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A pepsinogen from rainbow trout
Comp. Biochem. Physiol. Vol. 75B. No. 1, pp. 109 to 112, 1983 Printed in Great Britain
A PEPSINOGEN
0305-0491/83/050109-04503.00/'0 © 1983 Pergamon Press Ltd
FROM RAINBOW TROUT
SALLY S. TWINING, PATRICIA A. ALEXANDER, KENT HU1BREGTSE and DAVID M. GLICK* Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226 USA (Received 26 August 1982) A b s t r a c t - - l . A pepsinogen, Ia on the basis of its electrophoretic mobility, from rainbow trout stomach,
has an optimum pH near 2 for activation. 2. The cognate pepsin is denatured at pH values above 7, in contrast to the zymogen, which is slightly more alkali-stable. It has an optimum pH of 3 for proteolysis of denatured hemoglobin. 3. The intrinsic reactivity of the zymogen and pepsin (rates of activation and of proteolysis, respectively) are quite high, but as they operate at the environmental temperature of the fish, are remarkably similar to rates of activation and proteolysis by mammalian pepsinogens and pepsins.
INTRODUCTION
standard for comparison and in other studies. Rates of pepsinogen activation were determined according to A1Janabi et al. (1972). Alkaline inactivation was performed by diluting samples in 0.4 M sodium acetate, pH 5.5, into alkaline buffer for 30 rain at 3 7 C before assay. The milkclotting assay was performed at 2 5 C by the method of McPhie (1976). Amino acid analysis was performed on a Dionex instrument. Protein was hydrolyzed in 6 M HCI at 110'C for 24, 48 and 72 hr (Crestfield et al., 1963). Protein was determined according to Lowry et al. (1951) using bovine serum albumin (Miles Laboratories) as a standard. DEAE-Sephacel, Sephadex, Sepharose and DEAE Sepharose were products of Pharmacia. Polylysine was purchased from Sigma Chemical Co.
The gastric pepsinogens of many h o m e o t h e r m s have been isolated and their activations to cognate pepsins studied. We have been interested in the early stages of pepsinogen activation and wanted to extend our studies to a pepsinogen from a poikilotherm because long ago it had been reported that the pepsins of poikilotherms, in comparison with those of homeotherms, are adapted to functioning at low temperature by having lower Arrhenius activation energies. This was qualitatively demonstrated for pike pepsin as early as 1874 (Fick & Murisier). The data of Hykes et al. (1934) for perch pepsin indicate an activation energy of 4 kcal/mol, and of Norris & Elam (1940) for salmon pepsin, 3.5kcal/mol, assayed against protein substrates. These values compare with 18 2 0 k c a l / m o l for pig pepsin assayed against a dipeptide at pH 4, considerably above the o p t i m u m (Casey & Laidler, 1950). The conversion of pepsinogen to pseudopepsin is also a catalytic property, and might also show a kinetic adaptation to environmental temperature. We have proceeded to purify zymogen Ia from rainbow trout (Salmo gairdneri) and to characterize it and its potential activity.
RESULTS
MATERIALS AND METHODS
Polyacrylamide slab gel electrophoresis of pepsinogens was performed on a Bio Rad Laboratories Model 220 dual vertical slab cell with a VoKam SAE 2761 (Shandon Scientific Co.) power supply, according to Taggert et al. (1978). Electrophoresis chemicals and supplies were purchased from Bio Rad Laboratories. Protein was visualized by staining with Coomassie Brilliant Blue in perchloric acid according to Holbrook & Leaver (1976). Alternatively, in zymograms, the zymogen's potential activity was visualized by diffusing bovine hemoglobin (Sigma Chemical Co.) from a 0.66% solution in 0.l M HCI into the gel at 37°C for 15 min followed by digestion in 0.1 M HCI for 40min at 37c'C and staining for protein with Coomassie Brilliant Blue (Taggert et al., 1978). Pepsin activity and pepsinogen potential activity were determined according to Chow & Kassell (1968). Pig pepsin from Worthington Biochemical Corp. was used as a * To whom correspondence should be addressed.
