Camp. Biochem. Physiol. Vol. 98C, No. 213, pp. 337-343, 1991 0306-4492/91 $3.00 + 0.

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Printed in Great Britain 0 1991 Pergamon Press plc

THE ISOLATION AND PARTIAL CHARACTERIZATION

OF PROELASTASE FROM THE PANCREAS OF THE
OSTRICH (STRUTHIO CAA4ELUS)
JENNI L. SUTHERLAND,RYNO J. NAUDB* and WILLEM OELOFSEN
Department of Biochemistry, University of Port Elizabeth, P.O. Box 1600, Port Elizabeth 6000,
Republic of South Africa (Telephone: (041) 531-1928; Fax: (041) 531-1280)

(Received 3 May 1990)

Abstract-l. Cationic proelastase from the pancreas of the ostrich was purified by an ammonium acetate
extraction, (NH&SO, fractionation and SP-Sephadex C-50 chromatography.
2. Relative molecular weight of the proenzyme is about 25,000.
3. The amino acid composition of ostrich proelastase is similar to that of catfish elastase B, which is
a Ser protease.
4. The effects of pH, temperature and inhibition (LBTI, q-PI, PMSF and elastatinal) on elastolytic
activity were investigated. Elastatinal, a specific inhibitor of elastase, revealed a K, of 4.46 PM.

INTRODUCTION to compare the proelastase of a primitive bird with

that of mammals.
Elastases are a heterogeneous class of enzymes
comprising both Ser and metalloproteases that are
capable of solubilising fibrous elastin. MATERIALS AND METHODS

The mammalian exocrine pancreas synthesises, Chemicals

stores and secretes approximately 15 enzymes and Synthetic substrates, PPE, porcine enterokinase, DMSO,
proenzymes. A dominant fraction of these enzymes molecular weight marker proteins for SDS-PAGE, a,-PI,
are the Ser proteases, including several forms of and elastatinal were purchased from Sigma Chemical Co.,
trypsin, chymotrypsin and elastase. The members of U.S.A. Bovine trypsin was obtained from Miles Laboratory
this gene family are probably related by evolution Inc., U.S.A. and bovine chymotrypsin from Millipore Corp.
to a common ancestral protease (Neurath et al., LBTI was supplied by Worthington Biochemical Corp.,
1967). U.S.A. Carrie; gmphoiytes (PhaGalyte Tm) (pH 3-10) and
SP-Seohadex C-50 were obtained from Pharmacia Fine
Based on substrate specificities, Largman (1983)
Chemicals, Uppsala, Sweden, while PMSF was purchased
suggested the presence of 2 distinct groups of pan- from Boehringer-Mannheim, GmbH.
creatic elastases. PPE 1 is an example of the “classic”
Ser proteases, while the other group of elastases, Ostrich material
the “chymotrypsin-like” enzymes, are represented by Ostrich pancrei were excised 20-30min after the birds
PPE 2. among others. were killed. Visible fat was removed and pancrei were kept
More than one form of proelastase and elastase on ice for 4-8 hr, before being stored at - 20°C until use.
have been isolated from the pancrei of the following
Isolation
species: human (Largman et al., 1976), moose
(Stevenson and Voordouw, 1973, canine (Geokas The method of Naughton and Sanger (1961) was adapted
et al., 1980), porcine (Gertler and Birk, 1970) and rat appropriately. All steps were performed at 4°C unless
otherwise stated. Three hundred grams of frozen pancrei
(MacDonald et al., 1982). were thawed in 900ml cold acetone, homogenised in a
Ostrich trypsin (Hartley et al., 1987) and chymo- Waring blender for 1 min and filtered through a Buchner
trypsin (Van der Westhuizen et at., 1989) were isolated funnel. The residue was homogenised a further two times
and purified and shown to resemble the respective with 900 ml acetone and filtered. The remaining residue was
enzymes from other species. Very little is known dried over CaCl, in uacuo for 16 hr. Forty-three and a half
about avian elastase and this study was undertaken grams of acetone powder resulted, and was solubilised in
700 ml 0.1 M ammonium acetate (PH 4.5) with stirring for
4 hr. After centrifugation the pellet was resuspended in
*To whom all correspondence should be addressed. 300 ml 0.1 M ammonium acetate (pH 4.5), stirred for 1.5 hr
Abbreviations: ATEE, N-acetyl-tyrosine ethyl ester; a,- and centrifuged again. The two supematants were pooled
PI, alpha-1-protease inhibitor; BAPNA, benzoyl-Arg- and brought to 45% saturation with (NH& SO,. The pellet
paranitroanilide; BPE, bovine pancreatic elastase; CRE, resulting from centrifugation was washed 3 times with
Congo-Red elastin; DMSO, Dimethyl sulphoxide; LBTI, 100 ml 0.1 M ammonium acetate (PH 4.5) containing 45%
Lima bean trypsin inhibitor; PAG-IEF, Polyacrylamide (NH&SO,. Centrifugation yielded a pellet which was re-
gel isoelectrofocussing; PMSF, phenylmethanesulphonyl suspended in 150 ml Na,CO,-HCl buffer (PH 8.8), dialysed
fluoride; PPE 1 and 2, porcine pancreatic elastase 1 against 100 1running distilled H,O for 44 hr and centrifuged.
and 2; SAPLA, succinyl-Ala-Pro-Leu-paranitroanilide; The supernatant (275 ml) yielded 5.5 g of a lyophilised
SAPNA, succinyl-(Ala),-paranitroanilide. powder containing crude proelastase.

