Purification and Properties of a Novel Latent Proteinase Showing Myosin Heavy Chain-Degrading Activity from Threadfin-Bream Muscle'

Transcription

1 J. Biochem. 107, (1990) Purification and Properties of a Novel Latent Proteinase Showing Myosin Heavy Chain-Degrading Activity from Threadfin-Bream Muscle' Masato Kinoshita,2 Haruhiko Toyohara, and Yutaka Shimizu Department of Fisheries, Faculty of Agriculture, Kyoto University, Sakyo-ku, Kyoto, Kyoto 606 Received for publication, January 8, 1990 A novel latent proteinase of which activity was induced by heating in the presence of NaCI was purified to homogeneity from threadfin-bream muscle by a combination of DEAE cellulose, Con A-Sepharose, Arg-Sepharose, and Shim-pack HAC chromatographies. This proteinase was a glycoprotein having a monomeric subunit structure; Mr was estimated to be 77,000 on SDS-PAGE analysis. The proteinase hydrolyzed Boc-Leu-Thr-Arg-MCA as well as myosin heavy chain in the presence of 2-4% NaCl at ph 7.0 and at 60'C, optimally. The proteinase was classified as serine proteinase based on the effects of soybean trypsin inhibitor, leupeptin, and antipain. In search of a factor which causes the modori phenomenon, thermal gel degradation of fish jelly products, we found a novel latent proteinase in threadfin-bream muscle and showed that the addition of the partially purified proteinase caused the modori phenomenon (1, 2). So-called heat stable alkaline proteinase (HAP), which is now classified as a multicatalytic proteinase (3), has long been supposed to be a candidate to cause gel degradation, because HAP was latent, was changed into the active form by heating and showed a strong endopeptidase activity (4 7). The newly found proteinase was also latent, was changed into the active form by heating and showed strong endopeptidase activity, but was clearly different from HAP in its high Mr and strong myosin heavy chain (MHC)-degrading activity in the presence of NaCl (2). In the present study, we made a closer examination of the enzymatic properties of the novel latent proteinase using purified preparation from threadfin-bream (Nemipterus bathybius) muscle. MATERIALS AND METHODS Materials-Threadfin-bream (N. bathybius, 405 g in body weight and 28 cm in body length) was purchased in Kyoto wholesale market in a fresh state. The sources of other materials used in this work were as follows: DEAE cellulose (DE-52) from Whatman, Con A-Sepharose and Arginine-Sepharose from Pharmacia LKB Biotechnology, and Shimpack-HAC from Shimadzu. Other reagent-grade chemicals were purchased from Wako Pure Chemicals. Purification Procedures-All procedures were carried out at 4'C, except for Shimpack-HAC column chromatog This work was supported by a Grant-in-Aid for Scientific Research (No ) from the Ministry of Education, Science and Culture of Japan. 2 To whom correspondence should be addressed. Abbreviations: MHC, myosin heavy chain; PAGE, polyacrylamide gel electrophoresis; AMC, 7-amino-methylcoumarin; MCA, 4-methyl coumaryl-7-amide; Boc, tert-butoxycarbonyl; Suc, succinyl; Bz, ben zoyl; Z, benzyloxycarbonyl; BLTR, Boc-Leu-Tyr-Arg-MCA; E-64, L trans-epoxysuccinyl-leucylamido-(4-guanidinobutane). raphy which was carried out at room temperature. Step 1: Dorsal white muscle (120 g) was collected and homogenized with 480 ml of 20 mm Tris-HC1 buffer, ph 7.5, containing 1 mm EDTA in a non-bubbling homogenizer (Nihon Seiki) for 3 min at top speed. The homogenate was centrifuged at 12,000 x g for 15 min and the supernatant obtained was used as the crude extract for further purifi cation. On the other hand, the precipitate was further washed three more times with 4 volumes of 39 mm borate buffer, ph 7.0, containing 5 mm EDTA and 90 mm KCl to obtain myofibrils free from MHC-degrading activity and used as the substrate. Step 2: Crude extract was applied on a DEAE-cellulose column (2 x 10 cm) equilibrated with 20 mm Tris-HCl, ph 7.5, and washed thoroughly with the same buffer. Elution was performed with NaCl stepwise in 20 mm Tris-HCl, ph 7.5 (Fig. 1). Step 3: Active fractions from the DEAF-cellulose column (fraction number in Fig. 1) were concentrated by against 20 m Tris-HCI, ph 7.5, including 0.5 M NaCI, 1 mm CaC12, and MnCl2. The dialysate was applied to a column (1 x 5 cm) of Con A-Sepharose equilibrated with the same buffer. Elution was performed with a M methyl-a-d-pyranoside gradient in a total volume of 100 ml (Fig. 2). Step 4: Active fractions from Con A-Sepharose (fraction number in Fig. 2) were pooled, concentrated by against 20 mm Tris-HCI, ph 7.5. The dialysate was applied to a column (1 x 3 cm) of Arg-Sepharose equilibrated with the same buffer. Elution was performed with a M NaCl gradient in a total volume of 60 ml (Fig. 3). Step 5: Active fractions from Arg-Sepharose (fraction number in Fig. 3) were pooled and concentrated by against 1 mm phosphate buffer (ph 7.5) containing 0.1 M NaCl. The dialysate was subjected to high-performance liquid chromatography on Shim-pack HAC. The fraction at a retention time of around 17 min was used for the experi Vol. 107, No. 4,

