Purification and characterization of elastase from the pyloric caeca of rainbow trout (Oncorhynchus mykiss)

Comp. Biochem.Physiol.Vol. 106B,No. 2, pp. 331-336, 1993 Printed in Great Britain

0305-0491/93$6.00+ 0.00 © 1993PergamonPress Ltd

PURIFICATION A N D CHARACTERIZATION OF ELASTASE FROM THE PYLORIC CAECA OF RAINBOW TROUT (ONCORHYNCHUS MYKISS) M. BASSOMPIERRE,H. H. NIELSEN* and T. BORRESEN Technological Laboratory, Ministry of Fisheries, Technical University of Denmark, Building 221, 2800 Lyngby, Denmark (Fax 45 4288-4774)

(Received 12 February 1993; accepted 19 March 1993) Absa'aet--1. An elastase-like enzyme was purified from the pyloric caeca of rainbow trout by hydrophobic interaction, cation exchange and gel-filtration chromatography. 2. The approximate molecular weight of the elastase was 27 k_Da and the isoelectric point was remarkably basic. 3. The pH optimum of this enzyme was 8.0, when assayed with Suceinyl-Ala-Ala-Ala-p-Nitroanilide. 4. When assayed with Suecinyl-Ala-Ala-Ala-p-Nitroanilide, the enzyme activity had a temperature optimum of 45°C, and the enzyme was stable up to this temperature. 5. The trout elastase exhibited a higher specific activity than porcine elastase against Succinyl-Ala-Ala-Ala-p-Nitroanilide and elastin-orcein. 6. The trout elastase was inhibited by elastatinal, PMSF, TPCK, SBTI and Bowman-Birk inhibitor.

The aim of the present work was the study of a third serine protease, an elastase-like enzyme, purified from the pyloric caeca of Oncorhynchus mykiss. In this study we present data from the isolation and characterization of the elastase-like proteinase, and compare some of its catalytic properties with data from literature.

INTRODUCTION

Elastase is known to digest a large variety of protein substrates, but differs from the other serine proteases by its ability to digest elastin, as well as substrates with uncharged, non-aromatic side-chains (Lewis et al., 1956). Among the elastase-like proteases reported in the literature, these enzymes appear to be relatively small, 24-28 kDa, consisting of a single polypeptide chain, and most of them have a basic pI. Inhibition by PMSF and by elastatinal proves the affiliation, respectively, to the serine-protease family (EC 3.4.21.-) and to the elastase family. As expected, they have a special ability to digest elastin and other substrates with uncharged non-aromatic sidechains. Elastase-like activity has been found in extracts from several fish species: gummy shark, red stingray, rainbow trout, carp, catfish, eel, bluefin tuna, yellowtail, sea bass, angler (Yoshinaka et al., 1985). Various forms of purification and characterization of elastase have been reported for African lungfish (De Ha6n and G-ertler, 1974), carp (Cohen et aL, 1981a,b) catfish (Yoshinaka et al., 1982, 1983) Dover sole (Clark et al., 1985), and Atlantic cod (Raae and Walther, 1989; Gildberg and Overbe, 1990). The elastase isolated from Atlantic cod appears to be adapted to low temperatures (Gildberg and Overbo, 1990). This has previously also been reported to be the case for the digestive enzymes trypsin and chymotrypsin from rainbow trout (Oncorhynchus mykiss (Kristj/msson, 1991; Kristj/msson and Nielsen, 1992).