337
338 et al.
JENNIL. SUTHERLAND

Purification using a LKB constant power supply. The gels were poly-
Crude proelastase was dissolved in 8 ml 0.05 M ammonium merised in a LKB 2117-200 ultramould gel casting unit on
acetate (pH 5.0). The sample was cleared by centrifugation a sheet of cellophane (Giirg et al., 1977). Gels contained
and the supematant was applied to a SP-Sephadex C-50 7.3% acrylamide and 2% ampholine (PH 3-10). After
column (1.6 x 29 cm) at 20 ml/hr, previously equilibrated prefocusing for 30min, 5Opg Folin protein was loaded
with 0.05 M ammonium acetate (pH 5.0). After the break- and focused for 3-3.5 hr at 105OV. Gels were stained
through peak was eluted the column was developed with a with Coomassie Brilliant Blue G-250 (Blakesley and Boezi,
560ml linear gradient of increasing pH and salt concen- 1977). Marker proteins, with well-defined pH, values in
tration: 0.05 M ammonium acetate (pH 5.0) to 0.25 M parentheses, were: amyloglucosidase (3.55), trypsin inhibitor
ammonium acetate @H 6.0) at a flow rate of 40 ml/hr. After (4.55), a-lactoglobulin A (5.13), carbonic anhydrase (5.85),
completion of the gradient the column was washed with myoglobulin (7.16), L-lactic dehydrogenase (8.55) and
0.25 M ammonium acetate (pH 5.0), followed by 0.55 M trypsinogen (9.3).
ammonium acetate (PH 6.0). Fractions (7 ml) were monitored
for A,,, and 100 ~1 aliquots tested for tryptic, chymotryptic Amino acid analysis
and elastolytic activity, pooled and lyophilised. Amino acid analysis was performed by hydrolysing
samoles in 400 ul of 5.7 N azeotrooic HCl at 110°C for
Protein determination 20 hi in vacua,’ in the presence of’ 0.01 ml 5% phenol.
Protein concentration was determined calorimetrically by Hydrolysates were analysed on a Beckman 118 BL amino
the method of Lowry ef al. (1951). BSA was used to construct acid analyser (Spackman et al., 1958). Ser and Thr values
an appropriate calibration curve. were routinely corrected for hydrolytic losses of 10 and
8%, respectively. Tryptophan was determined by a similar
Enzyme assays hydrolysis using 0.2 ml 4 M methane sulphonic acid (Moore,
Elastase 1972).
Congo -Red elastin
The natural substrate, elastin, coupled to a colour reagent Activation of proelastase
was used, according to the method of Shotton (1970). The optimum trypsin concentration and incubation time
The amount of dye released into solution is determined for complete activation was determined by activating 100 fig
calorimetrically and is directly proportional to elastase ostrich proelastase with varying bovine trypsin concentra-
concentration. tions (2.5~40%, w/w) for 20 min at 37°C. Once the optimum
SAPNA trypsin concentration was established, incubation time was
varied from 5 to 40min. Activation of ostrich proelastase
The use of a synthetic substrate provides an accurate and with ostrich trypsin was also tested in order to observe
convenient means of determining elastolytic activity. The species specificity. Elastolytic activity was determined as
method of James et al. (1985) was used. The increase in described using SAPNA as substrate.
A 4,,,,,,,,due to the release of paranitroaniline was recorded as
a function of time. Specific activity was expressed as AA,,, EfSect of pH
x 103/min/ng Folin protein which was obtained from the
