Furthermore, all cellular membranes had the
same structure that led Robertson to conclude
that they were identical. This led him to his
"unit membrane hypothesis" which stated
simply that all membranes had essentially the
same sandwich like structure
74
Conflict – Problems with the Davson-Danielli Sandwich
and Unit Membrane models
 Despite the unifying effect of Robertson's unit membrane
hypothesis, problems developed when it was found that each
membrane-bound organelle had its own particular function.
 How could membranes vary in function if they all had the
same structure?
 Variations in membrane thickness were also observed which cast
further doubt on Robertson's generalization. Furthermore, the
Davson-Danielli model seemed inadequate to explain some
membrane properties,
 e.g. how could lipid soluble material pass through
membranes regardless of size if a protein cap protected the
lipid?

75
3. The Fluid Mosaic Model
 Proposed by Singer & Nicholson proposed in 1972
 This model, called the fluid mosaic model, emphasized the
dynamic nature of membranes in sharp contrast to the static
Davson-Danielli model.
 According to Singer & Nicholson:
 the molecular structure of the membrane is not rigid and
fixed, but rather fluid.
 The lipids are not capped with a solid protein coating. Instead,
protein molecules are dispersed throughout the membrane
leaving many portions of the lipid bare and exposed to the
extra-and intra-cellular environments.
 It is through these bare areas that lipid soluble molecules
pass.

76
  In this model, the
phospholipids bilayer is a
fluid matrix, in essence, a
two-dimensional solvent
for proteins.
 Both lipids and proteins are
capable of rotational and
lateral movement.
 Singer and Nicolson also
pointed out that proteins
can be associated with the
surface of this bilayer or
embedded in the bilayer to
varying degrees

77
The lipid bilayer
 The lipid bilayer:
 provides the basic fluid structure of the membrane
 serves as a relatively impermeable barrier to the
passage of most water-soluble molecules.
all of the other functions of the membrane, transporting
specific molecules across it, or catalyzing membrane-
associated reactions, such as ATP synthesis are provided by
membrane proteins

78
Properties of lipid bilayer
1. All of the lipid molecules in cell membranes are amphipathic
(or amphiphilic)—that is, they have a
 hydrophilic (“water-loving”) or polar end and a
 hydrophobic (“water-fearing”) or nonpolar end.
 The hydrophobic ends are composed of fatty acids
 FA differ in length (they normally contain between
14 and 24 carbon atoms).
 One of the tails is unsaturated while the other tail
is saturated.
 The hydrophilic head group is composed of alcohol
attached with phosphate

79
 Cont.
2. Self sealing property:
 Lipid molecules spontaneously aggregate to bury their
hydrophobic tails in the interior and expose their
hydrophilic heads to water.
 Depending on their shape, they can aggregate in either of
two ways:
1. They can form spherical micelles, with the tails
inward or
2. They can form bimolecular sheets, or bilayers, with
the hydrophobic tails sandwiched between the
hydrophilic head groups.

Micelles Liposome Bilayer

80
Cont.
3. The lipid component of a biological membrane is a two-
dimensional liquid in which the constituent molecules are free
to move laterally
4. Biological membranes are asymmetric structures.
Composition of lipid bilayer
 The lipid bilayer of many cell membranes is composed of
 Phospholipids
 Cholesterol
 Glycolipids.

81
Phospholipids:
 Composed of four components:
 2 fatty acid chains
 a phosphate,
 an alcohol (Glycerol or
sphingosine) attached to the
phosphate
 Head group(choline, serine,
ethanolamine….. etc)
 The fatty acid components
 provide a hydrophobic barrier
 A phosphate , head group and an
alcohol molecules
 has hydrophilic properties to
enable interaction with the
environment.
82
Cont.
 Phospholipids derived from glycerol
are called phosphoglycerides
(glycerophospholipids). Phospholipids
derived from sphingosine are called
spingophospholipids

 The four major phospholipids are:

•Only phosphatidylserine carries a net negative

charge,
•the other three are electrically neutral at
physiological pH, carrying one positive and one
negative charge.
83
Cholesterol
 It is a steroid, built from four linked hydrocarbon rings.
 Attached to the ring are a hydrocarbon tail and a hydroxyl group.
 In membranes,
 the hydrophobic part interact with the fatty acid chains of the
phospholipids
 the hydroxyl group interacts with the nearby phospholipid head
groups.
 absent from prokaryotes but is
found in all animal
membranes.
 constitutes almost 25% of the
membrane lipids in certain
nerve cells but is essentially
absent from some intracellular
membranes.
84
Glycolipids.
 Glycolipids, as their name implies, are sugar-containing lipids.
found exclusively on the extracellular layer of the membrane
 Constitute 2–10 percent of the total lipid in plasma membranes;
they are most abundant in nervous tissue.
.

85
Membrane fluidity
 Plasma membrane is a two dimensional fluid.
 It can change from a liquid state to a two-dimensional rigid
crystalline (or gel) state at a characteristic freezing point.
 This change of state is called a phase transition
 The temperature at which the structure undergoes the transition
from ordered to disordered (ie, melts) is called the transition
temperatures or melting temperature (Tm).

86
Factors affecting membrane fluidity
1. Fatty acid chain length
 A shorter chain length reduces the tendency of the
hydrocarbon tails to interact with one another thus increase
membrane fluidity
2. Degree of saturation of the hydrocarbon chain
 Presence of cis-double bonds produce kinks in the
hydrocarbon chains that make them more difficult to pack
together, so that the membrane remains fluid
3. Amount of Cholesterol
 because of its shape cholesterol prevents long-chain fatty
acids from packing close to each other at low temperature
and increase membrane fluidity.

 
87
Cont.
 Cholesterol at high temperature, decrease membrane
fluidity by making the lipid bilayer less deformable. In this
way, it inhibits possible phase transitions
4. Temperature
Fluidity increases with increased temperature

88
Membrane proteins
 There are two classes of membrane proteins.
1. Peripheral proteins (or extrinsic proteins)
2. Integral proteins (or intrinsic proteins or transmembrane
proteins)
Peripheral proteins
 do not penetrate the bilayer to any significant degree
 associated with the membrane by weak non-covalent
bonds at the surface of membrane
o can be dissociated from the membrane by treatment
with salt solutions or by changes in pH (treatments that
disrupt hydrogen bonds and ionic interactions).

89
Cont.
Integral proteins
 possess hydrophobic surfaces that can readily penetrate the lipid
bilayer itself
 can insert into the membrane or extend all the way across the
membrane and expose themselves to the aqueous solvent on both
sides.
Because of these intimate associations with membrane lipid,
integral proteins can only be removed from the membrane by
agents capable of breaking up the hydrophobic interactions
within the lipid bilayer itself (such as detergents and organic
solvents).