MATERIALSAND METHODS

Materials Rainbow trout viscera were obtained from a local fish farm in August 1990, frozen, and kept at - 4 0 ° C until use. Succinyl-L-Ala-L-Ala-L-Ala-p-nitroanilide (Suc-AAA-pNA), elastin-orcein, elastase from porcine pancreas (type IV), elastatinal, leupeptin, aprotinin, benzamidine, soybean trypsin inhibitor (SBTI), Bowman-Birk inhibitor, Suc-AAA-pNA, phenylmethanesulphonyl fluoride (PMSF), N-tosylL-phenylalanine chloromethyl ketone (TPCK), N-tosyl-L-lysyl-chloromethyl ketone (TLCK), trizma base, trizma-HC1, monoethanolamine were all purchased from Sigma Chemical Co. (St Louis, MO). Phenyl-Sepharose CL4B, CM-Sepharose Fast-Flow and Sephacryl high-resolution gel (S-200 HR), were obtained from Pharmacia LKB (Uppsala, Sweden). All other chemicals were of analytical grade. Purification procedure One hundred grams of pyloric caeca were dissected from the viscera, homogenized, and extracted with 6 vol weight of 50 mM Tris-HCl pH 7.5, containing 0.2 M NaCI and stirred for 3 hr. The extract was centrifuged at 16,300 g for 20 min. The supernatant was filtered and CaC12 added to a final concentration

*To whom all correspondence should be addressed 331

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M. BASSOMPIERREet al.

of 0.02 M. This solution was stirred for 16 hr and then clarified with tangential flow filtration on a Minitan unit (Millipore), using Durapore membranes (0.2 g m cut-off). The concentrated fraction was centrifuged at 17,761 g for 30 rain, and the supernatant added to the previously filtered fraction. This pool was concentrated down to 50 ml on Minitan with 10 kDa cut-off membranes. The crude extract was then applied to a phenyl-Sepharose CL4B column (Pharmacia XK50, 50 x 100 mm) and eluted at first with I M (NH4)2SO4, 25raM Tris-HC1, 10raM CaC12, pH 7.5, until the unbounded proteins had passed through the column. The column was then eluted with 25 mM Tris-HCl, 10 mM CaC12, pH 7.5, which released the trypsin and the elastase activity. The fractions containing the elastase activity were pooled and concentrated by ultrafiltration on a 10kDa cut-off membrane (Amicon, Diaflo, PMI0). The concentrate was then applied to a C M Sepharose Fast-Flow column (Pharmacia XK26, 26 × 200 mm) equilibrated in 25 mM ethanolamine, 10mM CaCI2, pH9.5 and eluted with the same buffer, until the unbounded proteins had passed through the column. The elastase activity was then released by 30 mM NaCI in the same buffer, and the fractions containing the elastase activity were pooled and concentrated by ultrafiltration on 10 kDa cut-off membrane (Amicon, Diaflo, PMI0). This enzyme solution was applied to a Sephacryl S-200 HR column (Pharmacia XK 16, 16 × 600 mm), and eluted with 20mM ethanolamine, 10mM CaC12, 10mM NaC1, pH 9.5. The enzyme collected was kept in the ethanolamine buffer, frozen in liquid nitrogen and stored at - 8 0 ° C until further use. All steps of the purification were carried out at 4°C and the reported pH values are those of the buffers at that temperature.

Activity measurements Elastase amidase activity was assayed using SucAAA-pNA as a substrate (Bieth et al., 1974). The substrate was dissolved in DMSO and diluted in 100 mM Tris-HC1, 10 mM CaCI2, pH 8.0, to a final DMSO concentration about 0.4%. The assay was performed at 25°C, 950/~1 of 1.1 mM Suc-AAA-pNA was added to 50/~1 of enzyme solution for the assay. The release of p-nitroaniline was monitored by the increase of absorbance at 410nm over l min (e410= 8800 M.-lcm-l) using a Shimadzu UV-160A recording spectrophotometer (Shimadzu Corp., Kyoto). The activity was obtained in U / m l = 2.3585 x AA410m/min. The Michaelis-Menten parameters, Km and kcat at 25 and 10°C, pH 8.0, were estimated from Hanes' plot. Activity towards elastin-orcein was determined according to the method of Sachar (1955). To 20 mg elastin orcein were added 200/~1 enzyme solution and 1800#1 of a 100 mM Tris-HC1 buffer pH 8.8. The tubes were agitated for 2.5 hr at 37°C. The reaction was stopped by adding 2 ml of sodium phosphate

buffer (0.7M, pH6), and then centrifuged at 3000 rpm for 10 min. A standard curve was obtained by measuring on total digestion of elastin-orcein, the hydrolysed elastin was detected in the supernatant by measuring the optical density at 590 nm.

