Presence of Cysteine Endopeptidase in the Pyloric Caeca of Pacific Mackerel Scomber japonicus and Its Purification and Characterization
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1 Fisheries Science 64(4), (1998) Presence of Cysteine Endopeptidase in the Pyloric Caeca of Pacific Mackerel Scomber japonicus and Its Purification and Characterization Yasuyuki Tsukamasa, Takayuki Nakagawa, Kazuhiro Masuda, Masashi Ando, and Yasuo Makinodan Department of Fisheries, Faculty of Agriculture, Kinki University, Nara , Japan (Received February 1, 1998) An endopeptidase which hydrolyzed myofibril strongly from the pyloric caeca of Pacific mackerel Scomber japonicas was purified to homogeneity electrophoretically. The molecular weight and the opti mum ph of the endopeptidase was about 30 k and around 9.5, respectively. Since the endopeptidase was not inhibited by serine protease inhibitors, e.g. PMSF and soybean trypsin inhibitor, but complete ly inhibited by E-64, a high specific cysteine protease inhibitor, it was considered to be a cysteine en dopeptidase. This is the first report on the existence of the cysteine endopeptidase in the pyloric caeca. The endopeptidase showed a hydrolytic specificity for MCA-synthetic peptide substrates which had a basic amino acid at P1 position and a hydrophobic amino acid at P2 or P3 position. It hydrolyzed vari ous proteins examined except for collagen. Key words: Pacific mackerel, pyloric caeca, cysteine endopeptidase, myofibril, protease The pyloric caeca, in which various kinds of digestive en zymes exist, is a specific digestive and absorptive organ of teleost fish. In general, the great part of external secreted proteases are classified into serine protease and the remain ing into metalloprotease. Thus, only trypsin,1) chymotryp sin-like external endopeptidase,2) aminopeptidase,3) and collagenase4) have been known as proteases in the pyloric caeca. In the current investigations on purification and characterization of endopeptidases from the pyloric caeca, only specific substrates for expected proteases such as ser ine and metallo-proteases were used to measure proteolyt ic activity. Therefore, unexpected endopeptidases which had weak proteolytic activity toward these substrates had been disregarded. In the research on trypsin,1) which hydro lyze casein and tosyl-l-arginine methyl ester (TAME), from the pyloric caeca of chum salmon, a fraction of column chromatography which showed hydrolytic activity toward casein and not toward TAME was found. However, details of the endopeptidase were not examined. Only a reports 5) on an endopeptidase from the pyloric caeca of anchovy indicated the existence of non-serine and non aspartic endopeptidase which hydrolyzed collagen at neu tral and alkaline conditions. It seems to be possible to find novel proteases if myofibril will be used as a substrate, be cause proteases from pyloric caeca are presumed to have a proteolytic activity to digest myofibril of muscle intaken as food. In the present study, we attempted to find and purify a novel protease existing in the pyloric caeca of Pacific mack erel Scomber japonicas strate. Fish and Reagents Materials by using fish myofibril as a sub and Methods Pacific mackerel was purchased in a local wholesale mar ket in Nara in rigor stage. The sources of other materials used in the present work were as follows: Sephacryl S-200 from Pharmacia biotech, DEAE-Toyopearl from Tosoh, hydroxyapatite from Seikagaku. MCA-synthetic peptide substrates were purchased from Peptide institute. Other analytical grade chemicals were purchased from Wako pure chemicals. Preparation of Substrate Myofibril which had been prepared from Pacific mack erel muscle was used as a substrate. Briefly, the ordinary muscle of Pacific mackerel was collected and homogenized with six volumes of 39 mm borate buffer, ph 7.0, contain ing 100 mm KCI and 4.6 mm EDTA and then centrifuged at 600 ~ g for 15 min. The precipitate was washed again with the same buffer, and then centrifuged. The obtained precipitate was suspended in the same buffer and used as the myofibril substrate. Casein and bovine serum albumin were