slope of a plot of AA.,,,, x lO)/min vs. enzyme concentration. The effect of pH on the activity of activated proelastase
The slope was obtained by calculating a least-squares linear at a concentration of 100 pg/assay and 21°C was examined
regression line for the linear portion of the progress curve. with SAPNA as substrate at pH values ranging from 5 to
11. Samples were activated with 10% (w/w) bovine trypsin
SAPLA for 20min at 37°C and lyophilised. Each sample (1OOpg
SAPLA works on the same principle as SAPNA, hence aliquot) was dissolved in the appropriate pH buffer and
similar conditions were employed as described above. This assayed. The pH of the contents of the assay cuvette was
also facilitated a comparison of substrate specificities. measured to obtain the actual pH at which activity occurred.
Relative activity values were based on the activity at pH 8.9,
Trypsin taken as 100%.
Erlanger et al. (1961) described an assay for trypsin
utilising the synthetic substrate BAPNA. The increase in Effect of temperature
A 410nm was recorded and expressed as AA,,, x lO’/min/ pg Thermal stability was examined at pH 8.9 at temperatures
Folin protein, calculated as for SAPNA. ranging from 15 to 70°C. Aliquots containing 100 pg pro-
elastase were activated (10% w/w trypsin, 20min, 37”(Z),
Chymotrypsin lyophilised and solubilised in buffer. Enzyme and substrate
The artificial substrate ATEE was used to measure were incubated separately until the required temperature
chymotryptic activity by a modified spectrophotometric was reached. This temperature was maintained for 5 min
procedure of Schwert and Takenaka (1955). The decrease before the enzyme was added to the substrate and the re-
in A237nmwas determined and the specific activity was maining activity was determined. The percentage remaining
expressed as AA,,, x 103/min/ pg Folin protein. activity was expressed relative to the activity at 5O”C, taken
as 100%.
SDS-polyacrylamide gel electrophoresis
Electrophoresis on 7% polyacrylamide gels in the presence Determination of K, values
of SDS was performed according to a modification of the K,,, values for SAPNA and SAPLA were determined for
methods of Weber and Osbom (1969) and Davies and Stark PPE and activated ostrich proelastase. Aliquots of 250 pg
(1970). The molecular weight marker proteins, with M, ostrich proelastase were activated as before, and 4 substrate
values in parentheses, were: a-lactalbumin (14,200), trypsin concentrations were used, namely 0.5,0.75, 1.0 and 2.0 mM.
inhibitor (20,100), trypsinogen (24,000), carbonic anhydrase The AA,,,/min values at each substrate concentration were
(29,000), glyceraldehyde-3-phosphate dehydrogenase used to construct Lineweaver-Burk plots. K, for CRE
(36,000), egg albumin (45,000) and bovine albumin (66,000). was determined for PPE and activated ostrich nroelastase.
Four substrate concentrations were used, namely 0.05, 0.1,
Polyacrylamide gel isoelectrofocusing 0.15 and 0.2%. A,,,., at 80. 100 and 200 min were used to
_I_.....

PAG-IEF was performed in gel slabs (250 x 150 x 0.5 construct three Lineweaver-Burk plots yielding a range of
mm) in the LKB 2117 Multiphor II electrophoresis unit K,,, values.
 Pancreatic proelastase from the ostrich 339