p H optimum determination The effect of pH on the activity of elastase towards Suc-AAA-pNA was determined using the following buffer: 200 mM acetate, 10 mM CaCI2, (pH 5.4--6.0), 50 mM bis-Tris Propane-HCl, 10 mM CaCI2 (pH 6.0 to 9.0) and 100mM ethanolamine, 10mM CaCI: (pH 9.0 to 11.4). The assay was carried out as described in Activity measurements. Temperature optimum determination The effect of temperature on the activity of elastase towards Suc-AAA-pNA was measured in the range of 10-65°C. The buffer, consisting of 100mM Tris-HCl and 10 mM CaCI2, was adjusted to pH 8.0 at each temperature. The other conditions were described in Activity measurements. Temperature stability determination The enzyme was preincubated for 30 min in 10 mM ethanolamine, 5 mM NaC1, 10 mM CaC12 at pH 9.5, at each different temperature (10-65°C) and kept 10 rain on ice, before measuring the activity towards Suc-AAA-pNA at 25°C as described in Activity measurements. Enzyme stored on ice was used as the 100% activity reference. Effect of inhibitors The enzyme was preincubated with the inhibitor in 10 mM ethanolamine, 5 mM NaCI, 10 mM CaCI2 at pH 9.5 and 30°C for 30min, then kept on ice for I min. The remaining activity towards Suc-AAApNA was measured at 30°C instead of 25°C. Enzyme preincubated without an inhibitor was used as reference. The other conditions were described in Activity measurements. Electrophoresis SDS-polyacrylamide gel electrophoresis (SDSPAGE) was carried out by using a discontinuous buffer system in 12.5% vertical slab gels, according to the procedure of Laemmli (1970). Gels were stained with silver nitrate (Blum et al., 1987). pI determination The isoelectric point was estimated by HPLC, using anion-exchange chromatography at pH 11.5 (Pharmacia Mono-Q, LKB), and by isoelectric focusing (Pharmacia Ampholine PAG-plates, pH 3.5-9.5). Protein determination All protein estimations were done according to the Lowry method (1951), with bovine serum albumin as standard.

Purification and characterization of elastase from the pyloric caeca of rainbow trout

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Table 1. Purificationof elastas¢from the pyloriccaeca of rainbow trout Total Protein, activity, Sp. act., Yield Purification, Purificationstep (rag) (units) (units/mg) (%) (facto0 Crude extract 1600 210 0.13 100 1 Phenyl-Sepharose 139 153 1.10 72.9 8.5 CM-Sepharose 25 95 3.80 45.2 29.2 SephacrylS-200 10 57 5.70 27.1 43.8

RESULTS

Effect of temperature on stability and activity

Purification and physical characteristics The results of the purification are summarized in Table 1. The final recovery was about 27%. The enzyme migrated as a single band on SDSpolyacrylamide gel electrophoresis (Fig. 1) positioned such that a molecular weight of 27,000 could be estimated. The electrophoretic behaviour of the enzyme was not affected by mercaptoethanol treatment, suggesting that the enzyme is composed of a single polypeptide chain. The position of the rainbow trout elastase was slightly changed by the absence of mercaptoethanol, resulting in a molecular weight of 25 kDa. The porcine elastase type IV showed more than one band; this could be due to isoenzymes or impurities. The isoelectric point was estimated to be higher than pH 11, based on the fact that the enzyme did not bind to the anion-exchange column when using an HPLC system at pH 11.5 with Na2HPO4 (Pharmacia Mono-Q, LKB). On isoelectric focusing with Ampholine PAG-plates (pH 3.5-9.5) the enzyme migrated to pH 9.5 and stopped at the cathode. No other bands could be detected (data not shown).

Effect of pH on activity at 25°C When assayed with Suc-AAA-pNA the activity increased from pH 5.4 to 8.0, then reach an optimum at 8.0 and decreased from 8.0 to 10. The increase of the amidase activity of the trout elastase from pH 6.0 to 8.0 (Fig. 2), seems to implicate the ionisation of a group in the active site with an apparent pKa about 7.