90
Functions of membrane proteins
Proteins of the plasma membrane provide 6 membrane functions:
1. Transport Proteins: transport lipid insoluble molecules and ions across
the membrane
2. Receptor Proteins: Bind to chemical messengers (Ex. hormones) which
sends a message into the cell
3. Enzymatic Proteins: Carry out enzymatic reactions right at the
membrane when a substrate binds to the active site
4. Cell Recognition Proteins: Glycoproteins (and glycolipids) on
extracellular surface serve as ID tags (which species, type of cell,
individual)
5. Attachment Proteins: Attach to cytoskeleton to maintain cell shape
6. Intercellular Junction Proteins: Bind cells together

91
 Distribution of membrane components in two layers
The lipid compositions of the two halves of the lipid bilayer are
different. i.e asymetrical
For example, in the human red blood cell membrane
 Phosphatidylcholine and sphingomyelin are located in the outer
half of the lipid bilayer,
 phosphatidylethanolamine and phosphatidylserine are located in
the inner half.
 Because the negatively charged phosphatidylserine is located
in the inner monolayer the inner is slightly negative in charge
than the outer
 Glycolipids are found exclusively in the non-cytoplasmic
half(outer) of the lipid bilayer

92
93
 TRANSPORT ACROSS BIOLOGICAL
MEMBRANES
 The internal composition of the cell is maintained because the
plasma membrane is selectively permeable to small molecules.
 Most biological molecules are unable to diffuse through the
phospholipids bilayer, so the plasma membrane forms a barrier
that blocks the free exchange of molecules between the
cytoplasm and the external environment of the cell.
 Specific transport proteins (carrier proteins and channel proteins)
then mediate the selective passage of small molecules across the
membrane, allowing the cell to control the composition of its
cytoplasm

94
In general
 Small non-polar molecules, such as O2
and N2 readily dissolve in lipid bilayers
and therefore diffuse rapidly across
them.
 Small uncharged polar molecules, such
as water and urea, also diffuse across a
bilayer, albeit much more slowly
 By contrast, lipid bilayers are highly
impermeable to charged molecules
(ions) such as Na+ and K+ Cl-, HCO3-,
small hydrophilic molecules like
glucose, macromolecules like proteins
and RNA can not freely diffuse through
the plasma membrane

95
Types of Membrane Transport
 Generally there are two membrane transport systems based on
energy requirements
1. Passive transport
 Simple Diffusion
 Facilitated Diffusion
2. Active transport
 Direct active transport
 Indirect Active transport

96
 Simple Diffusion
 The simplest mechanism by which molecules can cross
the plasma membrane is simple diffusion.
 Diffusion is the movement of molecules from region of
high concentration to the region of low concentration.
 During simple diffusion,
 A molecule dissolves in the phospholipid bilayer
and diffuses across it
 It does not need energy for the process.
 No membrane proteins are involved
 the direction of transport is determined simply by
the relative concentrations of the molecule inside
and outside of the cell.

97
 The net flow of molecules is always down their concentration gradient—
from a compartment with a high concentration to one with a lower
concentration of the molecule
 Molecules that can pass through plasma membrane by simple diffusion
are
 Gases (such as O2 and CO2)
 Hydrophobic molecules (such as benzene)
 Small polar but uncharged molecules (such as H2O and ethanol) are
able to diffuse across the plasma membrane.
 Other biological molecules, however, are unable to dissolve in the
hydrophobic interior of the phospholipid bilayer and can not diffuse
freely through the plasma membrane. This includes:
 Larger uncharged polar molecules such as glucose, proteins
 Charged molecules of any size (including small ions such as H +, Na+,
K+, and Cl-).

98
 Facilitated Diffusion
 Facilitated diffusion like simple diffusion, involves
 No external source of energy
 The direction of the transport is down hill
 Facilitated diffusion differs from simple diffusion in that
 The transported molecules do not dissolve in the phospholipid
bilayer. Instead, their passage is mediated by proteins that
enable the transported molecules to cross the membrane
without directly interacting with its hydrophobic interior.
 It allows the movement of polar and charged molecules, such
as carbohydrates, amino acids, nucleosides, and ions, to cross
the plasma membrane.
 The direction of their transport is determined both by the
concentration and voltage(electrical) differences-
Electrochemical gradient
99
 Types of proteins that mediate facilitated diffusion

100
  Uniporters: transport a single type
of molecule down its concentration
gradient via facilitated diffusion
 Symporters are also known as Co-
transporters
 They transport two different
solutes simultaneously in the
same direction
 Antiporters: are also known as
counter transporters
 They also transport two
different solutes simultaneously
but in opposite direction

101
 Mechanism of transport by carrier proteins
 Carrier proteins transport molecules by binding to specific molecules to
be transported on one side of the membrane.
 They then undergo conformational changes that allow the molecule to
pass through the membrane and be released on the other side.
 Are responsible for the facilitated diffusion of sugars, amino acids, and
nucleotides across the plasma membranes of most cells.
E.g. Glucose transporter - functions by alternating
between two conformational state In the first
conformation, a glucose-binding site faces the
outside of the cell. The binding of glucose to this
exterior site induces a conformational change in the
transporter, such that the glucose-binding site now
faces the interior of the cell. Glucose can then be
released into the cytosol, followed by the return of
the transporter to its original conformation s

102
 Channel proteins
 they form a hydrophilic passageway across the membrane
through which polar molecules or ions move
 In contrast to carrier proteins, Channel proteins simply form open
pores in the membrane, allowing small molecules of the
appropriate size and charge to pass freely through the lipid
bilayer. E.g.
 Porins: permit the free passage of ions and small polar
molecules through the outer membranes of bacteria.
 aquaporins: water channel proteins through which water
molecules are able to cross the membrane much more
rapidly than they can diffuse through the phospholipid
bilayer.
 Ion channels: are the best-characterized channel proteins
which mediate the passage of ions across plasma membrane
103
 Properties of ion channels
 Transport through ion channels is extremely rapid. More than a
million ions per second flow through open channels—a flow rate
approximately a thousand times greater than the rate of transport
by carrier proteins.
 Ion channels are highly selective because narrow pores in the
channel restrict passage of ions of the appropriate size and charge.
Thus, specific channel proteins allow the passage of Na+, K+, Ca2+,
and Cl- across the membrane.
 Some ion channels are open much of the time; these are referred
to as non-gated channels. Most ion channels, however, open only
in response to specific chemical or electrical signals; these are
referred to as gated channels.

104
 Examples of gated channels
1. Ligand-gated channels: open in response to the binding of
neurotransmitters or other signaling molecules; Example The
binding of the neurotransmitter acetylcholine at certain
synapses opens channels that admit Na+ and initiate a nerve
impulse or muscle contraction.
2. Voltage-gated channels: open in response to changes in
electric potential across the plasma membrane.

105
 Osmosis
Osmosis: is the diffusion of water through a
selectively permeable membrane
 Water moves from high water area to low
water area or from dilute(weak) solution to
concentrated(strong) solution
 If two solutions have unequal solute
concentration, the solution with higher solute
concentration is known as Hypertonic (hyper=
more than) and the one with low solute •In cells, plasma membrane
concentartion is known as hypotonic(Hypo= separates two aqueous
less than). solutions, one inside the
 If the solute concentration of two solutions cell(cytoplasm) and one
are equal the solutions are said to be Isotonic outside(extracellular fluid).
( Iso= same) •The direction of net diffusion
of water is determined by solute
concentration of either side

106
Direction of Osmosis
 The net direction of osmosis depends on the relative
concentration of solutes on the two sides of the membrane.
 When the concentration of solute molecules outside the cell is
lower than the concentration in the cytosol, the solution
outside is hypotonic to the cytosol. In this situation, water
diffuses into the cell until equilibrium is reached.
 When the concentration of solute molecules outside the cell is
higher than the concentration in the cytosol, the solution
outside is hypertonic to the cytosol. The water will diffuse out
of the cell until equilibrium is established.
 When the concentration of solutes outside and inside the cell
are equal, the outside solution is said to be isotonic to the
cytosol. The water diffuses into and out of the cell at equal
rates, so there is no net movement of water.
107
 Water balance of cells
Animal cells
1. If placed in Hypotonic solution:
 Cells swell and finally burst as water enters them by
osmotic flow.
 Rupture of the plasma membrane by a flow of water into
the cytosol is termed osmotic lysis.
2. Hypertonic solution
 causes them to shrink as water leaves them by osmotic
flow a condition called crenation.
3. Isotonic solution
 No net flow of water into or out of the cell. This maintains
normal animal cell volume

