Purification and characterization of two chymotrypsin-like proteases from the pyloric caeca of rainbow trout (Oncorhynchus mykiss)

Comp. Biochem. PhysioLVol. 101B,No. 1/2, pp. 24%253, 1992

0305-0491/92 $5.00+ 0.00 © 1992PergamonPresspie

Printed in Great Britain

PURIFICATION A N D CHARACTERIZATION OF TWO CHYMOTRYPSIN-LIKE PROTEASES FROM THE PYLORIC CAECA OF RAINBOW TROUT

(ONCORHYNCHUS MYKISS) MAGNUS M. KRISTJ~NSSON* and HENRIK H. NIELSEN Marine Biotechnology Center, Technological Laboratory, Ministry of Fisheries, Technical University of Denmark, Lyngby, Denmark

(Received 14 May 1991) Abstract--1. Two chymotrypsins, called chymotrypsin I and II, were purified from the pyloric caeca of rainbow trout, by (NH 4)2SO4 fractionation, hydrophobic interaction chromatography (phenyl-Sepharose) and ion-exchange chromatography (DEAE-Sepharose). 2. The approximate molecular weights of chymotrypsin I and II were 28,200 (+ 1200) and 28,800 (+900), respectively, as determined by SDS-PAGE and their isoelectric points were about 5. 3. The pH optima of the enzymes were centered around nine, when assayed for succinyl-L-Ala-L-Aia-LPro-L-Phe-p-nitroanilide (Suc-AAPF-NA) as substrate and both enzymes were unstable at pH values below 5. 4. The amidase activity of both enzymes increased with temperature up to about 55°C. Chymotrypsin I was found to be more heat stable than ehymotrypsin II, an effect most likely explained by stronger calcium binding of the former. 5. The trout chymotrypsins were significantly more active than bovine a-chymotrypsin when assayed against Suc-AAPF-NA at 25°C and casein at low temperatures (10-20°C), indicating an adaptation of the activities of the trout chymotrypsins to the habitation temperatures of the fish.

INTRODUCTION About 95% of the volume of the ocean is colder than 5°C (Hultin, 1980). F o r poikilothermic organisms, such as fish, an evolutionary adaptation of their enzymes was thus required for them to be able to maintain full physiological activities in their cold habitats. Enzymes from cold-adapted fish thus often have higher catalytic activities at low temperatures than their counterparts from warmblooded animals (Hultin, 1980; Hochaehka and Somero, 1984; Simpson and Haard, 1987). The structural aspects of cold adaptation of proteins are poorly understood. It is clear, however, that the chemistry of the reactions catalysed by a given enzyme, whether its source is a cold- or warm-adapted organism, is the same, but that the catalytic efficiency of the enzymes differ at the different temperatures (Hochachka and Somero, 1984). Higher catalytic efficiency of cold-adapted enzymes has been associated with higher molecular flexibility that would allow conformational changes to occur during catalysis requiring less energy input (Hultin, 1980; Hochachka and Somero, 1984). Most studies so far on cold adaptation of fish enzymes have focused on intracellular metabolic enzymes. Among extracellular enzymes, trypsin-like proteases have received most attention and have been isolated from several fish species. Much less information is available on fish chymotrypsins. Chymotrypsin or *Author to whom correspondence should be addressed. Present address: Science Institute, University of Iceland, Dunhagi 3, Reykjavik, IS-107, Iceland.

chymotrypsin-like enzymes have so far been isolated from dogfish (Prahl and Neurath, 1966; Racicot and Hultin, 1987), carp (Cohen et al., 1981a,b) and the Atlantic cod (Raae and Walther, 1989; Asgeirsson and Bjarnason, 1991). While many properties of the enzymes have been found to be similar to those of the better characterized bovine and porcine chymotrypsins, some important differences have been observed, such as in the polypeptide chain composition (Cohen et al., 1981a; Raae and Walther, 1989; Asgeirsson and Bjarnason, 1991). The fish enzymes also generally exhibit higher catalytic activities than the bovine enzyme (Cohen et al., 1981a; Ramakdshna et al., 1987a,b; Racicot and Hultin, 1987; Raae, 1990). In this report we present data on the isolation and characterization of two chymotrypsin-like proteases from the pyloric caeca of rainbow trout (Oncorhynchus mykiss) and compare some of its catalytic properties with those of bovine ~t-chymotrypsin. MATERIALS AND METHODS