2 588 M. Kinoshita et al. ment. The homogeneity of the purified proteinase was examined in 7.5% gel PAGE under non-denaturing condi tions by the method of Davis (8) and in 10% gel SDS-PAGE by the method of Laemmli (9). The molecular mass standards were rabbit myosin heavy chain (205 kda), /3-galactosidase (116 kda), phosphorylase B (97.4 kda), bovine plasma albumin (66 kda), egg albumin (45 kda), and carbonic anhydrase (29 kda). Both gels were stained with silver. Assay of MHC-Degrading Activity-The novel latent proteinase activity was measured as MHC-degrading ac tivity. The standard reaction mixture contained 50 mm phosphate buffer (ph 7.0), 3% NaCI, 20 mg threadfin bream myofibrils, and purified proteinase in a total volume of 1 ml. Reaction was performed at 60'C for 60 min and stopped by the addition of 0.5 ml of 10% SDS in Tris-HCI, ph 6.8 and subsequent heating at 100'C for 3 min. The degree of degradation of MHC was determined by SDS PAGE analysis. SDS-PAGE in 7.5 or 10% gel were per formed by the method of Laemmli (9) and gels were stained with Coomassie Brilliant Blue R-250. Substrate Specificity-Substrate specificity was inves tigated with synthetic fluorogenic peptides as substrates. The reaction mixture was composed of 50 mm phosphate buffer (ph 7.0), 3% NaCl, 0.5 mm substrate (previously dissolved in dimethylsulfoxide to obtain 10 mm stock solution), and the proteinase in a total volume of 0.1 ml. Reaction was performed at 60'C for 20 min and stopped by the addition of 2.6 ml of 1% SDS Tris-HCI (ph 9.0). The increase of fluorescence (excitation at 380 nm; emission at 460 nm) of AMC was measured with a Hitachi 204 fluores cence spectrophotometer. Effect of Heating on the Proteinase Molecule with or without Substrate-The proteinase was incubated at 60'C for 20 min with or without BUR in the reaction mixture described above. Reaction was stopped by the addition of 50,u1 of 10% SDS in Tris-HC1, ph 6.8, and subsequent heating at 100'C for 3 min. SDS-PAGE in 10% gel was performed by the method of Laemmli (9) and the gel was stained with silver. RESULTS Purification of a Novel Latent Proteinase-The elution profile from DEAE-cellulose is shown in Fig. 1A. Strong MHC-degrading activity was eluted around fraction num ber 59 (Fig. 1B). Several bands observed below the MHC band in fraction number 6 (Fig. 1B) were not degradation products of MHC but proteins contained in this fraction. From the Con A-Sepharose column (Fig. 2), MHC degrading activities were eluted in both the non-adsorbed fractions (fraction numbers 5 and 9) and the adsorbed fractions (fraction numbers 24 and 33). The latter showed much stronger MHC-degrading activity despite its lower protein concentration. Therefore, the latter fractions were used for further purification. As shown in Fig. 3, the latent proteinase was bound to Arg-Sepharose and eluted around fraction number 26. From Shim-pack HAC, the novel latent proteinase was eluted as a sharp single peak and it was judged to be homogeneous on the basis of electropho retic analysis under non-denaturing conditions (Fig. 4). The purified proteinase migrated as a single band in SDS-PAGE (Fig. 4) corresponding to the molecular weight of 77,000, Fig. 1. (A) Elution profile of the novel latent proteinase from a column (2. 10 cm) of DEAE-cellulose. (B) MHC-degrading activity of each fraction determined on SDS-PAGE. Activity was measured at 60'C in the presence of 3% NaCI. The arrow indicates Fig. 2. (A) Elution profile of the novel latent proteinase from a column (1 x 5 cm) of Con A-Sepharose. (B) MHC-degrading activity of each fraction determined on SDS-PAGE. Activity was measured at 60'C in the presence of 3% NaCI. The arrow indicates J. Biochem.