The stages of purification of a pepsinogen from 60 rainbow trout stomachs are summarized in Table 1. Frozen stomachs weighing 291 g were thawed and homogenized in 950 ml of 0.05 M sodium phosphate, pH 7.1, the buffer used t h r o u g h o u t the purification. This and subsequent operations were performed at 4°C. The homogenate was centrifuged at 27,000 O for 60min. The supernatant fluid (820 ml) was passed t h r o u g h glass wool a n d centrifuged at 90,000q for 90 min. The 800 ml of supernatant fluid was brought to 20% saturation by the addition of 88g of amm o n i u m sulfate. The pH was maintained at 7.1 by addition of dilute sodium hydroxide. After 16 hr the suspension was centrifuged at 90,000,q for 90 min. To the resulting supernatant fluid (760ml) was added 176 g a m m o n i u m sulfate to bring it to 50% saturation. After 16 hr this was again centrifuged at 90,000 ,q for 90min. The sedimented material was repeatedly extracted with buffer by centrifuging down the undissolved material after each resuspension. The pooled extracts, 605ml, were dialyzed against buffer to remove residual a m m o n i u m sulfate. The solution, 650 ml, was separated on polylysine Sepharose (Nevaldine & Kassell, 1971) by passing it over a 2 x 34 cm column. Little of the potential activity was retained. The protein, then in 1200 ml, was adsorbed batch-wise onto sufficient D E A E Sephacel to fill a 3.8 x 5 0 c m c h r o m a t o g r a p h y column a n d
109
110
SALLY S. TWINING et al. Table I. Purification of a rainbow trout pepsinogen
Total protein Total zymogen recovered* recoveredt Percent (mg) (mg) recovered
Step 27,000 y supernatant 90,000 y supernatant 20 50'!,i;(NH4)2 SO4 fraction Polylysine -Sepharose DEAE Sephacel Sephadex G-200 DEAE Sepharose
2493.0 2060.0 866.0 271.0 160.0 33.0 8.9
257.0 243.0 187.0 65.0 13.0 5.8 8.9
Sp. potential activity (/~g/mg protein)~
100.0 95.0 73.0 25.0 5.0 2.3 3.5
103.0 118.0 216.0 240.(I 82.(/ 175.0 1000.0
* TCA-precipitable protein determined by the Lowry (1951) method. 5 Potential activity is expressed as mg rainbow trout pepsin equivalents, assayed at 37C. + Specific potential activity is expressed as/~g potential rainbow trout pepsin activity per mg protein, calculated from E,80 = 93,500cm ~ M
washed with 41. of buffer. This removed a substantial fraction of the potential activity but on zymograms this activity was largely of group II (Rr wdues between 0.32 and 0.41 I. The retained potential activity was eluted with 560 ml buffer supplemented with 1 M NaCI. The eluate was desalted over an Amicon YM-10 m e m b r a n e and concentrated to 20ml. This was passed over a Sephadex G-200 column (4.3 × 50 cm), the potentially active material appearing between 460 and 6 6 0 m l of eluting buffer. The zymogen, Ia on the basis of its electropharetic mobility, was finally purified on a D E A E - S e p h a r o s e column (2.5 × 4 0 c m ) eluted by a (~1 M NaC1 gradient over 21. The potential activity eluting between 1115 and 1205 ml was pooled and found to
be electrophoretically homogeneous (Fig. 1t. It was dialyzed first against diluted buffer, then against water and lyophilized. The amino acid composition is presented in Table 2. Its molar extinction coefficient, calculated from the a m i n o acid analysis of a preparation of k n o w n absorbance at 2 8 0 n m , is 93,500 c m - 1 M - 1 The cognate pepsin was prepared by bringing 0.5 mg of the zymogen in 3 ml water to pH 2.1 with 3 m l 0.1 M HC1. After 5 min at 2°C, 6 ml of 0 . 4 M sodium acetate, p H 5.5, was added to raise the p H to 5.1. This was diluted with 20 ml water, then dialyzed to remove salts and activation peptides a n d concentrated over a YM-10 membrane.
Table 2. Amino acid composition of rainbow trout pepsinogen* Amino acid Asx Thr Set Glx Pro Ala Gly Cys Val Met Leu Ile Tyr Phe His Lys Arg Try
A
B
C
Fig. 1. Polyacrylamide slab gel electrophoresis of crude and purified rainbow trout pepsinogen. The 90,000 g supernatant (A) and the purified material (B) and (C) were separated and stained for potential proteolytic activity (A) and (B) according to Taggert et al. (1978) or for protein (C) according to Holbrook & Leaver (1976).
Rainbow trout 37.6 26.6 30.0 43.6 16.5 33.0 27.2 10.6 20.6 10.0 20.8 18.8 12.6 12.6 1.2 12.0 3.2 +~
38 27 30 44 17 33 27 10 21 10 21 19 13 13 2 12 3
Pigt 46 27 46 28 18 36 20 6 25 4 27 33 17 16 3 10 4 5
* Values are averages of 6 hydrolyzates or, for serine and threonine, are extrapolations to zero from 24, 48 and 72 hr hydrolyses. Cysteine was determined as carboxymethyl-cysteine according to Crestfield et al. (1963). i T h e composition of pig pepsinogen (Foltmann & Pedersen, 1977} is shown for comparison. ++Not determined.
A pepsinogen from rainbow trout 1.2
100
o.e
8o
o
111
0.4
50
C 0.0
o v
4o
4
-0.