Materials Rainbow trout viscera was obtained from a local fish farm, frozen, and kept at -30°C until use. Succinyl-L-AlaL-Ala-L-Pro-L-Phe-p-nitroanilide (Suc-AAPF-NA), phenylmethanesulphonyl fluoride (PMSF), tosyl-L-phenylalanine chloromethylketone (TPCK), soybean trypsin inhibitor (SBTI), Bowman-Birk inhibitor, aprotinin, chymostatin, bovine casein and bovine chymotrypsin (type II), were all purchased from Sigma Chemical Co., St Louis, MO. Phenyl-Sepharose CL-4B, DEAE-Sepharose Fast Flow, sodium dodecyl sulphate (SDS), N,N,N'N'-tetramethyl

247

248

MAGNUS M. KRISTJ~,NSSONand HENRIK H. NIELSEN

ethylenediamide (TEMED), acrylamide, N,N'-methylenebisacrylamide, ammoniumpersulphate, Coomassie Brilliant Blue R-250, electrophoresis low mol. wt calibration kit, Ampholine PAG-plates (pH 3.5-9.5) and isoelectric point markers (pI 2.4-5.65 and 4.7-10.6), were obtained from Pharmacia-LKB, Uppsala, Sweden. All other reagents used were of analytical grade.

Laemrrdi (1970). Gels were stained with Coomassie Brilliant Blue R-250. For SDS--PAGE analysis the proteases were first inactivated with PMSF, as described by Weber and Osbom (1975). Isoelectric focusing was carried out at 4°C in horizontal precast thin layer polyacrylamide gels, Ampholine PAG-plates (Pharmacia-LKB), containing Ampholine for the pH range 3.5-9.5.

Purification procedure Pyloric caeca, dissected from other parts of the viscera, were homogenized and extracted with 5 vols of 25 mM Tris-HC1 buffer, pH 8.1, containing 0.3 M NaC1 and stirred for 3 hr. The extract was centrifuged for 30 min at 19,600g and the supernatant adjusted to pH 7 and CaC1z added to a final concentration of 20mM. The mixture was stirred for 18 hr, then allowed to stand for 2 hr and finally centrifuged at 19,600g for 30 min (Asgeirsson et al., 1989). The supernatant was fractionated by (NH4)2SO 4 and the precipitate that formed at 30-70% saturation of (NH4) 2SO4 was collected and redissolved in a minimum volume of 25 mM Tris-HC1, pH 7.7, made one molar in (NH4)2SO 4 and applied to a phenyl-Sepharose CL-4B column (2.5 cm × 12 cm). The column was first eluted with the same buffer, until the non-binding proteins had passed through the column, followed by the buffer without (NH4)2 SO4, that among other proteins, eluted the trypsin activity from the column. The column was finally eluted with the original buffer containing 50% ethyleneglycol. This fraction, which contained all the chymotrypsin activity, was collected, dialysed against 25 mM Tris-HCl, pH 7.7, and applied to a DEAE-Sepharose Fast Flow column (1.5 x 25 cm), equilibrated in the same buffer and eluted with a linear NaCI gradient (0-0.15M). On this column the chymotrypsin activity was separated into two peaks that were collected, dialysed against 10mM Tris-HC1, pH 7.7, containing 10mM CaC12, concentrated by ultrafiltration and finally frozen in liquid nitrogen and stored at -80°C until use. All steps in the purification were carried out at 5°C and the reported pHs are those of the buffers at that temperature.