dissolved into 50 mm sodium bicarbonate buffer, ph 9.5. One gram of hemoglobin was dissolved into 20 ml of distilled water containing 0.1 g of NaOH and 6 g of urea, and the solution was incubated at 37 Ž for one hour. Af ter that, the hemoglobin solution was dialyzed overnight against 50 mm sodium bicarbonate buffer, ph 10, then the volume of the dialyzate was adjusted to 100 ml with the same buffer. Collagen solution was prepared by mixing Type I collagen from pig skin solution (3 mg/ml) and 50 mm sodium bicarbonate buffer, ph 9.5, at a ratio of one to four. Assay of Proteolytic Activity The proteolytic activity was measured by the method of Anson6) with a slight modification. Briefly, the reactive mixture was prepared with 500 ƒêl of 50 mm sodium bicar bonate buffer, ph 9.5, 250,ƒÊl of substrate solution and 250,ƒÊ1 of enzyme solution, and then incubated for 0-30 min at 37 Ž. The reaction was stopped by the addition of 5 ml of 6% trichloroacetic acid (TCA). After filtration,
2 628 Tsukamasa et al. TCA-soluble peptides in filtrate were measured by the method of Lowry et al.7) The proteolytic activity was ex pressed as pmol Tyr equivalent/min/ml. One unit of the endopeptidase which catalyzes the formation of l pmol Tyr equivalent/min at 37 Ž. Assay of Synthetic Substrate Hydrolyzing Activities The hydrolyzing activities of TAME and benzoyl-l-tyro sine ethyl ester (BTEE) was measured according to the method of Hummel8) at ph 8.5 and 30 Ž. MCA-synthetic substrate hydrolyzing activity was deter mined as follows. The reactive mixture was prepared with 500ƒÊ1 of 50 mm sodium bicarbonate buffer, ph 10.0, 250 l of 1 mm substrate solution, and 250p1 of enzyme solu ƒê tion, and then incubated for 10 min at 37 Ž. The reaction was stopped by the addition of 1 ml of 10% TCA. The fluorogenic intensity was measered with a fluorescence spectrophotometer (excitation at 380 nm; emission at 460 nm). Protein Determination Protein was measured by the method of Lowry et al.7) with bovine serum albumin as a standard. SDS polyacrylamide Gel Electrophoresis SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed by the method of Laemmli9) using 10% gels. The gels were stained with Coomassie brilliant blue R-250 or silver. Fig. 1. Effect of ph on the hydrolysis of myofibrillar protein by the crude extract from the pyloric caeca of Pacific mackerel., Sodium bicarbonate buffer;, Glycine-NaOH œ buffer;, Sodi um phosphate buffer;, Tris-HCI buffer. Table 1. Effect of various protease inhibitors on the myofibril hydrolyzing activity in the extracts from the pyloric caeca of Pacific mackerel Activity Staining Activity staining was performed by the method of Gar cia-carreno et al. 101 with a slight modification. After 10% gel PAGE of the purified endopeptidase under non denaturing condition at 4 Ž, the gel was immersed in the myofibril solution and incubated for 90 minutes at 25 Ž. Thereafter, the gel was stained with Coomassie brilliant blue R-250. Result An endopeptidase, which hydrolyzed myofibrillar pro tein but did not either TAME or BTEE, was purified. All the following procedures were carried out at 4 Ž. Preparetion of Crude Extract The pyloric caeca was removed from Pacific mackerel. Acetone powder prepared from the pyloric caeca was mix ed with five volumes of chilled distilled water, then cen trifuged at 20,000 ~ g for 30 minutes. The supernatant was filtered through glass wool and the filtrate was used as the crude extract. Proteolytic Activity of the Crude Extract Effect of ph on the myofibril hydrolyzing activity of the crude extract from the pyloric caeca was determined (Fig. 1). Strong proteolytic activity was detected in the alkaline condition and the optimum ph was around 9.5. The pro teolytic activity was measured at verious temperatures for 30 min. As shown in Fig. 1, the endopeptidase showed op timum temperature around 40 Ž. The effect of the pro tease inhibitors on the myofibril-hydrolyzing activity was shown in Table 1. Common inhibitors of serine and cys teine-proteases such as leupeptin and tosyl-l-lys-chlo romethyl ketone (TLCK) inhibited 100% and 32% of the activity, respectively. Specific serine