108
 Plant, algal, fungal, and bacterial cells
1. Hypotonic solution
Unlike animal cells they are surrounded by a rigid cell wall
Because of the cell wall, the osmotic influx of water that
occurs when such cells are placed in a hypotonic solution
(even pure water) leads to an increase in intracellular
pressure called osmotic pressure
The osmotic pressure also called turgor pressure, generated
from the entry of water into the cytosol pushes the cytosol
and the plasma membrane against the resistant cell wall. In
this case the cell get turgid and turgidity maintains normal
cell volume.
2. Hypertonic solution
The water will diffuse out of the cell and the protoplasm of
the cell shrink. The condition known as plasmolysis.
109
What will happen to plant and animal cells if placed in the following
solutions?

110
 Active transport

111
 Direct Active Transport: Example
The Na+/K+ ATPase
The cytosol of animal cells contains a concentration of
potassium ions (K+) as much as 20 times higher than that in the
extracellular fluid. Conversely, the extracellular fluid contains a
concentration of sodium ions (Na+) as much as 10 times greater
than that within the cell.
These concentration gradients are established by the active
transport of both ions. And, in fact, the same transporter,
called the Na+/K+ ATPase(Pump), does both jobs.
 It uses the energy from the hydrolysis of ATP to actively
transport 3 Na+ ions out of the cell for each 2 K+ ions pumped
into the cell.

112
 The process involve ATP-driven
conformational changes in the pump.
1. Na+ ions bind to sites inside the cell. This
binding stimulates the hydrolysis of ATP
and phosphorylation of the pump,
2. Phosphorylation induce a
conformational change that exposes the
Na+-binding sites to the outside of the
cell and reduces their affinity for Na+.
Consequently, the bound Na+ is released
into the extracellular fluids.
3. At the same time, K+-binding sites are 4. Hydrolysis of phosphate
exposed on the cell surface. The binding induces a second
of extracellular K+ to these sites then conformational change,
stimulates hydrolysis of the phosphate exposing the K+-binding
group bound to the pump sites to the cytosol and
lowering their binding
affinity so that K+ is
released inside the cell.
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 Secondary active transport: Active Transport driven by Ion
Gradients
 function as coupled carriers, in which the transfer of one solute
strictly depends on the transport of a second.
 Coupled transport involves either the simultaneous transfer of a
second solute in the same direction, performed by symporters or
the transfer of a second solute in the opposite direction,
performed by antiporters
 In this transport system, the free energy released during the
movement of an inorganic ion down an electrochemical gradient
is used as the driving force to pump other solutes uphill, against
their electrochemical gradient.

114
 Example of Indirect active transport
The Na+/glucose transporter(Co-transporter or symporter)
 the Na+/glucose transporter protein allows transport of
sodium ions and glucose to enter the cell together.
Glucose molecules are pumped actively into the cell using the
energy released from the flow of sodium ions down their
concentration gradient
Later the sodium is pumped back out of the cell by the Na+/K+
ATPase.
The Na+-H+ exchange protein(counter transporter or antiporter)
The Na+-H+ antiporter couples the transport of Na+ into the cell
with the export of H+, thereby removing excess H+ produced by
metabolic reactions and preventing acidification of the
cytoplasm.

115
 Endocytosis and Exocytosis
Endocytosis
 The carrier and channel proteins discussed in the preceding
section transport small molecules through the phospholipid
bilayer.
 Eukaryotic cells are also able to take up macromolecules and
particles from the surrounding medium by a distinct process
called endocytosis using ATP energy.
 In endocytosis, the material to be internalized is surrounded by an
area of plasma membrane, which then buds off inside the cell to
form a vesicle containing the ingested material.
 There are three types of Endocytosis
1. Phagocytosis
2. Pinocytosis
3. Receptor mediated endocytosis
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 Phagocytosis (cell eating)
 Phagocytosis is the process by which cells engulf large particles such as
bacteria, cell debris, or even intact cells.
 Mechanism of Phagocytosis
 The particle to be transported binds to receptors on the surface of the
phagocytic cell and triggers the extension of pseudopodia.
 The pseudopodia eventually surround the particle and their
membranes fuse to form a large intracellular vesicle called a
phagosome.
 The phagosomes then fuse with lysosomes, producing
phagolysosomes in which the ingested material is digested by the
action of lysosomal acid hydrolases.

117
Role of Phagocytosis
amoebas use phagocytosis to capture food particles
 provide a defense against invading microorganisms and to
eliminate aged or damaged cells from the body e.g
Macrophages
Pinocytosis or “cell drinking,” is the process of taking in droplets of
ECF containing molecules of some use to the cell. The process
begins as the plasma membrane becomes dimpled, or caved in, at
points. These pits soon separate from the surface membrane and
form small membrane-bounded vesicles in the cytoplasm.

118
 Receptor-Mediated Endocytosis
 Movement of very specific molecules
into the cell with the use of vesicles
coated with the protein clathrin.
 Coated pits are specific locations
coated with clathrin and receptors.
When specific molecules (ligands)
bind to the receptors, then this
stimulates the molecules to be
engulfed into a coated vesicle.
 Ex. Uptake of cholesterol (LDL) by
animal cells

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 Exocytosis
 Movement of large molecules
bound in vesicles out of the cell
with the aid of ATP energy.
 Vesicle fuses with the plasma
membrane to eject
macromolecules.
 Ex. Proteins, polysaccharides,
polynucleotides, whole cells,
hormones, mucus,
neurotransmitters, waste

120
 Cell Walls and The Extracellular Matrix
 Although cell boundaries are defined by the plasma membrane,
Cells of bacteria, fungi, algae, and higher plants are surrounded by
rigid cell walls
 Although animal cells are not surrounded by cell walls, most of
the cells in animal tissues are embedded in an extracellular matrix
consisting of secreted proteins and polysaccharides.
Cell Walls
 The rigid cell walls that surround bacteria and many types of
eukaryotic cells (fungi, algae, and higher plants) determine cell
shape and prevent cells from swelling and bursting as a result of
osmotic pressure. Despite their common functions, the cell walls
of bacteria and eukaryotes are structurally very different.

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Bacterial Cell Walls
 determine cell shape and prevent the cell from bursting as a
result of osmotic pressure.
 The structure of their cell walls divides bacteria into two
broad classes that can be distinguished by a staining
procedure known as the Gram stain, developed by Christian
Gram in 1884.
1. Gram-negative bacteria (Example: E. coli)
 have a two membrane system, the plasma membrane and
the outer lipopolysaccharide membrane.
 These bacteria have thin cell walls located between, the two
membrane systems, their inner and outer membranes.
1. Gram-positive bacteria (Example: Staphylococcus aureus)
 have only a single membrane, which is surrounded by a much
thicker cell wall.