Effects of p H on activity and stability The effect of pH on the activity of the trout chymotrypsins was determined by measuring initial rates against Suc-AAPF-NA, dissolved in either Universal buffer (Dawson et al., 1969) (pH 4-9) or 50 mM glycine/NaOH buffer (pH 8.5-10.6), at 25°C. The pH stability of the enzymes was determined by measuring activity before and after incubation for 30 min at 30°C in the following buffers (all at 0.1 M): Na-acetate (pH 4.1-5.5); bis-Tris (pH 5.8-6.8); bis-Tris-propane (pH 6.4-7.0); Tris-HC1 (pH 7.4-8.5) and glycine/NaOH (pH 8.7-9.9). All buffers also contained 10 mM CaC12.

Activity measurements The amidase activity of chymotrypsin was assayed using Suc-AAPF-NA as a substrate (DelMar et al., 1979). The assays were typically carried out at 25°C with 0.3 mM Suc-AAPF-NA dissolved in 0.1 M Tris-HC1, pH 7.8, containing 10mM CaC12 and the release of p-nitroaniline was followed by the increase in absorbance at 410nm (E410= 8480 M-1 cm-1 ) using a Shimadzu u.v. 160 A recording spectrophotometer (Shimadzu Corp., Kyoto, Japan). The Michaelis-Menten kinetic parameters for amidase activity of the trout and bovine chymotrypsins at 25°C were determined from Lineweaver-Burk plots at six substrate concentrations in the range 0.035-0.5 mM Suc-AAPF-NA. The caseinolytic activity of the enzymes at 10-20°C was determined with a Radiometer pH stat [consisting ofa PHM 82 pH meter, TTT 80 titrator and ABU 80 autoburette (Radiometer, Copenhagen, Denmark)]. In a typical assay the pH of the casein solution (5 ml of 18 mg/ml in 5 mM CaC12) was adjusted to pH 8.0 with 0.1 N NaOH and equilibrated at the desired temperature. To this was added 50 ~1 enzyme solution (approximately 0.5 mg/ml dissolved in 5 mM CaC12) to start the reaction. The time course of hydrolysis was monitored by measuring the amount of 0.01 N NaOH required to maintain the pH at 8.0. Activity was determined from initial rates of hydrolysis as micromoles NaOH added per minute per milligram enzyme in assay. Electrophoresis SDS-Polyacrylamide gel electrophoresis (SDS-PAGE) were carried out using a discontinuous buffer system in 12.5% (w/v) vertical slab gels, according to the procedure of

Effects of temperature on activity and stability To determine the temperature dependency of the chymotrypsin activity, the standard Suc-AAPF-NA assay mixture was equilibrated at selected temperatures between 10 and 70°C, before adding the enzyme and determining initial rates. The pH of the buffer was adjusted such that it was 7.8 at each temperature tested. Thermal stability profiles of the enzyme were determined by measuring remaining activity after incubating aliquots of the enzymes dissolved in 0.1 M Tris-HC1 and 10 mM CaClz, pH 8.0, for 30min in sealed tubes at selected temperatures between 20 and 65°C. The effect of CaCI2 on stability was determined by heating aliquots of the enzymes, dissolved in 0.1 M Tris-HC1, pH 8.0, containing 1 mM EDTA and with or without added 15mM CaCI z, at 50°C. After a specific heating time the samples were cooled on ice and assayed for remaining activity. The effect of CaC12 concentration was determined under the same conditions, except CaC12 concentrations were varied from 0 to 15 mM. Heating was for 30 min at 50°C and activity was measured before and after heating to determine residual activity at each calcium concentration. Protein determinations All protein estimations were done according to Lowry et al. (1951) with bovine serum albumin as a standard.

RESULTS Purification The results of the purification of chymotrypsin from the pyloric caeca of rainbow trout are summarized in Table 1. The overall recovery of activity was about 45%, divided approximately equally between two peaks separated on the D E A E - S e p h a r o s e column (Fig. 1). The specific activity (amidase activity) of the enzyme in peak II (chymotrypsin II) was about 1.5-fold higher than that in peak I (chymotrypsin I). Both enzymes migrated as single major bands on SDS-polyacrylamide gel electrophoresis, according to estimated mol. wt of 28,200 ( + 1200) for enzyme I and 28,800 (+900) for enzyme II (Fig. 2). The electrophoretic behaviour of the enzymes was not affected by treatment with mercaptoethanol, suggesting that the trout chymotrypsins are composed of single polypeptide chains. Both enzymes also migrated as single major bands on isoelectric focusing, with approximately the same pI of about 4.9-5.0 (data not shown).