20
J
0 6
7
g
8
10
~1
-0. 8 3.2
i
i
3.3
3.4
12
3~
.5
i 3.0
i
3.7
1000/T
pH Fig. 2. pH-Dependence of alkaline inactivation of rainbow trout pepsin and pepsinogen. The enzyme or zymogen was incubated before assay according to Chow & Kassell (1968) at pH 5.5 in 0.133 M sodium acetate, at pH 6, 7 or 11 in 0.167M sodium phosphate, or at pH 8.5 or 10 at 0.167 M Tris hydrocbloride.
The stabilities of the zymogen and enzyme to neutral and alkaline conditions was tested in a series of buffers of pH values 6-8.5 (Fig. 2). The results indicate it is possible to inactivate the pepsin while preserving the pepsinogen at pH 7.0. Therefore rates of activation at various pH values were determined by quenching pepsinogen solutions in 0.1-0.2 M sodium phosphate, pH 7, at intervals after acidification with 0.05-0.15 M HC1. The rate was so fast, even at 0°C, that at pH 2 we were unable to sample the acidified mixture soon enough to accurately determine a rate constant. We estimate the half-time to be no more than 4sec, making the first order rate constant at least 10 rain 1. At pH values above and below 2 we were able to determine rate constants (Table 3). For reasons already presented, we were interested in determining the temperature-dependence of the activation rate. Because it was too fast at pH 2, the measurements were made at pH 3 (Fig. 3). The Arrhenius activation energy was found to be 9.13 kcal/mol. For similar reasons we were interested in the temperature-dependence of the pepsin's proteolytic activity. This we observed and compared with the activities of pig and dog pepsins (Fig. 4). The Arrhenius energies calculated for them were: rainbow trout 7.56, pig 6.32 and dog 8.38 kcal/mol.
Table 3. pH-Dependence of activation of rainbow trout pepsinogen*
pH
First order rate constant (min - 1)
1.4 2.0 2.7 4.0
1.54 10.00 2.31 0.22
* Activations were performed at 0°C by the method of AI-Janabi et al. (1972).
Fig. 3. Temperature-dependence of activation of rainbow trout pepsinogen. The activation was performed at temperatures between 0 and 37°C in 0.1 M sodium formate, pH 3.
The pH-dependence of rainbow trout activity is shown in Fig. 5. The optimum pH is 3. At pH 2 (not even its optimum pH value), rainbow trout pepsin is almost 5 times as active against a hemoglobin substrate as is pig pepsin. In contrast, in the milkclotting assay, rainbow trout pepsin is only 25~0 as active. DISCUSSION The zymogram of the crude extract shows as many as 7 acid proteinases or their acid-activatable zymogens (Fig. 1A). Following the practice with other species, we can refer to the most mobile group (R s 0.44-0.53) as group I, and the other major group (R/ 0.32-0.41), as group II. The preparation of the major zymogen from group I follows familiar lines (Foltmann, 1981). The zymogen is rather less stable at alkaline pH values than is pig pepsinogen. Compared with the stability of its pepsin (Fig. 2), this leaves a fairly narrow pH range for preferential inactivation of pepsin in the presence of pepsinogen in conjunction with assays of rate of activation.
1.6
~ " "~.
•~. ~ a i n b o w ~" 1.
t Po,.at
"~'*""
0.
gl"13. 2
3.3
i
i
i
j
3.4
3.5
3.6
~,7
10001T
Fig. 4. Temperature-dependence of proteolytic activities of rainbow trout, dog and pig pepsins. Assays were performed at 0, 5, 10, 15, 20 and 37°C according to Chow & Kassell (1968) and are presented for 10/~g of each pepsin expressed as the amount of pig pepsin at 37°C with the same activity: • rainbow trout; × dog; © pig.
112
SALLY S. T W I N I N G et
100
~. 80 50
20 ~l 0
1
2
3
4
5
5
FH Fig. 5. pH-Dependence of rainbow trout pepsin activity. Assays were performed according to Chow & Kassell (1968) using HC1 (pH values 1-2.5), 0.1 M sodium formate (pH 3 and 3.3) and 0.02 M sodium acetate (pH values 3.5-5).