1

2

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The enzyme was stable during 30 min at pH 9.5 in the range of 10-45°C and the stability decreased drastically from 45 to 60°C (Fig. 3), when assayed against Suc-AAA-pNA at pH 8.0. The activity increased with temperature (Fig. 3), at first slowly, between 10 and 25°C, then rapidly until the protease lost its stability. The optimum temperature was reached at 45°C.

Comparison of catalytic activity of porcine and trout elastase The Michaeiis-Menten kinetic parameters, Km and kc= at 25 and 10°C, pH 8.0, for the amidase activity towards Suc-AAA-pNA, were estimated from Hanes' plot. Porcine eiastase and rainbow trout elastase had a Km of about 2 mM at 10 and 25°C, whereas the k~t values were three times higher at 25 than at 10°C for both enzymes (Table 2). Calculation of k~t/Km values thus indicated that rainbow trout elastase was about five times more efficient at both temperatures than porcine elastase (type IV) (Table 2). The trout enzyme appeared to be three times more efficient in digesting elastin-orcein than porcine elastase (type IV), when assayed at 37°C and pH 8.8 (Table 3).

Effect of inhibitors In order to determine the type of the purified enzyme, various proteinase inhibitors were used. An overview of their effects are summarized in Table 4. PMSF, a serine proteinase inhibitor, and elastatinal,

4

5 94.0 67.0 43.0 30.0 20.1 14.4

Fig. I. SDS-PAGE of elastase from rainbow trout and porcine elastase (type IV). Lane 1 and 2: elastase from porcine (type IV), with and without mereaptoethanol, respectively. Lane 3 and 4: elastase from rainbow trout, without and with mercaptoethanol, respectively. Lane 5: molecular weight markers. Indicated on the figure are molecular weights, in kDa, of protein standards.

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M. BASSOMPIERREet al. Table 2. Kineticparameters of the hydrolysisof Suc-AAA-pNAby trout elastase and porcineelastase Km k~at kcaJXm Enzyme (mM) (see-I) (sec-lmM-1) Rainbow trout elastase 10°C 2.0 63 31.5 25°C 2.2 184 83.6 Porcine elastase (type IV) 10°C 1.8 12 6.7 25°C 1.9 32 16.8 Enzymeswere assayed against1,0.5,0.25, 0.12mM Suc-AAA-pNA, at pH 8.0. Determinationof Kmand k=t was calculatedfrom Hanes' plot and linearregression.

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Fig. 2. Effect of pH on the activity of rainbow trout elastase against Suc-AAA-pNA. The assay was performed at 25°C. The substrate buffers used were 200 mM acetate, 10 mM CaC12 (1), 50 mM bis-Tris propane-HC1, 10 mM CaC12 (O), 100 mM ethanolamine, 10 mM CaC12 ( I ) .

must be considered with care, because this inhibitor seemed to be very unstable in aqueous solution. The 6.25% DMSO used to dissolve PMSF or TPCK appeared to be harmless to rainbow trout elastase, whereas 50% has previously been found to show an inhibitory effect on cod elastase (Gildberg and Overbo, 1990). DISCUSSION

a specific inhibitor of elastase, completely inhibited the trout enzyme confirming, respectively, the affiliation to the serine-proteinase family and to the elastase group. Benzamidine, leupeptin and TLCK, specific inhibitors of trypsin, did not have any effects. Aprotinin, a serine-protease inhibitor did not inhibit the trout enzyme, but a soybean trypsin inhibitor, such as Bowman-Birk inhibitor, did effect inhibition, as previously also described for cod elastase (Gildberg and Overbo, 1990) and catfish elastase (Yoshinaka et aL, 1982). Furthermore, TPCK, a specific inhibitor of chymotrypsin-like proteases, inhibited the trout enzyme activity. Partial TPCK inhibition has also been reported for cod elastase (Gildberg and Overbo, 1990). The results with TPCK