Effects of inhibitors Table I. Summary of elastolytic, tryptic and chymotryptic activities

of fractions obtained from SP-Sephadex C-50 chromatography of
A proelastase concentration of 50pg/assay was used in 500 mg of crude proelastase
all incubations and activation was as previously described.
Specificactivity (%)
Protein inhibitors AA/min/pg Folin protein
Yield of
The effect of LBTI was tested against activated ostrich Folin Chymo-
proelastase at an inhibitor-to-enzyme molar ratio of 0.25, protein Elastolytic Tryptic tryptic
0.5, 1,2 and 10. At each inhibitor concentration, assays were Fraction (ma) (SAPNA) (BAPNA) (ATEE)
performed at 2.5, 5, 15 and 30 min intervals using SAPNA Bovine trypsin - ND 100 ND
as substrate. Assays of activated proelastase without inhibi- Bovine chymotrypsin - ND ND 100
tor were also performed at each time interval, in order to PPE - 100 ND ND
obtain the 100% initial activity value. The initial slopes of B 121.0 - 43.8
the progress curves in the presence of inhibitor were plotted C 15.2 - 1.25t
as a % of the uninhibited slope vs. time for each inhibitor D 53.2 - - 3.25t
E 8.5 - - -
concentration. F 29.2 35.7’ -
q-PI, a natural protein inhibitor of elastase was tested on G 6.4 16.1. - -
activated proelastase with inhibitor-to-enzyme molar ratios
*Activated for 5 min/37”C/lO% (w/w) bovine trypsin.
of 0.005,0.025,0.05,0.25 and 0.5. All other conditions were
tActivated for 2 hr/37”C/2% (w/w) bovine trypsin.
as described for LBTI. ND. not determined.

Active site-directed irreversible inhibitor

PMSF was dissolved in isopropanol and the final concen-
could only be detected after activation by bovine
tration of isopropanol was 5% or less. The molar ratio of trypsin, while the trypsin fraction was completely
inhibitor-to-enzyme was 62.5, 125, 250, 500 and 1000. The autoactivated (Table 1).
assay conditions were similar to those described for LBTI. CRE was used to assay the ostrich proelastase
fraction after activation. PPE was also assayed to
Reversible inhibitor serve as a comparison to the other synthetic sub-
Elastatinal was dissolved in DMSO and the final DMSO strates. Figure 2 shows a characteristic hyperbolic
concentration in the cuvette was 10% or less. The inhibitor- curve for a highly active PPE and a sigmoidal shaped
to-enzyme molar ratio of0.75, 1.5,3,6 and 12 was used with
SAPNA concentrations of 0.5 and 0.75 mM. All other
conditions were as described for LBTI.

RESULTS

Proelastase was purified using a SP-Sephadex C-50

column (Fig. 1). CM-cellulose columns were used
successfully in the separation of the Ser proteases in
other species (Largman et al., 1976; Yoshinaka et al.,
1984; Largman, 1983; and Honda et al., 1986), but
were not successful in separating the crude ostrich
preparation. Since proelastase is more basic than
chymotrypsinogen and trypsin, the latter enzymes 0 20 40 60 80 100 120 140 160
eluted first from the cation exchange column (Fig. 1). Time (min)
Protease activity of fractions which eluted from the Fig. 2. Elastolytic assay progress curves (CRE as substrate)
column was tested by removal of 100 ~1 aliquots from for PPE and activated ostrich proelastase (fraction F).
every fifth tube. Elastolytic and chymotryptic activity A----A, PPE; and A-A, fraction F.

a b C
r--

1.0 30-

0.E

7
T 0.E

5
fJ 0.L
4

0.2

20 40 60 80 100 120 140 160 180 200 220

Tube no. (7nWtube)

Fig. 1. SP-Sephadex C-50 chromatography of 500 mg of crude proelastase. O-0, AA,,,, x lO’/min;
A-A, A&,,,,x IO/min; and A-A, AA4,0nm/min. (a) 0.05 M NH, acetate (PH 5); (b) 0.05 M NH,
acetate @H 5) to 0.25 M NH, acetate @H 6) (560 ml linear gradient); (c) 0.25 M NH, acetate @H 6); and
(d) 0.55 M NH, acetate (pH 6).
340 JENNI L. SUTHERLANDetal.