In determining the pH-dependence of rate of zymogen activation (Table 3) we became aware of its very rapid rate. Even at 0°C it was too fast to measure at pH 2, near its optimum, but it did show rates above and below pH 2 that we could determine. Because of this very fast activation, we could not measure the temperature dependence of activation at pH 2. Consequently this study was performed at pH 3 (Fig. 3). The Arrhenius activation energy for activation of the rainbow trout zymogen is 9.13kcal/mol, compared to 12 kcal/mol measured for pig and 17 kcal/mol for dog (unpublished observations) pepsinogens. However, its actual rate was surprising. If we compare rate constants for pH 2 at 37°C for the rainbow trout against the pig, the fish zymogen constant would be at least 75 m i n - l compared with 14 m i n - l for the pig. (We have assumed that the rate constant for the rainbow trout zymogen at pH 2, 0°C, is at least 10 m i n - t and that the energy of activation does not vary with pH.) However, a fairer comparison would be of rainbow trout at 15°C, its approximate environmental temperature, to the pig at 37°C: 24 vs 14 m i n - 2. Now we see that the rates of activation, properly viewed, are really not all that disparate. Rainbow trout pepsinogen was activated, and the pepsin was examined for the pH-dependence of its activity (Fig. 4). In contrast to the activation reaction, the pH optimum for proteolysis is 3. For the same reasons that we were interested in the temperature dependence of activation, we were interested in the temperature-dependence of proteolysis, in comparison with some other pepsins. Remarkably, comparison of proteolytic rates for rainbow trout pepsin at 15°C with dog and pig pepsins at 37°C (Fig. 4) shows them all to have similar proteolytic activities at the temperatures at which they actually operate. In the absence of data on other fish species, it would be rash to claim that the higher rate of activation and the greater proteolytic activity of trout pepsinogen and pepsin are either characteristic of poikilotherms or are an adaptation to the environmental temperature, but we are aware of the possibility these may prove to be true. Contrary to our expectations, rainbow trout showed an Arrhenius ac-
al.
tivation energy for proteolysis at pH 2, very much like that for pig and dog pepsins, despite the fact that pig pepsin has been reported to have an activation energy of about 20 kcal/mol when assayed against the dipeptide, carbobenzoxy-L-glutamyl-L-tyrosine (Casey & Laidler, 1950). Whether the difference is due to the fact that the earlier determination was done at pH 4, or whether the important distinction is that protein is a "good" substrate (one cleaved at a high rate) and the dipeptide is a poor substrate, is uncertain. If further experiments on temperature dependence of catalysis show that good substrates have low Arrhenius energies and poor substrates, high energies, it would suggest that activation of mammalian pepsinogens proceeds like hydrolysis of a poor substrate and activation of rainbow trout pepsinogen, like hydrolysis of a good substrate. In any case, comparison of the activation of the rainbow trout and pig zymogen will be very informative of the factors governing maintenance of the aspartate proteinase zymogens in an inactive state and those that on acidification lead to activation. Acknowledgements The rainbow trout stomachs were made available by the Center for Great Lakes Studies. This work was supported by USPHS grant AM-09827. REFERENCES
AL-JANAB1J., HARTSUCKJ. A. & TANG J. (1972) Kinetics and mechanism of pepsinogen activation. J, biol. Chem. 247, 4628~,632. CASEY E. J. & LAIDLER K. J. (1950) Molecular kinetics of pepsin-catalyzed reactions. J. Am. chem. Soc. 72, 2159-2164. CHOW R. B. & KASSELCB. (1968) Bovine pepsinogen and pepsin I. Isolation, purification and some properties of the pepsinogen. J. biol. Chem. 243, 1718 1824. CRESTFIELD A. M., MOORE S. & STEIN W. H. (1963) The preparation and enzymatic hydrolysis of reduced and S-carboxymethylated proteins. J. biol. Chem. 238, 622 627. FICK A. & MURISIER D. 0874) Uber das Magenferment kaltblutiger Tiere. Jber. Fortschr. Tierchem. 3, 162-163. FOLTMANN B. (1981) Gastric proteinases--structure, function, evolution and mechanism of action. Essays BiGchem. 17, 52 84. HOLBROOK I. B. & LEAVER A. G. (1976) A procedure to increase the sensitivity of staining by Coomassie brilliant blue G250-perchloric acid solution. Analyt. Biochem. 75, 634-636. HYKES O. V., MAZANELJ. S. • SZECSENYIL. (1934) Contribution a la connaissance des ferments digestifs des poissons. C.r. Soc. Biol. ll7, 166 168. LOWRY O. H., ROSEBROUGHN. J., FARR A. L. & RANDALL R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265 275. McPHIE P. (1976) A turbidimetric milk-clotting assay. Analyt. Biochem. 73, 258-261. NEVALDINEB. & KASSELLB. (1971) Bovine pepsinogen and pepsin IV. A new method of purification of the pepsin. Biochim. biophys. Acta 250, 207-209. NORRIS E. R. & ELAM D. W. (1940) Preparation and properties of crystalline salmon pepsin. J. biol. Chem. 134, 153 157. TAGGERT T., MILLER R. B., KARN R. C., TRIBBLE J. A.. CRAFT M., RIPBERGERJ. & MERRITTA. D. (1978) Vertical thin layer slab polyacrylamide get electrophoresis of selected human polymorphic proteins. In Electrophoresis '78 (Edited by CATSIMPOOLASN.), pp. 231-242. Elsevier, Amsterdam.