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An elastase-like protease was isolated from the pyloric caeca of rainbow trout. Treatment with mercaptoethanol did not reveal further bands on SDS-PAGE (Fig. 1), suggesting that the enzyme consists of one single polypeptide chain. The estimated molecular weight of 27 kDa and the very basic isoelectric point (pI > 11) of this trout enzyme, are in agreement with previous results concerning the elastase-like enzymes. Molecular weights of the purified elastases have been estimated by SDS-PAGE to be in the range of 24-28 kDa. Porcine, human, catfish, carp, and cod elastases show pI around 9.5. The elastase of Dover sole represents an exception by having a pI about 5.7 and a molecular weight of 19.5 kDa, which was determined by molecular sieve chromatography (Sephadex G-200) of intestine extracts (Clark et al., 1985). The pI of rainbow trout elastase was estimated to be higher than 11 by ion-exchange chromatography. By isoelectric focusing, the pI of the same enzyme was estimated to be 9.5 or higher. However, this system does not allow determination of pI-values higher than 9.5. In this connection, it is interesting to ask if the previously isolated elastases have a true pI of about 9.5, as isoelectric focusing has been the method generally used for pI determination. The following theory could be proposed to explain why a very high pI is useful for an elastase enzyme: most proteins have a pI lower than eight, thus being negatively loaded at pH 8, which is the actual pH in

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Fig. 3. Thermostability of rainbow trout elastase (~)-) at pH 9.5. The enzyme was kept in 10mM ethanolamine, pH 9.5 containing 10mM CaCIz, 5mM NaC1, and w a s preincubated for 30 min at different temperatures in the range 10--60°C.Remaining activity towards Suc-AAA-pNA was measured at 25°C, pH 8.0. Effect of temperature on activity of rainbow trout elastase (i-). Activity was assayed at different temperatures in the range 10-65°C, in 100 mM Tris-HCl, 10 mM CaCI2, at pH 8.0.

Table3. Digestionof elastin-oreeinby troutelastase and porcine elastase.

Elastin-orcein(mg) min-I nag enzyme Enzyme solution-! Rainbowtrout elastase 2.2 Porcine elastase (type IV) 0.6 Enzymes were assayed against 20mg elastin--orcein, at 37°Cat pH8.8, in 100mM Tris-HCl.

Purification and characterization of elastase from the pyloric caeca of rainbow trout Table4. Effectof inhibitorson the activityof an elastase-likeenzyme from rainbow trout Preineubation Assay Residual Inhibitor concentration concentration activity(%)