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Composition of Bacterial cell wall
 The cell wall of both gram positive and gram negative bacteria are
composed of a polysaccharide known as peptidoglycan
 Peptidoglycan is made up of two monomeric subunits-
N-acetylglucoseamine (NAG) and
N-acetylmuramic acid (NAM)
 These subunits are cross linked with a short peptide that provide
strength to the bacterial cell wall
 The unique structure of their cell
walls also makes bacteria vulnerable
to some antibiotics. Penicillin, for
example, inhibits the enzyme
responsible for forming cross-links
between different strands of the
peptidoglycan, thereby interfering
with cell wall synthesis and blocking
bacterial growth.
 Eukaryotic Cell Walls
 In contrast to bacteria, the cell walls of eukaryotes (including fungi, algae,
and higher plants) are composed principally of polysaccharides
 The basic structural polysaccharide of fungal cell wall is chitin (a polymer of
N-acetylglucosamine residues), which also forms the exoskeleton of
arthropods (e.g., the shells of crabs).
 The cell walls of most algae and higher plants are composed principally of
cellulose (a polymer of β-glucose).
 Cellulose is the most abundant polymer on earth.
 One of the critical functions of plant cell wall is to prevent cell swelling as a
result of osmotic pressure.

126
The Extracellular Matrix of Animal cells
 Although animal cells are not surrounded by cell walls, many of the cells
in tissues of multi-cellular organisms are embedded in an extracellular
matrix consisting of secreted proteins and polysaccharides. It fills the
spaces between cells and binds cells and tissues together
 Extracellular matrices are composed of
Tough fibrous proteins(collagen) – provide structural support
A stretchable protein (elastin)- provide flexibility
Polysaccharide: glycosaminoglycans(GAGs) such as N-
acetylglucosamine or N-acetylgalactosamine.
 GAGs are negatively charged modified sugars and they bind
positively charged ions and trap water molecules to form hydrated
gels, thereby providing mechanical support to the extracellular
matrix.
Adhesion proteins: are responsible for linking the components of the
matrix to one another and to the surfaces of cells. Examples:
Fibronectins, entactin and Laminins
 The major cell surface receptors responsible for the attachment of cells
to the extracellular matrix are the integrins. In addition to attaching cells
to the extracellular matrix the integrins serve as anchors for the
cytoskeleton
Cell-Cell Interactions
Direct interactions between cells, as well as between cells and the
extracellular matrix, are critical to the development and function of
multicellular organisms.
Some cell-cell interactions are transient, such as the interactions
between cells of the immune system and the interactions that direct
white blood cells to sites of tissue inflammation. In other cases, stable
cell-cell junctions play a key role in the organization of cells in tissues.
For example, several different types of stable cell-cell junctions are
critical to the maintenance and function of epithelial cell sheets.
The connections between one cell and another are called intercellular
junctions. These attachments enable the cells to resist stress and
communicate with each other.
The principal types of intercellular junctions are
tight junctions, gap junctions, adhesion junctions.

128
 Tight Junctions
 A tight junction joins two neighboring cells
tightly to each other by proteins that hold
two adjacent cells by forming a zipperlike
pattern.
 This seals off the intercellular space and
provide a tight barrier for some substances
to pass between the cells.
 For example, In the stomach and intestines,
tight junctions prevent digestive juices from
seeping between epithelial cells and
digesting the underlying connective tissue.
 They also help to prevent intestinal bacteria
from invading the tissues, and they ensure
that most digested nutrients pass through
the epithelial cells and not between them.

129
 Gap (Communicating) Junctions
 A gap junction is formed by a ringlike
proteins known as connexin, which
consists of six transmembrane proteins
surrounding a water-filled channel.
 Gap junctions provides direct
connections between the cytoplasms of
adjacent cells. Gap junctions are open
channels through the plasma membrane,
allowing ions and small molecules (less
than approximately a thousand daltons)
to diffuse freely between neighboring
cells, but preventing the passage of
proteins and nucleic acids.
 Plasmodesmata are the only intercellular
junctions in plants; they function like gap
junctions

130
3. Adhesion junction (desmosome).
 In an adhesion junction
(desmosome), the adjacent plasma
membranes do not touch but are held
together by intercellular filaments or
adhesion proteins which can be
divided into four major groups: the
selectins, the integrins, the
immunoglobulin (Ig) super family and
the cadherins
 The cell-cell interactions mediated by
the selectins, integrins, and most
members of the Ig superfamily are
transient
 Stable adhesion junctions are based
largely on cadherins.
 The neighboring cells are separated
by a small gap, which is spanned by
dhesion proteins that hold the cells
together.
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 The Cytoskeleton
 The cytoplasm of eukaryotic cells is crisscrossed by a network of
protein fibers called cytoskeleton.
 There are three types of cytoskeleton that can be distinguished
on the bases of their diameter, type of their subunit, and subunit
arrangment
1. Actin filaments
2. Intermediate filaments
3. Microtubules
1. Actin Filaments
 Are thin, flexible fibers approximately 8nm in diameter
 They are the thinnest of the cytoskeletons and also called
microfilaments
 Formed from monomeric subunits called actin proteins.
Actin proteins polymerize to form actin filaments.
 Each filament is composed of two protein chains that form a
twisted two-stranded structure.
132
 Some functions of Actin filaments:
 Provides mechanical strength to the cell
 Links transmembrane proteins to cytoplasmic proteins
 anchors the centrosomes at opposite poles of the cell during
mitosis
 Pinches dividing animal cells apart during cytokinesis
 Generate cytoplasmic streaming in some cells and generate
locomotion in cells such as white blood cells and the amoeba
 Interact with myosin ("thick") filaments in skeletal muscle fibers
to provide the force of muscular contraction

133
2. Intermediate filaments
 Diameter – about 10nm- intermediate in diameter between actin
filaments(8nm) and microtubules(25nm)
 Whereas actin filaments and microtubules are polymers of single types of
proteins (actin and tubulin, respectively), intermediate filaments are composed
of a variety of proteins that are expressed in different types of cells.
 More than 65 different intermediate filament proteins have been identified and
classified into six groups based on similarities between their amino acid
sequences Intermediate filaments found in
 the nucleus are composed of Lamins.
 epithelial cells is composed of Keratin
 mesenchymal cells are composed of Vimentin
 muscle cells are composed of Desmin
 long axons of neurons are composed of Neurofilaments-
Functions
 They provides mechanical support to cells and organelles.
 They mediate cell–cell adhesion and cell–matrix adhesion

134
3. Microtubules
 Are hollow tubes of about 25nm in diameter
 Composed of a ring of 13 protein protofilaments
 formed by the polymerization of a molecule that consists of two
subunits, called alpha and beta tubulin
 Polymerization starts from the centrosome, which is the primary
microtubule-organizing center (MTOC) in animal cells.
Functions
 Used as a “railroad tracks" for movement of organelles and
molecules with in the cell.
 The motion is provided by protein "motors" that use the
energy of ATP to move along the microtubule.
 The two major motor proteins that drag themselves along
microtubules and move molecules are kinesins and dyneins
 The migration of chromosomes in cell division takes place on
microtubules that make up the spindle fibers.