A PEPSINOGEN
0305-0491/83/050109-04503.00/'0 © 1983 Pergamon Press Ltd
FROM RAINBOW TROUT
SALLY S. TWINING, PATRICIA A. ALEXANDER, KENT HU1BREGTSE and DAVID M. GLICK* Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226 USA (Received 26 August 1982) A b s t r a c t - - l . A pepsinogen, Ia on the basis of its electrophoretic mobility, from rainbow trout stomach,
has an optimum pH near 2 for activation. 2. The cognate pepsin is denatured at pH values above 7, in contrast to the zymogen, which is slightly more alkali-stable. It has an optimum pH of 3 for proteolysis of denatured hemoglobin. 3. The intrinsic reactivity of the zymogen and pepsin (rates of activation and of proteolysis, respectively) are quite high, but as they operate at the environmental temperature of the fish, are remarkably similar to rates of activation and proteolysis by mammalian pepsinogens and pepsins.
INTRODUCTION
standard for comparison and in other studies. Rates of pepsinogen activation were determined according to A1Janabi et al. (1972). Alkaline inactivation was performed by diluting samples in 0.4 M sodium acetate, pH 5.5, into alkaline buffer for 30 rain at 3 7 C before assay. The milkclotting assay was performed at 2 5 C by the method of McPhie (1976). Amino acid analysis was performed on a Dionex instrument. Protein was hydrolyzed in 6 M HCI at 110'C for 24, 48 and 72 hr (Crestfield et al., 1963). Protein was determined according to Lowry et al. (1951) using bovine serum albumin (Miles Laboratories) as a standard. DEAE-Sephacel, Sephadex, Sepharose and DEAE Sepharose were products of Pharmacia. Polylysine was purchased from Sigma Chemical Co.
The gastric pepsinogens of many h o m e o t h e r m s have been isolated and their activations to cognate pepsins studied. We have been interested in the early stages of pepsinogen activation and wanted to extend our studies to a pepsinogen from a poikilotherm because long ago it had been reported that the pepsins of poikilotherms, in comparison with those of homeotherms, are adapted to functioning at low temperature by having lower Arrhenius activation energies. This was qualitatively demonstrated for pike pepsin as early as 1874 (Fick & Murisier). The data of Hykes et al. (1934) for perch pepsin indicate an activation energy of 4 kcal/mol, and of Norris & Elam (1940) for salmon pepsin, 3.5kcal/mol, assayed against protein substrates. These values compare with 18 2 0 k c a l / m o l for pig pepsin assayed against a dipeptide at pH 4, considerably above the o p t i m u m (Casey & Laidler, 1950). The conversion of pepsinogen to pseudopepsin is also a catalytic property, and might also show a kinetic adaptation to environmental temperature. We have proceeded to purify zymogen Ia from rainbow trout (Salmo gairdneri) and to characterize it and its potential activity.
RESULTS
MATERIALS AND METHODS
Polyacrylamide slab gel electrophoresis of pepsinogens was performed on a Bio Rad Laboratories Model 220 dual vertical slab cell with a VoKam SAE 2761 (Shandon Scientific Co.) power supply, according to Taggert et al. (1978). Electrophoresis chemicals and supplies were purchased from Bio Rad Laboratories. Protein was visualized by staining with Coomassie Brilliant Blue in perchloric acid according to Holbrook & Leaver (1976). Alternatively, in zymograms, the zymogen's potential activity was visualized by diffusing bovine hemoglobin (Sigma Chemical Co.) from a 0.66% solution in 0.l M HCI into the gel at 37°C for 15 min followed by digestion in 0.1 M HCI for 40min at 37c'C and staining for protein with Coomassie Brilliant Blue (Taggert et al., 1978). Pepsin activity and pepsinogen potential activity were determined according to Chow & Kassell (1968). Pig pepsin from Worthington Biochemical Corp. was used as a * To whom correspondence should be addressed.