protease inhibitor, phenylmethansulfonyl fluoride (PMSF), also inhibited 33% of the activity. Moreover, specific cysteine protease in hibitors such as N-ethylmaleimide (NEM) and [L-3-trans Carboxirane-2-Carbonyl]-L-Leucyl-Agmatin (E-64) in hibited 26% and 32% of the activity, respectively. These result suggested the existence of not only serine endopepti dase but also cysteine endopeptidase which had not been detected from the pyloric caeca. Therefore, we performed further purification of the cysteine endopeptidase. Purification of the Cysteine Endopeptidase Solid ammonium sulfate was added to the crude extract to give 70% saturation. After standing overnight, the precipitate was collected by centrifugation and dissolved in 10 mm Tris-HCl buffer, ph 7.5, containing 1 mm 2-mer captoethanol. The solution was applied to a column (2.8 ~ 95 cm) of Sephacryl S-200 equilibrated with the same buffer. The objective endopeptidase fractions were applied on a DEAE-Toyopearl column (2.8 ~ 45 cm) equilibrated with the same buffer and washed thoroughly
3 Cysteine Endopeptidase in the Pyloric Caeca 629 with the same buffer. Elution was performed with M gradient in a total volume of 1000 ml. The active NaCl fractions were dialyzed against 5 mm sodium phosphate buffer, ph 7.5, containing 1 mm 2-mercaptoethanol over night. The dialysate was applied to a column (2.8 ~ 18 cm) of hydroxyapatite equilibrated with the same buffer. Elu tion was performed with 5 mm and 20 mm phosphate step wise. The obtained objective endopeptidase fractions were pooled and dialyzed against 5 mm sodium phosphate buffer, ph 7.5, containing 1 mm 2-mercaptoethanol. Two active peaks, fraction number and , which showed myofibril-hydrolyzing activity, were ob tained by the gel-filtration using Sephacryl S-200 column (Fig. 2). However, the latter peak also showed strong TAME and BTEE hydrolyzing activity and assumed to be trypsin or chymotrypsin. Therefore, the former peak frac tions which had weak TAME and BTEE hydrolyzing activ ity were pooled and subjected to the ion-exchange column chromatography. Active fractions showing myofibril hydrolyzing activity was obtained around the position of Fig. 2. Sephacryl S-200 column chromatography of the crude extract. The flow rate was 60 ml/h and 4.0 ml of fractions were collected. Fig. 3. DEAE-Toyopearl column chromatography of the fractions obtained from Sephacryl S-200 column.
4 630 Tsukamasa et al. Fig. 4. Hydroxyapatite column chromatography of the fractions obtained from DEAE-Toyopearl column. and 20 mm phosphate, which showed myofibril-hydrolyz ing activity were obtained (Fig. 4). Because the hydrolyz ing activities to TAME and BTEE of the former fractions was weaker than those of latter, the former fractions were collected and used to characterize the enzymatic proper ties. The purification process so far noted is summarized in Table 2. An endopeptidase was purified 19-fold with an yield of 23%. The purified endopeptidase was migrated as a single band in SDS-PAGE corresponding to the molecu lar weight of about 30 k (Fig. 5). The proteolytic activity of the purified endopeptidase was analyzed on the PAGE gel by activity staining (Fig. 6). A clear zone was observed on the same position as the purified endopeptidase. Fig. 5. SDS-polyacrylamide gel electrophoresis of the purified endopep tidase from the pyloric caeca of Pacific mackerel. A: the purified endopeptidase, B: molecular weight marker. 0.1 M NaCl in DEAE-Toyopearl column chromatography (Fig. 3). TAME and BTEE hydrolyzing activities of these fractions were remarkably low. The active fractions were subjected to hydroxyapatite column chromatography. Two active peaks, around the positions of 5 mm phosphate Properties of Purified Protease Effect of Protease Inhibitors As shown in Table 3, the myofibril-hydrolyzing activity of this purified endopepti dase was inhibited by cysteine protease inhibitors such as E-64, IAA, and NEM and common inhibitors to serine and cysteine protease such as leupeptin, TLCK, and TPCK. On the other hand, serine protease inhibitors such as PMSF, SBTI, and 1, 10-phenanthroline and aspartic pro tease inhibitor, pepstatin A, did not inhibit the proteolytic activity at all. These results clearly suggested that this en dopeptidase should be classified as a cysteine endopepti dase. Effect of ph As shown in Fig. 7, the cysteine endopepti dase showed that the optimum ph was about 9.5. The en dopeptidase was preincubated at 37 Ž for 30 minutes at various phs, from 6 to 12, and remaining activities were measured. The endopeptidase was stable in the ph range Table 2. Purification chart of the endopeptidase from pyloric caeca of Pacific mackerel