% bovine trypsin (w/w)

Fig. 3. Activation of ostrich proelastase (fraction F) with PH

bovine trypsin. Fig. 5. The effect of pH on the hydrolysis of SAPNA by
activated ostrich proelastase. A, 50mM sodium acetate
curve for fraction F (low activity) which illustrates buffer; 0, 50 mM Tris-HCl buffer; and A, 50 mM glycine-
the presence of a co-operative effect. The sigmoidal NaOH buffer.
shape is caused by heavy cross-linking in the elastin
molecule, with progressively more Ala and Leu bonds comparison between the two enzymes. The ostrich
being exposed to the enzyme as time proceeds. enzyme appears to have a similar affinity for the
SDS-PAGE and isoelectric focusing showed that Ala residue as does PPE (Km values of 3.04 and
fraction F was homogeneous, with a M, value on 3.088 mM, respectively). Activated ostrich pro-
SDS-PAGE of 28,000. Isoelectric focusing showed a elastase has a lower affinity for Leu than PPE (K,,,
single component with a pH, > 9.3, trypsinogen being values of 1.68 and 0.606 mM, respectively) (Table 2).
the most basic standard available. Lewis et al. (1956) k,,, and k,,/K,,, values were also determined for the
reported a pH, of 9.6 for PPE. The PPE standard 2 enzymes, using SAPNA and SAPLA as substrates.
revealed a pH, of > 9.3, but was more acidic in Table 2 shows the catalytic constant of PPE with the
nature when compared to ostrich proelastase. 2 substrates to be similar, indicating similar catalytic
Figure 3 shows the effect of varying bovine trypsin activities. Activated ostrich proelastase, however,
concentrations on the activation of proelastase, reveals much lower catalytic activities for both sub-
whereas Fig. 4 illustrates the effect of incubation time strates. The k,,,/K,,, value of PPE with SAPLA reveals
on proelastase activation. Ostrich and bovine trypsin the highest catalytic efficiency followed by PPE with
were compared and ostrich trypsin activated ostrich SAPNA, whereas activated ostrich proelastase, with
proelastase more effectively than bovine trypsin. The SAPLA and SAPNA as substrates, revealed a rela-
conditions for maximum activation were thus taken
as 10% (w/w) trypsin for 20min at 37°C. Table 2. Summary of K,, k,,, k,,,/K,,, values for PPE and fraction F
Figure 5 describes the effect of pH on the activity with various substrates
of activated ostrich proelastase. The pH optimum of k k,lKm
8.9 corresponds well with that of other mammalian Fraction Substrate (setca’I) (set - ’M ”
enzymes (Lamy et al., 1961; Gertler, 1971). PPE SAPNA 3.088 8.142 2 636.70
Figure 6 indicates that activated ostrich proelastase SAPLA 0.606 7.777 12 833.00
has maximum thermal stability at 50°C. Ostrich CRE 0.666 ND ND
elastase remains active at lower temperatures as well, 1.000*
F SAPNA 3.040 0.283 93.09
e.g. 25% activity remains at 21°C. Above 51°C SAPLA 1.680 0.261 155.06
however, enzyme stability decreased considerably. CRE 0.138- ND ND
Km values for activated ostrich proelastase are 0.444’
reported in Table 2. Both PPE and activated ostrich *% (w/v) and range of K,,,.
proelastase (fraction F) were analysed to allow a ND. not determined.

0 10 20 30 40 50 60
Tme (mm)
Temperature (*C1
Fig. 4. Activation of ostrich proelastase (fraction F) with
10% (w/w) bovine and ostrich trypsin. A---A, ostrich Fig. 6. The effect of temperature on the stability of activated
trypsin; and o-0, bovine trypsin. ostrich proelastase.
 Pancreatic proelastase from the ostrich 341

Inhibitor/ enzyme (mol/mol)

Fig. 7. Inhibition of activated fraction F by LBTI.