136
Cilia and Flagella
 Cilia and flagella are microtubule-based projections of the plasma
membrane
 responsible for movement of a variety of eukaryotic cells.
Bacteria flagella
 are protein filaments projecting from the cell surface, rather than
projections of the plasma membrane supported by microtubules.
Eukaryotic cilia and flagella
 are very similar in structures, each with a diameter of approximately
0.25µm
 Cilia are responsible both for cell motility and for sweeping food
organisms over the cell surface and into the oral cavity. In animals, an
important function of cilia is to move fluid or mucus over the surface of
epithelial cell sheets. A good example is provided by the ciliated cells
lining
 Flagella differ from cilia in their length (they can be as long as 200 µm)
and in their wavelike pattern of beating. Cells usually have only one or
two flagella, which are responsible for the locomotion of a variety of
protozoans and of sperm.
Structure of cilia and flagella
 The fundamental structure of both cilia and flagella is the axoneme
 Axoneme is composed of microtubules and their associated proteins
 In their structures the microtubules are arranged in a characteristic "9 + 2"
pattern in which a central pair of microtubules is surrounded by nine outer
microtubule doublets.
 The two fused microtubules of each outer doublet are distinct: One (called
the A tubule) is a complete microtubule consisting of 13 protofilaments; the
other (the B tubule) is incomplete, containing only 10 or 11 protofilaments
fused to the A tubule.
 The outer microtubule doublets are connected to each other by links of a
protein called nexin. In addition, two arms of dynein are attached to each A
tubule, and it is the motor activity of these axonemal dyneins that drives the
beating of cilia and flagella.
Cell Metabolism
 Metabolism: refers to the sum of all the enzyme-catalyzed reactions in
a living organism
 There are two types metabolic reactions in the body
1. Catabolism: The energy releasing (Exergonic rxn) process in which a
chemical or food is used (broken down) by degradation or
decomposition, into smaller pieces.
2. Anabolism: is just the opposite of catabolism. In this portion of
metabolism, the cell consumes energy (Endegonic rxn) to produce
larger molecules from smaller ones. Anabolism is driven by the energy
that catabolism releases, so endergonic and exergonic processes,
anabolism and catabolism, are inseparably linked.
 All the chemical reactions that take place in the cell require catalysis.
 The agents that carryout catalysis in living organisms are called enzymes

141
Enzyme properties
 Catalysts for biological reactions
 Most are proteins
 Are highly specific
 Work by lowering the activation energy
 Increase the rate of reaction without being changed themselves
 Their activity is lost if denatured
 May be simple proteins or may contain cofactors such as metal ions or
organic (vitamins)
Enzyme Nomenclature and classification
 The name of an enzyme has two parts. The first part indicates name of
its substrate and second part ending in ‘ase’
 Example:
 sucrase – reacts sucrose
 lipase - reacts lipid

142
 Systematic Name
 The International Union of Biochemistry (IUB) assigned a name called a
systematic name
 In this scheme an enzyme is assigned a four-number (Enzyme
commission number or E.C number) classification and a two-part name
 In each enzyme code (EC) number.
The first digit indicates major class
second digit indicates sub class
third digit denotes sub-sub class and
final digit indicates specific enzyme
 For example, the enzyme commonly called “hexokinase” is designated
“ATP: D-hexose-6-phospho-transferase E.C. 2.7.1.1.”
 This identifies hexokinase as a member of class 2 (transferases), subclass 7
(transfer of a phosphoryl group), sub-subclass 1 (alcohol is the phosphoryl
acceptor). Finally, the term “hexose-6” indicates that the alcohol
phosphorylated is that of carbon six of a hexose.

143
 Enzyme classification
 According to the IUB there are six major classes of enzymes
1. Oxidoreductases.
 Oxidoreductases catalyze oxidation-reduction reactions.
 Subclasses of this group include the dehydrogenases, oxidases,
oxygenases, reductases, peroxidases, and hydroxylases.
2. Transferases.
 Transferases catalyze reactions that involve the transfer of groups
from one molecule to another. Examples of such groups include
amino, carboxyl, carbonyl, methyl, phosphoryl, and acyl (RC=O).
 Examples include the transcarboxylases, transmethylases, and
transaminases.
3. Hydrolases.
 Hydrolases catalyze reactions in which the cleavage of bonds is
accomplished by adding water.
 The hydrolases include the esterases, phosphatases, and peptidases.

144
4. Lyases
 Lyases catalyze reactions in which groups (e.g., H2O, CO2, and NH3)
are removed to form a double bond or are added to a double bond.
 Decarboxylases, hydratases, dehydratases, deaminases, and
synthases are examples of lyases.
5. Isomerases
 They catalyze inter conversion of optical, functional and geometrical
isomers.
6. Ligases
 Ligases catalyze bond formation between two substrate molecules.
 The energy for these reactions is always supplied by ATP hydrolysis.
 The names of many ligases include the term synthetase.

145
 How enzymes work
 A catalyst does not change the chemical reaction but it accelerates the
reaction. They are not consumed in overall reaction. But they undergo
chemical or physical change during reaction and returns to original
state at the end of reaction.
 For a chemical reaction A → B to occur, energy is required. When
enough energy is supplied. A undergoes to transition state which is an
unstable state. So, it gets converted to product B which is more stable.
 The amount of energy needed to convert a substance from ground
state to transition state is called activation energy.
 In presence of catalyst, A undergoes to transition state very fast and
requires less energy. Hence, a catalyst accelerates the rate of reaction
by decreasing the energy of activation.
 Likewise enzymes also speed up reaction by lowering energy of
activation. Further, the activation energy is very much less for a
reaction in presence of enzyme than non-enzyme catalyst. Therefore
enzymes are more efficient than non-enzyme catalyst.
146
147
 Enzymes Active site
 The active site is a specialized region of the enzyme where the enzyme
interacts with the substrate.
 The active site of an enzyme is generally a pocket or cleft that is specialized to
recognize specific substrates and catalyze chemical transformations. It is
formed in the three-dimensional structure by a collection of different amino
acids (active-site residues) that may or may not be adjacent in the primary
sequence
 There are two models that describe the binding interaction between the
enzyme and substrate.
 The “lock-and-key” Model
 The “induced-fit”model for substrate
Lock-and-key model for substrate binding
 Proposed by Emil Fischer in 1894
 The lock and key model for enzyme-substrate binding uses
complementarities between the enzyme active site (the lock) and the
substrate (the key). Simply, the substrate must fit correctly into the active
site—it must be the right size and shape.
 The two shapes are considered as rigid and fixed
148
In the induced-fit model
 Proposed by Daniel E. Koshland in 1958
 In this model, the structure of the enzyme
is different depending on whether the
substrate is bound or not.
 The enzyme changes shape (undergoes a
conformation change) on binding the
substrate. This conformation change
converts the enzyme into a new structure
in which the substrate and catalytic
groups on the enzyme are properly
arranged to accelerate the reaction.
 The induced fit model recognizes that the
substrate binding site is not a rigid “lock”
but rather a dynamic surface created by
the flexible overall three-dimensional
structure of the enzyme.