Chymotrypsin-like proteases

249

Table 1. Purification of chymotrypsin from the pyloric caeca of rainbow trout Protein Total activity Specific activity R e c o v e r y Purification Step (rag) (gmol min-1) (#tool min-1 mg l) (%) (-fold) Crude extract 4380 2792 0.64 100 1 (NH4)2SO4 (30-70%) 859 2710 3.2 97.1 5.0 PhenyI-Sepharose Peak C 98 1810 18.5 64.8 28.9 DEAE-Sepharose Peak I 20 605 30.3 21.7 47.3 Peak II 14 641 45.8 23.0 71.6

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Fig. 1. Purification of chymotrypsin I and II from rainbow trout on DEAE-Sepharose Fast Flow. The column was eluted with a linear gradient of 0-0.15 M NaC1 in 25 mM Tris-HC1 pH 7.7. Fraction volume was 6.6ml. Symbols: absorbance at 280nm ( - ) ; Suc-AAPF-NA activity (O), NaC1 concentration (O).

Inhibitors Both trout enzymes were fully inhibited by the serine protease inhibitor, P M S F , and their activity was significantly decreased after treatment with T P C K , a specific inhibitor of chymotrypsin (Table 2).

1

2

A striking difference was observed in the effect of aprotinin on the activity of the enzymes. While about 86% of the activity of enzyme I was inhibited, the activity of enzyme II was apparently increased in the presence of this inhibitor. Both enzymes were inhibited by soybean trypsin inhibitor, as well as by Bowman-Birk inhibitor and the chymotrypsin specific inhibitor, chymostatin, completely inactivated the enzymes, suggesting their identity as true chymotrypsins.

Effects of pH on activity and stability The amidase activity of the trout chymotrypsins increased with increasing pH, from p H 4 up to about p H 9 (Fig. 3). The increase in activity in this p H range seems to be associated with ionization of a group in the proteins with an apparent p K of about 6.5-7. On the alkaline side of the p H optimum the two enzymes show different behaviour in that chymotrypsin I retains more of its relative activity at higher p H values. Both enzymes were stable in the p H range 5-10 when incubated for 3 0 m in 30°C (data not shown), thus this difference in activity of the enzymes at pHs above nine cannot be attributed to differences in their p H stability. Both enzymes were unstable at acidic pHs ( < p H 5).

Effects of temperature on activity and stability The activity of the trout chymotrypsins increased with temperature up to about 55°C, when assayed

3

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Fig. 2. SDS-PAGE of chymotrypsin I and II from rainbow trout. Samples were prepared either in the presence (lanes 2 and 3) or absence (lanes 4 and 5) of mercaptoethanol in the sample buffer. Lane 1: mol. wt markers. Lanes 2 and 4: chymotrypsin I. Lanes 3 and 5: chymotrypsin II. Indicated on figure are mol. wts of protein standards.

250

MAGNUSM. KRISTJANSSONand HENRIKH. NIELSEN Table 2. Effect of inhibitors on the activity of chymotrypsin-likeproteases from rainbow trout Residual activity (%) Inhibitor Concentration* Chymotrypsin I ChymotrypsinII PMSF I mM 0 0 TPCK 1 mM 38.8 49.7 Aprotinin 100#g/ml 14.1 109.5 SBTI 100/zg/ml 1.3 0.4 Bowman-Birk inhibitor 100#g/ml 0.7 0.0 Chymostatin 100# g/ml 0 0 *Incubationwas for 30min at 30°C.