The stages of purification of a pepsinogen from 60 rainbow trout stomachs are summarized in Table 1. Frozen stomachs weighing 291 g were thawed and homogenized in 950 ml of 0.05 M sodium phosphate, pH 7.1, the buffer used t h r o u g h o u t the purification. This and subsequent operations were performed at 4°C. The homogenate was centrifuged at 27,000 O for 60min. The supernatant fluid (820 ml) was passed t h r o u g h glass wool a n d centrifuged at 90,000q for 90 min. The 800 ml of supernatant fluid was brought to 20% saturation by the addition of 88g of amm o n i u m sulfate. The pH was maintained at 7.1 by addition of dilute sodium hydroxide. After 16 hr the suspension was centrifuged at 90,000,q for 90 min. To the resulting supernatant fluid (760ml) was added 176 g a m m o n i u m sulfate to bring it to 50% saturation. After 16 hr this was again centrifuged at 90,000 ,q for 90min. The sedimented material was repeatedly extracted with buffer by centrifuging down the undissolved material after each resuspension. The pooled extracts, 605ml, were dialyzed against buffer to remove residual a m m o n i u m sulfate. The solution, 650 ml, was separated on polylysine Sepharose (Nevaldine & Kassell, 1971) by passing it over a 2 x 34 cm column. Little of the potential activity was retained. The protein, then in 1200 ml, was adsorbed batch-wise onto sufficient D E A E Sephacel to fill a 3.8 x 5 0 c m c h r o m a t o g r a p h y column a n d
109
110
SALLY S. TWINING et al. Table I. Purification of a rainbow trout pepsinogen
Total protein Total zymogen recovered* recoveredt Percent (mg) (mg) recovered
Step 27,000 y supernatant 90,000 y supernatant 20 50'!,i;(NH4)2 SO4 fraction Polylysine -Sepharose DEAE Sephacel Sephadex G-200 DEAE Sepharose
2493.0 2060.0 866.0 271.0 160.0 33.0 8.9
257.0 243.0 187.0 65.0 13.0 5.8 8.9
Sp. potential activity (/~g/mg protein)~
100.0 95.0 73.0 25.0 5.0 2.3 3.5
103.0 118.0 216.0 240.(I 82.(/ 175.0 1000.0
* TCA-precipitable protein determined by the Lowry (1951) method. 5 Potential activity is expressed as mg rainbow trout pepsin equivalents, assayed at 37C. + Specific potential activity is expressed as/~g potential rainbow trout pepsin activity per mg protein, calculated from E,80 = 93,500cm ~ M
washed with 41. of buffer. This removed a substantial fraction of the potential activity but on zymograms this activity was largely of group II (Rr wdues between 0.32 and 0.41 I. The retained potential activity was eluted with 560 ml buffer supplemented with 1 M NaCI. The eluate was desalted over an Amicon YM-10 m e m b r a n e and concentrated to 20ml. This was passed over a Sephadex G-200 column (4.3 × 50 cm), the potentially active material appearing between 460 and 6 6 0 m l of eluting buffer. The zymogen, Ia on the basis of its electropharetic mobility, was finally purified on a D E A E - S e p h a r o s e column (2.5 × 4 0 c m ) eluted by a (~1 M NaC1 gradient over 21. The potential activity eluting between 1115 and 1205 ml was pooled and found to
be electrophoretically homogeneous (Fig. 1t. It was dialyzed first against diluted buffer, then against water and lyophilized. The amino acid composition is presented in Table 2. Its molar extinction coefficient, calculated from the a m i n o acid analysis of a preparation of k n o w n absorbance at 2 8 0 n m , is 93,500 c m - 1 M - 1 The cognate pepsin was prepared by bringing 0.5 mg of the zymogen in 3 ml water to pH 2.1 with 3 m l 0.1 M HC1. After 5 min at 2°C, 6 ml of 0 . 4 M sodium acetate, p H 5.5, was added to raise the p H to 5.1. This was diluted with 20 ml water, then dialyzed to remove salts and activation peptides a n d concentrated over a YM-10 membrane.
Table 2. Amino acid composition of rainbow trout pepsinogen* Amino acid Asx Thr Set Glx Pro Ala Gly Cys Val Met Leu Ile Tyr Phe His Lys Arg Try
A
B
C
Fig. 1. Polyacrylamide slab gel electrophoresis of crude and purified rainbow trout pepsinogen. The 90,000 g supernatant (A) and the purified material (B) and (C) were separated and stained for potential proteolytic activity (A) and (B) according to Taggert et al. (1978) or for protein (C) according to Holbrook & Leaver (1976).
Rainbow trout 37.6 26.6 30.0 43.6 16.5 33.0 27.2 10.6 20.6 10.0 20.8 18.8 12.6 12.6 1.2 12.0 3.2 +~
38 27 30 44 17 33 27 10 21 10 21 19 13 13 2 12 3
Pigt 46 27 46 28 18 36 20 6 25 4 27 33 17 16 3 10 4 5
* Values are averages of 6 hydrolyzates or, for serine and threonine, are extrapolations to zero from 24, 48 and 72 hr hydrolyses. Cysteine was determined as carboxymethyl-cysteine according to Crestfield et al. (1963). i T h e composition of pig pepsinogen (Foltmann & Pedersen, 1977} is shown for comparison. ++Not determined.
A pepsinogen from rainbow trout 1.2
100
o.e
8o
o
111
0.4
50
C 0.0
o v
4o
4
-0.