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In the kinetic study, the rainbow trout elastase expressed a five-times higher catalytic efficiency (kc~t/Km) than did porcine elastase (type IV) with the substrate, Suc-AAA-pNA, at 25 and 10°C (Table 2). PMSF* I mM 0.05 mM 0 The Km of both enzymes was the same at 10 and 25°C, Elastatinal 100#g/ml 5 #g/ml 0 50 #g/ml 2.5 #g/ml 0 about 2 mM. The k~t of both enzymes was three 10#g/ml 0.5 #g/ml 2.5 times higher at 25 than at 10°C; on the other hand, SBTI 100# g/ml 5/~g/ml 51 the increase in activity for rainbow trout elastase is Bowman-Birk I. 100#g/ml 5 #g/ml 83 TPCK* 5 mM 0.25 mM 33 remarkably small, being between 10 and 25°C TLCK I mM 0.05 mM 101 (Fig. 3). Those results are interesting because they are Benzamidine 5 mM 0.25 mM 96 Aprotinin 100/zg/ml 5 #g/ml 100 observed within the interval of habitat temperature of Leupeptin 100 #g/ml 5 #g/ml 99 the rainbow trout. Against elastin-orcein, too, the CaCI2 500 mM 35mM 91 rainbow trout elastase had a higher catalytic activity NaCI 500 mM 25 mM 91 DMSO 6.25% 0.7% 98 (Table 3). Similar results have been shown for cod *Preincubated with 6.25% DMSO. Preincubationswere performed elastase, known to have the highest specific activity at 30°C, pH 9.5, for 30 rain. Measurementof remainingactivity among purified fish elastases (Gildberg and Overbo, towards Suc-AAA-pNAwas carried out at 30°C at pH 8.0. 1990). Yet it is important to point out that the comparison should be considered with care due to the the intestine (Grabner, 1985). Proteinases with differences in assay conditions and, in particular, in higher pI than eight, would be positively loaded. A degree of purification of the enzymes studied. non-specific binding could then occur between a Like all other elastases, the trout enzyme is inhibpositively loaded protein, being the enzyme, and a ited by elastatinal, PMSF, and is not inhibited by negatively loaded protein, being the substrate, inhibitors like benzamidine, leupeptin, aprotinin, thus accommodating a more specific binding of the TLCK (Table 4). However, the rainbow trout elastase substrate also in the active site. If this hypothesis is has shown a broader inhibitor sensitivity than valid, it could explain the pI evolution of ser- porcine elastase. The inhibitions of rainbow trout ine-proteinases from acid (some fishes) to basic elastase, catfish elastase (Yoshinaka et al., 1985) and (mammals). cod elastase (Gildberg and Overb~, 1990) by soybean When assayed with Suc-AAA-pNA, the purified inhibitor (SBTI or Bowman-Birk) as well as the trout elastase had a pH optimum of 8.0 (Fig. 2), partial inhibition of rainbow trout elastase and cod which is the pH of the in vivo condition. Similar elastase (Gildberg and Overb~, 1990) by TPCK are in results were reported for most of the elastases charac- contrast to the insensitivity of porcine elastase toward terized (Cohen et al., 1980a,b; Yoshinaka et al., 1982; this type of inhibitor (Gildberg and ~verb~J, 1990; Clark et al., 1985). The pH-dependent inactivation Raae and Walther, 1989). It is known that some fish involved a group with a p K a between 6.5 and 7, serine-proteinases are capable of binding inhibitors suggesting the p K a of the whole charge-relay system not recognised by their mammalian homologues, but characteristic for the serine--proteinase family (Kraut, which are specific for other mammalian serine1977). proteinases (Yoshinaka et al., 1985; Gildberg and At pH 8.0 the rainbow trout elastase has an opti- I~verbo, 1990; A.sgeirsson and Bjarnason, 1991; mal activity at 45°C and at pH 9.5 this elastase was Kristjfinsson and Nielsen, 1992). found to be heat-stable up to 45°C (Fig. 3), which is The behaviour of the rainbow trout elastase lower than for porcine and catfish elastases, which towards SBTI and TPCK seems to point out a remain stable up to 60°C (Yoshinaka et al., 1982), but common evolution between trypsin, chymotrypsin higher than for cod elastase which was unstable over and elastase. In the African lungfish, a proelastase A 35°C (Gildberg and Overb~, 1990). Concurrently, has been found, combining elastase specificity with rainbow trout trypsin and chymotrypsin were found structural similarity of chymotrypsinogens (De Ha6n to follow the same heat stability profile as rainbow and Gertler, 1974). The rainbow trout could obey to trout elastase (Kristjfinsson, 1991; Kristjfinsson and the same model of evolution, but only a sequence Nielsen, 1992). Thermal stability of enzymes from analysis will certify this hypothesis. An explanation poikilotherms is correlated to the habitat temperature could be found in the evolutionary strategy. To (Hochaehka and Somero, 1984). The previous results compensate for the low temperature of the habitat, agree with this, if one considers that the habitat cold-adapted enzymes may have a more flexible temperature of the Atlantic cod is about 5°C, that the structure than their homologues from warm-blooded habitat temperature of the farmed rainbow trout of animals (Simpson and Haard, 1989), which might this study is about 20°C, and that the porcine elastase induce a broader substrate specificity (Hultin, 1980) functions in vivo about 37°C. The habitat temperature and a higher sensitivity towards different inhibitors. of farmed trout can vary from 0 to 25°C; it could This seems to be the case for rainbow trout elastase. therefore be of interest to study the influence of the habitat temperature on the characteristics of rainbow Acknowledgements--The authors would like to thank Mrs trout elastase. Hanne Jacobsen and Ms Lotte Eriksen for excellent techni-

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cal assistance in part of this study, and Dr Bo J~rgensen for constructive criticism of the manuscript.

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