149
Co-enzymes and prosthetic groups
 Many enzymes requires the presence of small , non protein units
or cofactors to carryout their particular reaction
 Cofactors can be either inorganic ions such as Zn2+, Fe2+ or
complex organic molecules called coenzymes (e.g NAD+, NADP+
FAD+)
 A metal or coenzyme that is covalently attached to enzyme is
called prosthetic group.
 A complete catalytically active enzyme together with its
prosthetic group is called holoenzyme
 A protein part of the enzyme on its own without its cofactor is
termed apoenzyme
 Many coenzymes are derived from vitamins

150
 Enzyme Kinetics
  The rate of an enzyme catalyzed reaction is often called its velocity.
 The velocity of all enzymes is dependent on Substrate and enzyme
concentration, temperature, and pH
1. Substrate and enzyme concentration
 The normal pattern of dependence of enzyme rate on substrate
concentration ([S]) is that at low substrate concentrations a doubling of [S]
will lead to a doubling of the initial velocity (V0).
 However, at higher substrate concentrations the enzyme becomes
saturated and further increases in [S] lead to very small changes in V0. This
occurs because at saturating substrate concentrations effectively all of the
enzyme molecules have bound substrate. The overall enzyme rate is now
dependent on the rate at which the product can dissociate from the
enzyme, and adding further substrate will not affect this. The shape of the
resulting graph when V0 is plotted against [S] is called a hyperbolic curve

151
 In situations where the substrate concentration is saturating (i.e. all the
enzyme molecules are bound to substrate), a doubling of the enzyme
concentration will lead to a doubling of V0. This gives a straight line graph
when V0 is plotted against enzyme concentration.
 The dependence of enzyme rate (activity) on substrate concentration has
been provided in a quantitative way. The simplest of these equations, the
Michaelis-Menten equation, relates the initial velocity (V0) to the
concentration of substrate [S] and the two parameters Km and Vmax.
Michaelis-Menten equation
 The Michaelis-Menten model of enzyme kinetics applies to a simple reaction
in which the enzyme and substrate form an enzyme–substrate complex (ES)
that can dissociate back to the free enzyme and substrate.

 The enzyme (E), combines with its substrate (S) to form an enzyme–substrate
complex (ES). The ES complex can dissociate again to form E S, or can proceed
chemically to form E and the product P. The rate constants k1, k2 and k3
152
describe the rates associated with each step of the catalytic process.
 From the observation of the properties of many enzymes it was known that the
initial velocity (V0) at low substrate concentrations is directly proportional to
[S], while at high substrate concentrations the velocity ends towards a
maximum value that is the rate becomes independent of [S]. This maximum
velocity is called Vmax. Michaelis and Menten derived an equation to describe
these observations

 The equation describes a hyperbolic curve. In deriving the equation, Michaelis

and Menten defined a new constant, Km, the Michaelis constant
 For many enzymes k2 is much greater than k3. Under these circumstances Km
becomes a measure of the affinity of an enzyme for its substrate since its value
depends on the relative values of k1 and k2 for ES formation and dissociation,
respectively.
 A high Km indicates weak substrate binding (k2 predominant over k1), a low Km
indicates strong substrate binding (k1 predominant over k2).
 Km can be determined experimentally by the fact that its value is equivalent to
the substrate concentration at which the velocity is equal to half of V max.
153
154
 2. Temperature
 Enzymes have little activity at low temperature
 Rate of enzyme catalyzed rxn increases with temperature
 Enzymes are most active at optimum temperatures (usually
37°C in humans)
 Activity lost with denaturation at high temperatures

155
3. pH
 Each enzyme has an optimum pH at which the rate of reaction that
it catalyzes is maximum
 Deviations from optimum pH value decrease activity
 Larger deviation in pH lead to denaturation

156
Enzyme inhibition
 Many types of molecules exist which are capable of interfering with
the activity of an individual enzyme
 Any molecule which act directly on an enzyme to lower its catalytic
rate is called inhibitor
 Enzyme inhibition may be of two types
1. Irreversible inhibition
2. Reversible inhibition which in turn can be divided into two
 Competitive
 Non-competitive
 Irreversible inhibition
 Inhibitor binds tightly by covalent bond to amino acid residue of
enzyme active site and permanently inhibit the enzyme
 E.g. a nerve gas diisopropylphosphofluoride(DIPF) react with serine
residue in the active site of enzyme actylcholinesterase irreversibly
and prevent transmition of nerve impulses
157
Reversible inhibition can be
1. Competitive Inhibitor
 A competitive inhibitor has a structure similar to substrate and
occupies active site
 Inhibitor competes with substrate for active site
 Has effect reversed by increasing substrate concentration
2. A noncompetitive inhibitor
 Does not have a structure like substrate
 Binds to the allosteric site of enzyme (site other than active site)
 Changes the shape of enzyme and active site so that substrate
cannot fit altered active site, No reaction occurs
 Effect is not reversed by adding substrate
 Enzyme Regulation
 In biological systems the rates of many enzymes are altered by the
presence of other molecules such as activators and inhibitors
(collectively known as effectors).
1. Feedback inhibition:
 The end product in a metabolic pathway inhibit steps earlier in
the same pathway
 The end product bind to the enzyme at the control point at site
other than active site. Such enzymes are called allosteric
enzymes

159
2. Reversible covalent modification
 The activity of many enzymes is altered by the reversible making and
breaking of a covalent bond between the enzyme and a small non-
protein group.
 The most common such modification is the addition and removal of a
phosphate group; phosphorylation and dephosphorylation, respectively.
 Phosphorylation is catalyzed by protein kinases, often using ATP as the
phosphate donor, whereas dephosphorylation is catalyzed by protein
phosphatases.
 The addition and removal of the phosphate group causes changes in the
tertiary structure of the enzyme that alter its catalytic activity.
 For example, glycogen phosphorylase, an enzyme involved in glycogen
breakdown, is active in its phosphorylated form, and glycogensynthase,
involved in glycogen synthesis, is most active in its unphosphorylated
form

160
3. Proteolytic activation
 Several enzymes are synthesized as larger inactive precursor forms called
proenzymes or zymogens. Activation of zymogens involves irreversible
hydrolysisof one or more peptide bonds.
 Example: Pancreatic proteases
 The digestive enzymes trypsin, chymotrypsin and elastase are produced
as zymogens in the pancreas. They are then transported to the small
intestine as their zymogen forms and activated there by cleavage of
specific peptide bonds.
 Trypsin is synthesized initially as the zymogen trypsinogen. It is cleaved
(and hence activated) in the intestine by the enzyme enteropeptidase
which is only produced in the intestine. Once activated, trypsin can
cleave and activate further trypsinogen molecules as well as other
zymogens, such as chymotrypsinogen and proelastase

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 Many tasks that a cell must perform, such as movement and the synthesis of
macromolecules, require energy. A large portion of the cell's activities is
therefore devoted to obtaining energy from the environment and using that
energy to drive energy-requiring reactions.

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163
 Glycolysis
 Glycolysis is a series of reactions that takes place in the cytoplasm of all
prokaryotes and eukaryotes.
 Glycolysis converts one molecule of glucose into two molecules of
pyruvate [which are then converted to acetyl coenzyme A(CoA) ready for
entry into the citric acid cycle].
 Overall, glycolysis has a dual role.
 The first is to generate ATP. it also feeds substrates into the citric acid
cycle and oxidative phosphorylation, where most ATP is made.
 The second role is to produce intermediates that act as precursors for
a number of biosynthetic pathways. Thus acetyl CoA, for example, is
the precursor for fatty acid synthesis
 In the process of glycolysis, two ATP molecules are needed for early
reactions in the glycolytic pathway but four ATPs are generated later,
giving a net yield of two ATPs per molecule of glucose degraded.
 In addition to producing ATP, glycolysis converts two molecules of the
coenzyme NAD+ to NADH.
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 Steps of glycolysis