100

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8

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Fig. 3. Effect of pH on the activity of chymotrypsin I (O) and II (©) against Suc-AAPF-NA at 25°C. against S u c - A A P F - N A at pH 7.8 (Fig. 4). At temperatures above the optimum, the activity of enzyme II decreased at a faster rate than that of enzyme I, suggesting different stabilities of the two enzymes. The thermal stability profile of the enzymes confirmed this, showing chymotrypsin I to be

significantly more stable at temperatures above 40°C, when heated for 30 min at pH 8.0 (Fig. 5). Thermal stabilities of both enzymes were highly calcium dependent (Fig. 6a). When their stability at 50°C was measured in the presence of 15 m M CaCI2, chymotrypsin I displayed higher stability than enzyme II. In the absence of calcium, on the other hand, enzyme I was found to be less stable than enzyme II. These results suggest that the difference in thermal stability between the two trout chymotrypsins might be explained by their different binding affinities for calcium. This was supported by results showing that enzyme I reached maximum stability at 50°C, at significantly lower CaCI2 concentrations than enzyme II (Fig. 6b), suggesting stronger calcium binding by the former. As in the previous experiment, however, enzyme II showed higher thermal stability in the absence of CaC12.

Comparison of catalytic activity of bovine and trout chymotrypsins The Michaelis-Menten kinetic parameters for the amidase activity of bovine c~-chymotrypsin and cbymotrypsin I and II from trout at 25°C are listed in Table 3. Both trout enzymes were found to have lower Km values than the bovine enzyme and their turnover numbers (kca t) were also higher by factors of 2.6 and 3 for chymotrypsins I and II, respectively. In terms of catalytic efficiency (kcat/gm) , chymotrypsin I

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30 50 70 Temperature (°C) Fig. 4. Effect of temperature on the amidase activity of chymotrypsin I (D) and II (O) from rainbow trout. Activity was measured in the standard amidase assay, equilibrated at each temperature.

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Temperature (°C)

Fig. 5. Thermal stability of chymotrypsin I (O) and II (O) from trout at pH 8.0. Aliquots of the enzymes in 0.1 M Tris--HC1, pH 8.0, containing 10 mM CaCI 2, were heated for 30 min at the appropriate temperature before assaying for remaining amidase activity in the standard assay.

Chymotrypsin-like proteases (a)

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DISCUSSION

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CaCl2 added (mM) Fig. 6. Effect of calcium on thermal stability of trout chymotrypsins at 50°C. (a) Time dependence of thermal inactivation of enzyme I (A, A) and II (@, (3) in the absence (open symbols) and presence (closed symbols) of 15 mM CaC12. (b) Effect of CaCI2 concentration on thermal stability of chymotrypsin I (@) and II ((3). Heating was for 30 min at 50°C. See Methods for experimental details. was therefore about 4 times and chymotrypsin II about 6.8 times as active as bovine g-chymotrypsin in hydrolysing Suc-AAPF-NA at 25°C and pH 7.8. Comparison between the trout chymotrypsins shows that the Km value for enzyme II was about 1.5 times lower than that for enzyme I. This corresponds approximately to, and may explain, the difference in specific activities of the two enzymes against this substrate (Table 1). The trout chymotrypsins were also significantly more active than their bovine counterpart, when assayed against casein at 10, 15 and 20°C, at pH 8.0 (Fig. 7). Under these conditions, the caseinolytic activities of chymotrypsin I and II were about 3 and 3.3 times higher than that of bovine chymotrypsin. In all cases the difference in caseinolytic activity of the enzymes corresponded approximately to the differences in their turnover numbers against the amide substrate at 25°C. Table 3. Kineticparametersfor the amidaseactivityof ehymotrypsin I and II from rainbowtrout againstSue-AAPF-NA,comparedwith those of bovine ,,-chymotrypsinat 25°C and pH 7.8 Chymotrypsin Trout enzyme I Trout, enzymeII Bovine

'

Fig. 7. Comparison of the caseinolytic activities of bovine ~-chymotrypsin (open bars) and chymotrypsin I (dark bars) and II (shaded bars) from trout at low temperatures. Activity is expressed as micromoles of OH- consumed per min per milligram enzyme at each temperature at pH 8.0, measured with a pH stat.