5 Cysteine Endopeptidase in the Pyloric Caeca 631 Fig. 7. Effect of ph on the activity (A) and the stability (B) for my ofibril-hydrolysis of the purified endopeptidase from the pyloric cae ca. Fig. 6. Proteolytic activity staining of the purified endopeptidase sepa rated by polyacrylamide gel electrophoresis. Gel A was stained with silver, and gel B was stained for determination of proteolytic activity by the method of Garcia-Carreno et al. 10) of 6.5 to 9.5. Substrate Specificity Proteolytic activities toward syn thetic fluorogenic peptide substrates are shown in Table 4. The cysteine endopeptidase showed high hydrolyzing activ ity toward synthetic peptide substrate which was com posed of a basic amino acid of Arg or Lys for P1 position and a hydrophobic amino acid such as Phe or Leu for either P2 or P3 position. Whereas, Bz-Arg-MCA (sub strate of trypsin), Suc-Ala-Pro-Ala-MCA (substrate of chymotripsin), Suc-Gly-Pro-Leu-Gly-Pro-MCA (substrate of collagenase) and Suc-Leu-Leu-Val-Tyr-MCA (substrate of elastase) were not hydrolyzed by the cysteine endopepti dase. The proteolytic activities toward various protein sub strates are shown in Table 5. This endopeptidase hydro lyzed many kinds of proteins with high activity. As shown in Fig. 8, degradation of myosin heavy chain was observed clearly on the 30 min-incubation at 37 Ž. On the other Table 3. Effect of various protease inhibitors on the myofibril hydrolyzing activity of the endopeptidase from pyloric caeca Fig. 8. Proteolysis of myofibrillar fragments with the purified endopep tidase at 37 Ž. MHC, myosin heavy chain; A, actin. hand, most of other myofibrillar proteins were not degrad ed. In addition, this endopeptidase did not hydrolyze colla gen at all (Data not shown). Table 4. MCA-substrate specificity of the pyloric caeca endopepti dase
6 632 Tsukamasa et al. Table 5. Protein substrate specificity of the pyloric caeca endopep tidase Discussion In the present study, we have purified the cysteine en dopeptidase to homogeneity electrophoretically from the pyloric caeca of Pacific mackerel by monitoring the my ofibril-hydrolyzing activity. A cysteine endopeptidase has not been known in the pyloric caeca up to now. In the cur rent research on purification and characterization of pro tease from the pyloric caeca, only specific substrates for ex pected proteases, e.g. TAME and BTEE for trypsin and chymotrypsin, respectively, were used to measure their pro tease activity. Therefore, unexpected proteases which had weak hydrolytic activity toward these substrates had been disregarded. As for the report on an endopepttidase of pyloric caeca from anchovy,sl the non-serine and non aspartic endopeptidase is considered to be different from the cysteine endopeptidase because of the presence of col lagenase activity. In this study, the purified endopeptidase was partially in hibited by TLCK and TPCK (Table 3). They are often used as inhibitors to serine proteases such as trypsin and chymotrypsin.11) However cysteine proteases are also in hibited by these inhibitors 11,12) And homogeneity of this endopeptidase was identified by SDS-PAGE analysis. It is considered TLCK and TPCK inhibited the purified cys teine endopeptidase. The substrate specificity on synthetic peptide substrates of the purified cysteine endopeptidase, a basic amino acid for P1 position and a hydrophobic amino acid for either P2 or P3, was different from those of cysteine endopepti dase, cathepsin B, H, and L,13) but similar to those of the cysteine endopeptidase from the mantle of squid14) and cathepsin L from the muscle of chum salmon.12) This purified cysteine endopeptidase many kinds of proteins except collagen. It is similar to cathepsin L-like endopeptidase. 