165
166
 Fates of Pyruvate
1. Entry into the citric acid cycle.
Glycolysis releases relatively little of the energy present in a
glucose molecule; much more is released by the subsequent
operation of the citric acid cycle and oxidative phosphorylation.
Following this route under aerobic conditions, pyruvate is
converted to acetyl CoA by the enzyme pyruvate
dehydrogenase and the acetyl CoA then enters the citric acid
cycle.
2. Conversion to fatty acid or ketone bodies.
When the cellular energy level is high(ATP in excess), the rate
of the citric acid cycle decreases and acetyl CoA begins to
accumulate. Under these conditions, acetyl CoA can be used for
fatty acid synthesis or the synthesis of ketone bodies

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 3. Conversion to lactate.
 The NAD+ used during glycolysis must be regenerated if glycolysis is to
continue.
 Under aerobic conditions, NAD+ is regenerated by the re-oxidation of NADH
via the electron transport chain
 When oxygen is limiting, as in muscle during vigorous contraction, the re-
oxidation of NADH to NAD+ by the electron transport chain becomes
insufficient to maintain glycolysis. Under these conditions, NAD+ is
regenerated instead by conversion of the pyruvate to lactate by lactate
dehydrogenase
4. Conversion to ethanol.
 In yeast and some other microorganisms under anaerobic conditions, the
NAD+ required for the continuation of glycolysis is regenerated by a process
called alcoholic fermentation.
 The pyruvate is converted to acetaldehyde (by pyruvate decarboxylase) and
then to ethanol (by alcohol dehydrogenase), the latter reaction reoxidizing
the NADH to NAD+

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169
Tricarboxylic Acid Cycle
 is also called the Krebs cycle or the citric acid cycle
 Occurs in the mitochondria of eukaryotes and in the cytosol of prokaryotes.
 is used to oxidize the pyruvate formed duringthe glycolytic breakdown of
glucose into CO2 and H2O.
 It also oxidizes acetylCoA arising from fatty acid degradation and amino acid
degradation products
 In addition, the cycle provides precursors for many biosynthetic pathways.
Oxidative decarboxylation of pyruvate
 Pyruvate, the end-product of aerobic glycolysis, must be transported into the
mitochondrion before it can enter the TCA cycle.
 This is accomplished by a specific pyruvate transporter that helps pyruvate
cross the inner mitochondrial membrane. Once in the matrix, pyruvate is
converted to acetyl CoA by the pyruvate dehydrogenase
 During oxidative decarboxylation of pyruvate, one carbon of each pyruvate is
released as C02, and the remaining two carbons are added to CoA to form
acetyl CoA.
170 In the process, one molecule of NAD+ is reduced to NADH for each pyruvate.
Reactions of TCA cycle
 The citric acid cycle is composed of eight reactions
 The two-carbon acetyl group combines with oxaloacetate (four carbons) to
yield citrate (six carbons).
 Through eight further reactions, two carbons of citrate are completely oxidized
to C02 and oxaloacetate is regenerated.
 Output of TCA cycle for oxidation of one glucose molecule are
 Two GTP
 Six molecules of NADH and
 Two FADH2
 Total economy of complete oxidation of glucose
 Six molecules of C02.
 Four molecules of ATP - two from glycolysis and two from the citric acid
cycle
 Ten molecules of NADH (two from glycolysis, two from the conversion
of pyruvate to acetyl CoA, and six from the citric acid cycle)
 Two molecules of FADH2
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 Electron transport chain
 In eukaryotes, electron transport and oxidative phosphorylation occur in
the inner membrane of mitochondria. These processes re-oxidize the
NADH and FADH2 that arise from the citric acid cycle (located in the
mitochondrial matrix) and NADH of glycolysis (located in the cytoplasm)
 Oxidative phosphorylation is by far the major source of ATP in the cell.
In prokaryotes, the components of electron transport and oxidative
phosphorylation are located in the plasma membrane.
 The oxidation of a molecule involves the loss of electrons. The reduction
of a molecule involves the gain of electrons.
 In electron transport system electrons are transferred from NADH and
FADH2 to oxygen along the electron transport chain (also called the
respiratory chain).
 The components of electron transport chain are arranged according to
their increasing redox potential (affinity to gain electron). Oxygen is the
strongest oxidizing agent and has a tendency to accept electrons.

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  The electron transport chain exist as four large protein complexes
embedded in the inner mitochondrial membrane called:
 NADH-Q reductase (Complex I)
 Succinate-Q reductase (Complex II)
 Q-cytochrome c reductase (Complex III)
 Cytochrome c oxidase (Complex IV)
 In ETS, NADH passes its electrons to NADH-Q reductase (Complex I) and
FADH2 to succinate-Q reductase (Complex II).
 Energy liberated by electron transport in ETC is used to pump H ions
out of the mitochondrion to create an electrochemical proton (H)
gradient. The protons flow back into the mitochondrion through the
ATP synthase located in the inner mitochondrial membrane, and this
drives ATP synthesis.
 Approximately three ATP molecules are synthesized per NADH oxidized
and approximately two ATPs are synthesized per FADH2 oxidized.

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175
Cell cycle and cell cycle regulation
 All cells arise by the division of an existing cell.
 During cell cycle the cell doubles in mass, duplicates its genome and organelles,
and partitions these between two new progeny cells.
 Events in cell cycle have to be carried out with great precision and in the
correct order, and cells have established sophisticated control processes to
ensure that the cell cycle proceeds with the required accuracy
 To ensure the accuracy of cell cycle, cells contain genes known as “cell cycle
control genes.”
 The proper functioning of such genes not only determines how big we are, it
also prevents cell division becoming “out of control” and leading to cancer
 In humans the cells of some tissues, such as the skin, the lining of the gut, and
the bone marrow continue to divide throughout life.
 Others, such as the light-sensitive cells of the eye and skeletal muscle cells,
show almost no replacement.
.

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The cell cycle
 The cell cycle is an ordered series of events leading to cell replication
 The cell cycle consists of four coordinated processes
 Cell growth
 DNA replication
 Distribution of duplicated chromosomes to daughter cells and
 Cell division
 In eukaryotes cell cycle consists of four major phases.
 G1 phase(gap1)
 is the period between mitosis and phase of DNA replication
 It is the longest and most variable cell cycle phase
 During G1 phase cells synthesize RNAs and proteins required for DNA
synthesis
 Most cells in multicellular organisms are differentiated to carryout specialized
functions and no longer divide
 Such cells are considered to be in special compartment of G1 phase called the
G0 phase. Example nerve cells
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S Phase(Synthesis)
 The period during which chromosomes duplicate in number
G2 Phase(gap2)
 During G2 phase cells proof
read the DNA structure and
make preparations for mitosis
 The G1, S, and G2 phases are

collectively referred to as interphase,

the period between one mitosis and
the next.
The M phase( Mitosis)
 During mitosis and subsequent
cytokinesis, chromosomes and
cytoplasm partitioned into two
daughter cells
 Divided into four phases
 Prophase, Metaphase, Anaphase
and Telophase

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
1. Prophase.
 Chromosome condensed and become visible under microscope as
distinct paired threads termed sister chromatids
 Chromosome condensation reduces the chance of long DNA
molecules becoming tangled and broken
 Nucleoli and nuclear envelop disappear
 Microtubules grow outward from spindle poles and attach on the
sister chromatids at specialized structures called Kinetochores at
the centromere
2. Metaphase
 Chromosomes organized at equatorial plate(metaphase plate)
3. Anaphase
 Each chromatid is now considered a separate chromosome; there
are two complete and separate sets.
 The spindle fibers contract and pull the chromosomes, one set
toward each pole of the cell.