20
J
0 6
7
g
8
10
~1
-0. 8 3.2
i
i
3.3
3.4
12
3~
.5
i 3.0
i
3.7
1000/T
pH Fig. 2. pH-Dependence of alkaline inactivation of rainbow trout pepsin and pepsinogen. The enzyme or zymogen was incubated before assay according to Chow & Kassell (1968) at pH 5.5 in 0.133 M sodium acetate, at pH 6, 7 or 11 in 0.167M sodium phosphate, or at pH 8.5 or 10 at 0.167 M Tris hydrocbloride.
The stabilities of the zymogen and enzyme to neutral and alkaline conditions was tested in a series of buffers of pH values 6-8.5 (Fig. 2). The results indicate it is possible to inactivate the pepsin while preserving the pepsinogen at pH 7.0. Therefore rates of activation at various pH values were determined by quenching pepsinogen solutions in 0.1-0.2 M sodium phosphate, pH 7, at intervals after acidification with 0.05-0.15 M HC1. The rate was so fast, even at 0°C, that at pH 2 we were unable to sample the acidified mixture soon enough to accurately determine a rate constant. We estimate the half-time to be no more than 4sec, making the first order rate constant at least 10 rain 1. At pH values above and below 2 we were able to determine rate constants (Table 3). For reasons already presented, we were interested in determining the temperature-dependence of the activation rate. Because it was too fast at pH 2, the measurements were made at pH 3 (Fig. 3). The Arrhenius activation energy was found to be 9.13 kcal/mol. For similar reasons we were interested in the temperature-dependence of the pepsin's proteolytic activity. This we observed and compared with the activities of pig and dog pepsins (Fig. 4). The Arrhenius energies calculated for them were: rainbow trout 7.56, pig 6.32 and dog 8.38 kcal/mol.
Table 3. pH-Dependence of activation of rainbow trout pepsinogen*
pH
First order rate constant (min - 1)
1.4 2.0 2.7 4.0
1.54 10.00 2.31 0.22
* Activations were performed at 0°C by the method of AI-Janabi et al. (1972).
Fig. 3. Temperature-dependence of activation of rainbow trout pepsinogen. The activation was performed at temperatures between 0 and 37°C in 0.1 M sodium formate, pH 3.
The pH-dependence of rainbow trout activity is shown in Fig. 5. The optimum pH is 3. At pH 2 (not even its optimum pH value), rainbow trout pepsin is almost 5 times as active against a hemoglobin substrate as is pig pepsin. In contrast, in the milkclotting assay, rainbow trout pepsin is only 25~0 as active. DISCUSSION The zymogram of the crude extract shows as many as 7 acid proteinases or their acid-activatable zymogens (Fig. 1A). Following the practice with other species, we can refer to the most mobile group (R s 0.44-0.53) as group I, and the other major group (R/ 0.32-0.41), as group II. The preparation of the major zymogen from group I follows familiar lines (Foltmann, 1981). The zymogen is rather less stable at alkaline pH values than is pig pepsinogen. Compared with the stability of its pepsin (Fig. 2), this leaves a fairly narrow pH range for preferential inactivation of pepsin in the presence of pepsinogen in conjunction with assays of rate of activation.
1.6
~ " "~.
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t Po,.at
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0.
gl"13. 2
3.3
i
i
i
j
3.4
3.5
3.6
~,7
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Fig. 4. Temperature-dependence of proteolytic activities of rainbow trout, dog and pig pepsins. Assays were performed at 0, 5, 10, 15, 20 and 37°C according to Chow & Kassell (1968) and are presented for 10/~g of each pepsin expressed as the amount of pig pepsin at 37°C with the same activity: • rainbow trout; × dog; © pig.
112
SALLY S. T W I N I N G et
100
~. 80 50
20 ~l 0
1
2
3
4
5
5
FH Fig. 5. pH-Dependence of rainbow trout pepsin activity. Assays were performed according to Chow & Kassell (1968) using HC1 (pH values 1-2.5), 0.1 M sodium formate (pH 3 and 3.3) and 0.02 M sodium acetate (pH values 3.5-5).