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Two anionic chymotrypsin-like proteases were isolated from the pyloric caeca of rainbow trout. Treatment of the enzymes with mercaptoethanol did not affect their eleetrophoretic behaviour on SDS-PAGE, suggesting that both chymotrypsin I and II consist of single polypeptide chains. This is in contrast with bovine ~-chymotrypsin, which contains three polypeptide chains linked together by disulphide bonds (Blow, 1971). Similar results to those reported here have also been observed for the chymotrypsins from carp (Cohen et al., 1981a) and Atlantic cod (Raae and Walther, 1989; Asgeirsson and Bjarnason, 1991). For the carp enzyme the possible existence of a second small chain was not excluded, but suggested that the activated enzyme remained in either the n or the 6 form (Cohen et al., 1981a). Similar conclusions were reached for the cod enzyme (Asgeirsson and Bjarnason, 1991). Chymotrypsins I and II from trout have similar mol. wts of approximately 28,200 ( _ 1200) and 28,800 (+900), respectively, as determined by SDS-PAGE. When chymotrypsin I was not previously inhibited with PMSF, however, it migrated as a somewhat smaller (by 1000-2000 mol. wt) protein, suggesting that during sample preparation for electrophoresis the active enzyme may undergo a partial autolytic cleavage. Both chymotrypsin I and II were inhibited by standard inhibitors of the serine proteases and chymotrypsin. A marked difference was observed in the behaviour of the enzymes towards aprotinin. The inhibitor caused about 86% loss of activity of enzyme I, but apparently increased the activity of enzyme II. The reason for this difference in behaviour is not known, but as these inhibitors bind to the active sites of the proteases they inhibit, these results suggest that some structural differences may exist in the active site regions of the two chymotrypsin forms. Aprotinin also had a different inhibitory activity against cod

252

MAGNUSM. KRISTJ.~.NSSONand HENRIKH. NELSEN

and bovine chymotrypsins, in that the fish enzyme was less sensitive to inhibition by the inhibitor than the bovine enzyme (Asgeirsson and Bjarnason, 1991). Like the trout chymotrypsins, the cod enzyme was inhibited by soybean trypsin inhibitor, whereas the activity of the bovine ~t-chymotrypsin was not affected by this inhibitor (Asgeirsson and Bjarnason, 1991). Both chymotrypsin I and II have a pH optimum centred around pH 9, when assayed against SucAAPF-NA. On the acidic side of the optimum the increase in activity appears to coincide with ionization of a group in the protein with an apparent pKa of about 6.5-7, which most probably is the active site histidine residue, common to all serine proteases (Hess, 1971). The activity of bovine ~-chymotrypsin is also dependent on an amino group with a pKa of 8.5, which has been assigned to the free ct-NH; group of Ile-16, which forms a saltbridge to Asp-194, required for structural integrity of the active site of the enzyme (Hess, 1971). The trout chymotrypsins differ from the bovine enzyme in that they remain active at more alkaline pHs ( p K > 10.5). Higher activity at alkaline pHs was also reported for chymotrypsin from carp, where the alkaline activity was associated with a pK of 10.16 (Cohen et al., 1981b). This suggests that other groups may be involved in stabilizing the active site conformation of the fish and the bovine chymotrypsins. It is of interest that in the chymotrypsin-like protease B from Streptomyces griseus, which is a single chain protease, the carboxylate group of Asp-194 forms a saltbridge with Arg-169 (Delbaere et al., 1975), but the pH dependence was associated with a pK of 10.84 that was assigned to the E-amino-group of lysine 125, which forms a saltbridge to the ct-carboxyl-groups of the C-terminal Tyr-242 (Delbaere et al., 1975; Shinar and Gertler, 1979). The trout chymotrypsins were stable at pH 5-10 at 30°C, for at least 30 min, but were unstable at pHs below 5. Such instability at acidic pHs was also reported for the chymotrypsins from carp (Cohen et al., 1981a) and the Atlantic cod (Raae and Walther, 1989; Asgeirsson and Bjarnason, 1991). The activity of the trout chymotrypsins increased to a similar extent with temperature up to about 55°C. At higher temperatures, the enzymes were inactivated, but chymotrypsin II at a faster rate, due to its lower thermal stability. Thermal stability of both enzymes was highly dependent on the presence of calcium ions, but different in that enzyme I was more heat-stable than enzyme II in the presence of calcium, whereas the opposite was observed when the enzymes were incubated without calcium (Fig. 6A). The stabilizing effect of CaCI: on enzyme I was also exerted at significantly lower concentrations than on enzyme II (Fig. 6B), suggesting that chymotrypsin I has a stronger calcium binding affinity than chymotrypsin II. Stronger calcium binding thus probably explains the higher thermal stability of enzyme I as compared with enzyme II. Bovine chymotrypsins A and B and their zymogens bind calcium at a single binding site that stabilizes the conformation of these enzymes (Delaage et aL, 1968; Abita and Lazdunski, 1969; Chiancone et al., 1985). This binding site, situated in a loop comprising residues 70-80, appears to be a