131 However, the active ph range and opti mum ph were significantly different from other cysteine en dopeptidases. The optimum ph of the cysteine endopepti dase from the mantle of squid and cathepsin L from the muscle of chum salmon were 3.0 and 5.6, respectively, and that of the purified cysteine endopeptidase was 9.5. Z-Phe Arg-MCA, synthetic peptide substrate for cathepsin L, was hydrolyzed by this purified endopeptidase at ph 9.5. These results suggested that the purified cysteine endopepti dase was different from cathepsin L. The optimum ph of this cysteine endopeptidase is higher than those of trypsin and chymotrypsin. It is known that ph in the upper intes tines which includes the pyloric caeca in the starved state before feeding was more basic than that after feeding.15-16) Furthermore it is also known that proteolytic activities of trypsin and chymotrypsin are significantly low in the starved state and that those activities gradually rise by feed ing.16) If this high proteolytic activity of this purified cys teine endopeptidase is observed in the early state of feed ing, this endopeptidase is assumed to have the role of the initial digestion. As mentioned above, we found a cysteine endopeptidase different from known endopeptidases such as trypsin and chymotrypsin from the pyloric caeca of Pacific mackerel for the first time in this research. References 1) N. Uchida, K. Tsukayama and E. Nishide: Purification and some properties of trypsins from the pyloric caeca of chum salmon, Nip pon Suisan Gakkaishi, 50, (1984). 2) K. Muraki and M. Noda: Studies on proteinases from the digestive organs of sardine. 1. Purification and characterization of three alka line proteinases from pyloric caeca. Biochim. Biophys. Acta, 658, (1981). 3) M. Hajjou and Y. L. Gal: Purification and characterization of aminopeptidase from tuna pyloric caeca. Biochim. Biophys. Acta, 1204, 1-13 (1994). 4) R. Yoshinaka, M. Sato, and S. Ikeda: Studies on collagenase in fish, purification and properties of a collagenase from the pyloric caeca of yellowtail. Nippon Suisan Gakkaishi, 43, (1977). 5) A. Martinez and A. Gildberg: Autolytic degradation of belly tissue in anchovy (Engraulis encrasicholus). Int. J. Food Sci. Technol., 23, (1988). 6) M. L. Anson: The estimation of pepsin, trypsin, papain and cathep sin with hemoglobin. J. Gen. Physiol., 22, (1938). 7) O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall: Protein measurement with Folin phenol reagent. J. Biol. Chem., 193, (1951). 8) B. C. W. Hunmmel: A modified spectrophotometric determination of chymotrypsin, trypsin, and thrombin. Can. J. Biochem. Phys iol., 37, (1959). 9) U. K. Laemmli: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, (1970). 10) F. L. Garcia-Carreno, L. E. Dimes, and N. F. Haard: Substrate-gel electrophoresis for composition and molecular weight of pro teinases or proteinaceous proteinase inhibitors. Anal. Biochem., 214, (1993). 11) T. Koide: Serine protease inhibitors, in "Proteases and their inhibi tors" (ed. by 0. Hayaishi), Medicalview, Tokyo, 1993, pp (in Japanese). 12) M. Yamashita and S. Konagaya: Purification and characterization of cathepsin L from the white muscle of chum salmon, Oncorhyn chus keta. Comp. Biochem. Physiol., 96B, (1990). 13) A. J. Barrett and H. Kirschke: Cathepsin B, cathepsin H, and cathepsin L. Methods in EnZymology, 80, (1981). 14) J. Sakai-Suzuki, M. Tobe, T. Tsuchiya and J. J. Matsumoto: Purification and characterization of acid cysteine proteinase from squid mantle muscle. Comp. Biochem. Physiol., 85B, (1986). 15) K. J. Maier and R. E. Tullis: The effects of diet and digestive cycle on the gastroitestinal tract ph values in the goldfish, Carassius aura tus L., Mozambique tilapia, Oreochromis mossambicus (Peters), and channel catfish, Ictalurus punctatus (Rafinesque), J. Fish. Biol., 25, (1984). 16) T. Takeuchi: Digestion and nutrition, in "Fish physiology" (ed. by Y. Itazawa and I. Hanyu), Koseisya-Koseikaku, Tokyo, 1991, PP (in Japanese).
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