tively low catalytic efficiency. Both enzymes displayed @,-PI inhibits ostrich elastase implies a structural
a greater catalytic efficiency with SAPLA. similarity between the human and avian enzyme.
PPE and activated fraction F revealed K,,, values PMSF reacts directly with the active site Ser of
for CRE of 0.666-1.0 and 0.138-0.444%, respect- an enzyme. Since complete inhibition of elastase
ively. The affinity of the pancreatic elastases for the occurred at a lOOO-fold molar excess of PMSF, it is
natural substrate CRE is difficult to relate to that of implied that activated ostrich proelastase contains a
the artificial substrates since the molecular weight of Ser in its active site and is thus classified as a Ser
CRE is not known. Elastin contains very few Leu protease.
residues, but many Ala and Val residues. The ostrich Elastatinal, an inhibitor of microbial origin,
enzyme appears to have a higher affinity for elastin contains an Ala analog which is recognised by the
compared to PPE. enzyme. A 1Zfold molar excess of elastatinal inhibits
The effect of inhibitors on activated ostrich pro- activated ostrich proelastase completely after 15 min
elastase was similar to that obtained for ostrich of incubation (Fig. 9). Initial inhibition is rapid since
chyniotrypsin (Van der Westhuizen et al., 1989). all inhibitor : enzyme molar ratios displayed an in-
Various types of inhibitors were investigated. LBTI, hibitory effect at 2.5 min (Fig. 10). Ki of elastase with
a double-headed inhibitor of the Ser proteases, trypsin elastatinal was determined using 0.5 and 0.75 mM
and chymotrypsin (Krahn and Stevens, 1970), contains SAPNA. The K, value of 4.46pM, calculated by
a leucyl-seryl peptide bond that binds the elastase least-squares linear regression from a Double-Dixon
molecule. Lievaart and Stevenson (1974) showed plot can be compared to a value of 0.3 PM reported
elastase to bind to the “chymotrypsin site” on the by Zimmerman and Ashe (1977) for human leukocyte
inhibitor molecule, thus implying similarity between elastase.
chymotrypsin and elastase with regard to their active Amino acid composition of ostrich proelastase
site. Figure 7 shows that LBTI caused more than (fraction F) compares favourably with those of other
85% inhibition of activated ostrich proejastase at species (Table 3) especially catfish pancreatic elastase
molar excesses between 2 : 1 and 10 : 1. B (Yoshinaka et al., 1984). Since the catfish and the
q-PI is a natural protease inhibitor, which is ostrich are both primitive species, the similarity in
present in the serum of most mammals. The potency composition is expected. M,,,,, calculated from amino
of this inhibitor is high since total inhibition is evident acid composition is 25,249 Da (average of HCI and
at a molar excess of 0.25 (Fig. 8). The fact that human MSA hydrolyses).

0 0.1 02 0.3 04

Inhibitor/enzyme (md/mol)

Fig. 8. Inhibition of activated fraction F by q-PI.

342 JENNIL. SUTHERLAND
et al.

3 I 2 3 4 5 6 7 6 9 IO II 12
Inhibitor/enzyme (mol/moO

Fig. 9. Inhibition of activated fraction F by elastatinal; 0, 0.5 mM SAPNA and A, 0.75 mM SAPNA.

20 -

I I I I I I
0 ’
0 5 IO 15 20 25 30

lime (min)

Fig. 10. Inhibition of activated fraction F by elastatinal (0.75 mM SAPNA).

Table 3. Amino acid composition (molar ratios) of fraction F, BPE, insoluble components are removed and thus do not
PPE and catfish &stases A and B
interfere with later column steps. An ammonium
Catfish acetate (pH 4.5) extraction is commonly used to
Amino Fraction F elastase~
acid HCI MSA BPE* PPEt A B
prepare elastase/proelastase and is not as acidic as the
H,SO, extraction procedure used for chymotrypsino-
LYs 9.30(9) 8.78(9) 3 1 6 3
His 6.68(7) 6.50(7) 6 6 8 7
gen (Van der Westhuizen et al., 1989). The purification
A% 11.67(12) 10.61(11) 12 12 15 II of proelastase was achieved with a single ion-exchange
Asx 23.15(23) 22.69(23) 24 24 29 22 chromatography step, on a SP-Sephadex C-50 column.
Thr 13.04(13) 15.38(15) 19 19 9 18 The gel had been swollen in an ostrich acid-ethanol
Ser 20.71(21) 19.41(19) 22 22 18 24
Glx 22. I6(22)
pancreatic extract (Evans et af., 1988). SP-Sephadex
18.92(19) 19 19 19 17
Pro 10.15(10) 14.25(14) 7 7 7 8 swollen normally in distilled H,O does not produce a
Gly 24.15(24) 27.33(27) 25 25 11 25 complete separation of the three major Ser proteases.
Ala 18.08(18) 14.72(15) 17 17 15 I8 The elastase and chymotrypsin fractions from the
Cyh 5.43(5) 3.84(4) 8 8 7 9
val 19.55(20)
column were inactive whereas trypsin was present in
19.74(20) 27 27 14 22
Met 2.80(3) 2.50(3) 2 2 4 3 its active form. This is possible since trypsin requires
Ile 11.18(11) 10.68(11) IO 10 I2 IO only a small amount of enzyme for autoactivation to
Leu 17.50(18) 17.45(17) 18 18 13 15 occur. Once this process begins, activation can be
TYr 10.96(11) 10.86(11) 11 11 12 12
Phe 5.95(6) 5.59(6) 3 3 8 2
completed. Chymotrypsinogen requires a low trypsin
TOP O(4) 3.91(4) 7 6 4 11 concentration for activation, but a relatively long
Total No. activation time at 37°C (Van der Westhuizen et al.,
of residues 231 235 240 237 211 237 1989). Elastase, on the other hand, only requires a 20
*Walsh and Neurath (1964). min incubation time, but a high trypsin concentration
tShotton and Hartley (1970). for activation is required.
IYoshinaka er al. (1984).
Because ostrich proelastase is activated by ostrich
and bovine trypsin, its pH and temperature optima
and activity profiles are typical of elastases and the
DISCUSSION
evidence that the inhibitors studied inactivate it and
other elastases, all support the idea that the activated
The preparation of a pancreatic acetone powder form of proelastase contributes to protein digestion
was found to be advantageous in that all H,O in the ostrich.
 Pancreatic proelastase from the ostrich 343