common feature of the pancreatic serine proteases (Bode and Schwager, 1975) and it is not unreasonable to assume its presence in the trout enzymes. The different calcium-binding properties of the two chymotrypsins from trout, therefore suggest that some structural differences may exist in or around the calcium binding loops of these enzymes. It is noteworthy that despite close structural similarities, the effect of calcium binding to chymotrypsinogen A and B was found to be different, in that calcium stabilized chymotrypsinogen B less against urea denaturation than it did chymotrypsinogen A (Delaage et al., 1968). The catalytic efficiencies (keat/gm) of the trout chymotrypsins against Suc-AAPF-NA at 25°C, were significantly higher than that of bovine ~t-chymotrypsin. The higher activity of the trout enzymes resulted from both lower Km values, and higher turnover numbers (kcat) (Table 3). The trout chymotrypsins also exhibited about 3-fold higher activity against casein than the bovine enzyme. The relative differences in the caseinolytic activity between the three enzymes corresponded approximately to the differences in the kcat values for their amidase activities. Such correlation has also been found between the amidase and caseinolytic activities of trout and bovine trypsins (Kristj~insson, 1991). It appears therefore that kcat values obtained for the amidase reaction of those proteases, predict well their relative proteolytic activities, when assayed under substrate saturation conditions, as in the casein assays described in this study. Higher catalytic efficiency, in comparison with the bovine enzyme, has also been reported for carp chymotrypsin when assayed against several amide and ester substrates at 30°C (Cohen et al., 1981 b). Cod chymotrypsin was also reported to have a higher activity than bovine chymotrypsin when assayed against benzoyl tyrosine ethyl ester (3-15°C) (Raae, 1990). Asgeirsson and Bjarnason (1991) measured the kinetic parameters for both the esterolytic and amidolytic activites of the two chymotrypsins isolated from Atlantic cod at 10, 25 and 35°C as well as for the bovine enzyme. The kcat values for the cod enzymes were typically found to be two to three times higher than that of bovine chymotrypsin and their catalytic efficiencies were about 2-5-fold higher (Asgeirsson and Bjarnason, 1991). Dogfish chymotrypsin has also been found to hydrolyse several protein substrates, including casein, at a higher rate than its bovine counterpart (Ramakrishna et al., 1987a). In hydrolysing various p-nitrophenylesters of fatty acids, the major difference between the dogfish and bovine enzymes was 2-3 fold higher keat values of the fish enzyme (Ramakrishna et al., 1987b). The higher catalytic activity observed for the trout enzymes, as well as the other fish chymotrypsins mentioned, reflects an adaption of these enzymes to maintain high activities at the low temperatures of their habitats. This adaptation of catalytic efficiencies may be achieved by either lowering the Km or by raising keat values, or both parameters may be changed. The structural basis for this temperature adaption is not understood, but it may be that these enzymes have developed protein structures less rigid than their counterparts from warmblooded animals, which would allow them to maintain the

Chymotrypsin-like proteases conformational flexibility required to maintain efficient catalysis at low temperatures. In return for more flexible protein structures, their conformational stability at higher temperatures may be diminished, which is a frequent observation with enzymes from cold-adapted organisms.

Acknowledgements--The authors would like to thank Mrs Hanne Jacobsen and Mrs Karin H. Reimers for excellent technical assistance in parts of this study. This work was supported by the Danish Biotechnological Research and Development Program.

REFERENCES

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