In determining the pH-dependence of rate of zymogen activation (Table 3) we became aware of its very rapid rate. Even at 0°C it was too fast to measure at pH 2, near its optimum, but it did show rates above and below pH 2 that we could determine. Because of this very fast activation, we could not measure the temperature dependence of activation at pH 2. Consequently this study was performed at pH 3 (Fig. 3). The Arrhenius activation energy for activation of the rainbow trout zymogen is 9.13kcal/mol, compared to 12 kcal/mol measured for pig and 17 kcal/mol for dog (unpublished observations) pepsinogens. However, its actual rate was surprising. If we compare rate constants for pH 2 at 37°C for the rainbow trout against the pig, the fish zymogen constant would be at least 75 m i n - l compared with 14 m i n - l for the pig. (We have assumed that the rate constant for the rainbow trout zymogen at pH 2, 0°C, is at least 10 m i n - t and that the energy of activation does not vary with pH.) However, a fairer comparison would be of rainbow trout at 15°C, its approximate environmental temperature, to the pig at 37°C: 24 vs 14 m i n - 2. Now we see that the rates of activation, properly viewed, are really not all that disparate. Rainbow trout pepsinogen was activated, and the pepsin was examined for the pH-dependence of its activity (Fig. 4). In contrast to the activation reaction, the pH optimum for proteolysis is 3. For the same reasons that we were interested in the temperature dependence of activation, we were interested in the temperature-dependence of proteolysis, in comparison with some other pepsins. Remarkably, comparison of proteolytic rates for rainbow trout pepsin at 15°C with dog and pig pepsins at 37°C (Fig. 4) shows them all to have similar proteolytic activities at the temperatures at which they actually operate. In the absence of data on other fish species, it would be rash to claim that the higher rate of activation and the greater proteolytic activity of trout pepsinogen and pepsin are either characteristic of poikilotherms or are an adaptation to the environmental temperature, but we are aware of the possibility these may prove to be true. Contrary to our expectations, rainbow trout showed an Arrhenius ac-
al.
tivation energy for proteolysis at pH 2, very much like that for pig and dog pepsins, despite the fact that pig pepsin has been reported to have an activation energy of about 20 kcal/mol when assayed against the dipeptide, carbobenzoxy-L-glutamyl-L-tyrosine (Casey & Laidler, 1950). Whether the difference is due to the fact that the earlier determination was done at pH 4, or whether the important distinction is that protein is a "good" substrate (one cleaved at a high rate) and the dipeptide is a poor substrate, is uncertain. If further experiments on temperature dependence of catalysis show that good substrates have low Arrhenius energies and poor substrates, high energies, it would suggest that activation of mammalian pepsinogens proceeds like hydrolysis of a poor substrate and activation of rainbow trout pepsinogen, like hydrolysis of a good substrate. In any case, comparison of the activation of the rainbow trout and pig zymogen will be very informative of the factors governing maintenance of the aspartate proteinase zymogens in an inactive state and those that on acidification lead to activation. Acknowledgements The rainbow trout stomachs were made available by the Center for Great Lakes Studies. This work was supported by USPHS grant AM-09827. REFERENCES
AL-JANAB1J., HARTSUCKJ. A. & TANG J. (1972) Kinetics and mechanism of pepsinogen activation. J, biol. Chem. 247, 4628~,632. CASEY E. J. & LAIDLER K. J. (1950) Molecular kinetics of pepsin-catalyzed reactions. J. Am. chem. Soc. 72, 2159-2164. CHOW R. B. & KASSELCB. (1968) Bovine pepsinogen and pepsin I. Isolation, purification and some properties of the pepsinogen. J. biol. Chem. 243, 1718 1824. CRESTFIELD A. M., MOORE S. & STEIN W. H. (1963) The preparation and enzymatic hydrolysis of reduced and S-carboxymethylated proteins. J. biol. Chem. 238, 622 627. FICK A. & MURISIER D. 0874) Uber das Magenferment kaltblutiger Tiere. Jber. Fortschr. Tierchem. 3, 162-163. FOLTMANN B. (1981) Gastric proteinases--structure, function, evolution and mechanism of action. Essays BiGchem. 17, 52 84. HOLBROOK I. B. & LEAVER A. G. (1976) A procedure to increase the sensitivity of staining by Coomassie brilliant blue G250-perchloric acid solution. Analyt. Biochem. 75, 634-636. HYKES O. V., MAZANELJ. S. • SZECSENYIL. (1934) Contribution a la connaissance des ferments digestifs des poissons. C.r. Soc. Biol. ll7, 166 168. LOWRY O. H., ROSEBROUGHN. J., FARR A. L. & RANDALL R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265 275. McPHIE P. (1976) A turbidimetric milk-clotting assay. Analyt. Biochem. 73, 258-261. NEVALDINEB. & KASSELLB. (1971) Bovine pepsinogen and pepsin IV. A new method of purification of the pepsin. Biochim. biophys. Acta 250, 207-209. NORRIS E. R. & ELAM D. W. (1940) Preparation and properties of crystalline salmon pepsin. J. biol. Chem. 134, 153 157. TAGGERT T., MILLER R. B., KARN R. C., TRIBBLE J. A.. CRAFT M., RIPBERGERJ. & MERRITTA. D. (1978) Vertical thin layer slab polyacrylamide get electrophoresis of selected human polymorphic proteins. In Electrophoresis '78 (Edited by CATSIMPOOLASN.), pp. 231-242. Elsevier, Amsterdam.