SUMMARY Honda T.. Fuiita A.. Tsubakihara Y. and Morihara K.

(1986) AlTiniiy purification of kallikrein and elastase from
By preparing an acetone powder and performing a hog pancreas powder. J. Chromatogn. 376, 385-393.
mild acid extraction, salt fractionation and subsequent James H. L., James P. L., Painter R. G. and Cohen A. B.
ion-exchange chromatography on a SP-Sephadex C-50 (1985) Location of elastase in human blood platelets.
column, proelastase could be isolated from the pan- Thromb. Haemat. 54, 70-79.
creas of the ostrich. SDS-PAGE revealed a h4, value Krahn J. and Stevens F. C. (1970) Lima bean trypsin
of 28,000, while M,,,i, calculated from amino acid inhibitor. Limited proteolysis by trypsin and chymo-
trypsin. Biochemistry 9, 2646-2652.
composition data was 25,249 Da. The pH, of ostrich
Lamy F., Craig C. P. and Tauber S. (1961) Studies on
proelastase was found to be > 9.3. The elastolytic
elastase and elastin 1. Assay and properties of elastase.
activity of fractions was determined with CRE, J. biol. Chem. 236, 86-91.
SAPNA and SAPLA, thus utilising both natural Largman C., Brodrick J. W. and Geokas M. C. (1976)
and synthetic substrates. Optimum conditions for the Purification and characterization of two human pancreatic
activation of ostrich proelastase turned out to be 20 elastases. Biochemistry 15, 2491-2500.
min at 37°C in the presence of 10% (w/w) ostrich or Largman C. (1983) Isolation and characterization of rat
bovine trypsin. The ostrich enzyme showed maximum pancreatic elastase. Biochemistry 22, 3763-3770.
activity at 50°C and a stable pH optimum of 8.9. The Lewis U. J., Williams D. E. and Brink N. G. (1956)
Pancreatic elastase: purification, properties and function.
effects of inhibitors (LBTI, X,-PI, PMSF and elasta-
J. biol. Chem. 222, 705-720.
tinal) on elastolytic activity was examined and the Lievaart P. and Stevenson K. J. (1974) The isolation of
results obtained were more or less similar to those trypsin and elastase from moose pancreas (Alces alces)
obtained for elastase purified from various other by affinity chromatography on Lima-bean protease
species. inhibitor-Sepharose resin. Can. J. Biochem. 52, 637-644.
The properties of the ostrich elastase are similar to Lowry 0. H., Rosebrough N. H., Farr A. L. and Randall
those of other mammalian species, therefore it can R. J. (1951) Protein measurement with the Folin phenol
be concluded that the proelastase enzyme performs reagent. J. biol. Chem. 193, 265-275.
the same role in intestinal digestion in ostriches as is MacDonald R. J., Swift G. H., Quint0 C., Swain W., Pictet
R. L., Nikovits W. and Rutter W. J. (1982) Primary
known for proelastases in mammals.
structure of two distinct rat pancreatic prepro-elastases
determined by sequence analysis of the complete cloned
Acknowledgements-The authors gratefully acknowledge messenger ribonucleic acid sequences. Biochemistry 21,
the financial support granted by the South African Council 1453-1463.
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