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5. Telophase
 The sets of chromosomes reach the poles of the cell and become
indistinct as their DNA uncoils to form chromatin.
 A nuclear membrane re-forms around each set of chromosomes.
Cytokinesis
 During the last stages of telophase, the cell cytoplasm itself divides in
to two.
 In animal cells, a cleavage furrow made of actin and its motor protein
myosin constricts the middle of the cell
 In plants, a structure called the phragmoplast forms at the equator of
the spindles where it directs the formation of a new cell wall.

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Cell cycle control
 Progression of the cell cycle is regulated at several checkpoints, which
ensure that all cellular components are present and in good working
order before the cell proceeds to the next stage.
 The checkpoints are necessary to prevent cells with damaged or
missing chromosomes from proliferating
 the major control points are
 the G1/S boundary at which point the cell is committed to DNA
replication.
 the G2/M transition when it is committed to mitosis.
 At G1/S, the cell must decide whether
 it is big enough and
 nutritional conditions are appropriate to begin the crucial process of
replicating its genome.

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• In G2 the primary concern is
 Whether its DNA is in perfect condition before entering mitosis.
 There are sensitive mechanisms for detecting the presence of unreplicated
or damaged DNA, and cells will not commit themselves to mitosis until any
defects have been corrected.
 Both the G1/S and the G2/M checkpoints are regulated by a mechanism in
which two proteins interact.
 The Cyclin and cyclin dependent kinase (CDK)
1. the G2/M checkpoint.
 This checkpoint is regulated by cyclin B, which combines with CDK1 to form M-
phase promoting factor (MPF).
 After MPF is formed, it must be activated by the addition of a phosphate group
to one of the amino acids of CDK
 MPF senses unreplicated or damaged DNA and generate a signal that lead to
cell cycle arrest
 Operation of G2 check point therefore prevent initiation of M phase before
completion of S phase . This allow time for damage to be repaired rather than
being passed on to daughter cells

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2. The G1/S checkpoint
 This check point is known as START in yeast and restriction point in
animals
 At this check point cells evaluate its internal as well as external
enviroment and asses if it is in a position to enter S phase
 If it find the condition suitable, it decide to enter the S phase. Otherwise it
arrest the cell cycle at G1 and enter to Go phase
 entry into S phase is regulated by three cyclin-dependent kinases called
Cdk2, Cdk4, and Cdk6,which are activated by binding with cyclin D
proteins

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 Regulation of cell number
 cell division occurs constantly through out the life of an individual
and yet all multicellular organisms have limited size. E.g. Humans
can never be as big as elephant!
 The reason that multicellular organisms do not become infinitely
large is because the proliferation of cells is balanced by cell death.
 Cells die for two quite different reasons.
 One is accidental, the result of mechanical trauma or exposure
to some kind of toxic agent, and often referred to as
necrosis(pathological death). This is the only type of death
seen in unicellular organisms.
 The other type of death is deliberate, the result of an built-in
suicide mechanism known as apoptosis or programmed cell
death.

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 Features of cell death by necrosis
 Cells that die by necrosis swell and then burst.
 The cell contents then leak out, causing the surrounding tissues to become
inflamed.
Features of cell death by apoptosis
 Cells that die by suicide shrink, and their cell contents are packaged into
small membrane-bound packets called blebs.
 The nuclear DNA becomes chopped up into small fragments, each of which
becomes enclosed in a portion of the nuclear envelope.
 The dying cell modifies its plasma membrane, signaling to macrophages,
which respond by engulfing the blebs and the remaining cell fragments and
by secreting cytokines that inhibit inflammation.
 Apoptosis begin with the signal that can come from within cell
(E.g. detection of radiation induced DNA break) or from outside (e.g. decrease
in the level of growth factors or hormones)
These signals induce the cell to make a decision to commit suicide

186
 The changes that occur during apoptosis are the result of hydrolysis of cellular
proteins by a family of proteases called caspases
 All the cells of our body contain caspases, but they are normally locked in an
inactive form by an integral inhibitory domain of the protein
 Caspases can be activated by proteolytic removal of inhibitory domain
releasing the active caspase
 Cells are instructed to die when a ligand binds to one of a family of death
domain receptors.
 This occurs, for example, If a cell is infected by a virus, white blood cells
recognize viral proteins on the cell surface and activate Fas, a death domain
receptor on the surface of the unlucky cell.
 On binding its ligand, a death domain receptor causes caspase 8 to activate. In
turn, caspase 8 can hydrolyze and hence activate the effector caspases that
begin the processes of cell destruction.

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Cancer and control of cell proliferation
 Cell growth is a carefully regulated process that respond to specific
need of the body
 In multicellular organisms, the process of cell birth and cell death
are balanced
 If the control that regulate cell multiplication break down, cells
begin to grow and divide indefinitely and results in cancer.
 Cancer is unregulated cell proliferation often accompanied by
abnormal differentiation (neoplasia)
 Cancers are generally caused by the disruption of cell proliferation
mechanism either through the activity of virus or mutation of
critical growth regulation genes.
 There are two classes of such genes
1. Oncogenes
2. Tumor suppressor genes
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 Oncogenes
 Genes that normally stimulate cell division are called proto-
oncogenes
 Mutation that cause proto-oncogenes to be over expressed or
hyperactive convert them to oncogenes(onco=cancer) leading to
excessive cell proliferation that is characteristic of cancer
 Oncogenes can also be activated by retroviruses
Tumor suppressor genes(TSG)
 In normal cells TSG control cell cycle by blocking passage through
the G1 check point by preventing cyclin from binding to CDK thus
inhibiting cell division
 Genes that normally inhibit cell division are called tumor
supressor genes
 When TSG genes are mutated, they can also lead to uncontrolled
cell proliferation
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Tumor
 Tumor is any abnormal proliferation of cells which may be either a
benign or malignant tumor
1. Benign tumor:
 remains confined to its original location, neither invading
surrounding tissue or spreading to distant body sites.
 Such tumor can be removed surgically. E.g. skin wart
2. Malignant tumor:
 Tumor capable of both invading surrounding normal tissue or
spreading throughout the body via circulatory
systems( Metastasis)
 Due to spreading ability their treatment is usually difficult
 Only malignant tumors are properly referred to as cancer

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 Types of cancer
 There are more than 100 distinct types of cancers
 Most cancers fall into three main groups based on the type of cell
from which they arise
1. Carcinomas: cancer of epithelial tissue. Include about 90% of
human cancers
2. Sarcomas: tumors of connective tissues such as muscle, bone,
cartilage etc. it is rare in humans
3. Leukemias (Lympomas): cancer that arise from blood forming
cells and cells of immune system. Account for about 7% of human
cancers
 Tumors are further classified according to tissue of origin. E.g
lung cancer, breast cancer etc

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 Causes of cancer
 Substances that cause cancer are called carcinogenes
 There are many carcinogens that cause cancer
1. Radiation
 Radiation act by damaging DNA and inducing mutation
 E.g Radiation of short wave length: UV radiation, gamma radiation
2. Chemicals: can act either by damaging DNA and inducing mutation or by
stimulating cell proliferation
 E.g. chemicals in tobacco smoke( Lung cancer), aflatoxin from small
molds(Liver cancer) induce cancer though mutation
 Tumor promoters such as estrogen hormone cuase cancer through
stimulation of cell proliferation
3. Tumor Viruses:
 E.g. Hepatitis B and C(liver cancer), Papillomavirus(cervical cancer),
Herpesvirus, Retroviruses( blood cancer)

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