3 Latent Fish Muscle Proteinase 589 TABLE I. Substrate specificity of the novel latent proteinase on synthetic fluorogenic substrates. Activities were measured at 60 C in the presence of 3% NaCl. Fig. 3. (A) Elution profile of the novel latent protease from a column (1 x 3 cm) of Arg-Sepharose. (B) MHC-degrading ac tivity of each fraction determined on SDS-PAGE. Activity was measured at 60 C in the presence of 3% NaCI. The arrow indicates Fig. 5. Effect of temperature on MHC-degrading activity (A) and BLTR-degrading ac tivity (B) of the novel latent proteinase. Activities were measured in the presence of 3% NaC1. The arrow indicates Fig. 4. (A) Elution profile of the novel latent proteinase from a column (0.4 x 5 cm) of Shim-pack HAC. Inset: a and b show PAGE analysis of purified proteinase (retention time 17 min) without and with SDS, respectively. (B) MHC-degrading activity of each fraction determined on SDS-PAGE. Activity was measured at 60 C in the presence of 3% NaCI. The arrow indicates Details are described in "MATERIALS AND METHODS." while the value of 70,000 was obtained based on the elution position from a calibrated TSK gel G3000SWXL column (data not shown). This proteinase was a glycoprotein based on its adsorption to Con A-Sepharose (Fig. 2) and its positive reaction in periodic acid-schiff staining (data not shown). Substrate Specificity-Activities toward synthetic fluoro genic peptide substrates are shown in Table I. The novel latent proteinase hydrolyzed Boc-Leu-Thr-Arg-MCA (BLTR) most effectively among the peptides tested. This proteinase hydrolyzed some peptidyl Arg-MCA derivatives such as Boc-Leu-Gly-Arg-MCA, Z-Phe-Arg-MCA to some degree, while peptidyl Pro-MCA derivatives (substrates of collagenase), peptidyl Phe or Tyr derivatives (substrates of chymotrypsin), and peptidyl Ala derivatives (substrates of elastase) were not hydrolyzed at all. These results clearly demonstrated that this proteinase has a trypsin-like sub strate specificity. In addition, this proteinase showed little activity on casein in the presence or absence of NaCI despite its strong MHC-degrading activity. Effect of Temperature-The effect of temperature is Vol. 107, No. 4, 1990

4 590 M. Kinoshita et al. Fig. 6. Effect of ph on MHC degrading activity (A) and BLTR-degrading activity (B) of the novel latent proteinase. The arrow indicates Activ ities were measured at 60'C in the presence of 3% NaCl. Fig. 8. Effect of inhibitors on MHC-degrading activity of the novel latent proteinase. Activ ities were measured at 60 C in the presence of 3% NaCl. A, MHC+purified proteinase at 0 h incubation; B, MHC+purified proteinase; C, MHC+purified proteinase +soybean trypsin in hibitor (1 mg/ml); D, MHC+ purified proteinase+leupeptin (10,ug/ml); E, MHC+purified proteinase+antipain (10 jig/ ml); F, MHC+purified pro teinase+e-64 (100,ug/ml); G, MHC+purified proteinase + EDTA (10 mm). TABLE II. Effect of inhibitors on BLTR-degrading activity of the novel latent proteinase. Activities were measured at 60 C in the presence of 3% NaCl. Fig. 7. Effect of NaCI on MHC degrading activity (A) and BLTR degrading activity (B) of the novel latent proteinase. The arrow indicates Activities were measured at 60 C. Fig. 9. SDS-PAGE analysis of the heating effect on the proteinase molecule. a, before incubation without BLTR; b, before incubation with BLTR; c, incubated at 60 C for 20 min without BLTR; d, incubated at 60 C for 20 min with BLTR. Details are described in "MATERIALS AND METHODS." shown in Fig. 5. At near-physiological temperature (around C), no MHC or BLTR-degrading activity was detected, but strong activities were observed at 60 C. However, these activities were remarkably reduced above 70 C. This result can be explained by assuming that this proteinase existed as a latent form and was changed into the active form by heating. Effect of ph-the effect of ph on MHC and BLTR degrading activities is shown in Fig. 6. This novel latent proteinase showed these activities around ph 6-8. There fore, this proteinase is considered to be a neutral pro teinase. Effect of NaCl-The novel latent proteinase revealed the optimum activities toward MHC and BLTR in the presence of 2-4% NaCl (Fig. 7). Both activities were markedly decreased in the presence of lower concentrations of NaCl. Effect of Reagents-The results are shown in Fig. 8 and Table II. Soybean trypsin inhibitor and leupeptin effec tively inhibited both activities, while E-64 (inhibitor of cysteine proteinases) showed no effect. These results clearly suggested that this proteinase should be classified as a serine proteinase (10). EDTA inhibited only MHC degrading activity. This was probably due to the stabilizing effect of EDTA on MHC, because no inhibitory effect was observed on BLTR-degrading activity. Effect of Heating on the Proteinase Molecule-As shown in Fig. 9, the mobility of the proteinase molecule was not changed by heating, regardless of the presence of substrate. J. Biochem.

5 Latent Fish Muscle Proteinase 591 DISCUSSION In the previous report (1, 2), we clearly showed that the proteolytic fish gel degradation accompanied by the degra dation of MHC was not caused by HAP, but by the novel latent serine proteinase. The enzymatic properties of this latent proteinase presented herein support the involve ment of this proteinase in the modori phenomenon, which specifically occurs around C in the manufacturing process of fish jelly products, for the following reasons: (1) the novel latent proteinase revealed MHC-degrading ac tivity at unphysiologically high temperature, around 60 C (Fig. 5); (2) this proteinase degraded MHC in the neutral ph range (Fig. 6); (3) this proteinase revealed MHC degrading activity optimally only in the presence of 2-4% NaCl (Fig. 7), around which concentration fish gel products are made. The novel latent proteinase was classified as a serine proteinase from the inhibition spectra (Fig. 8 and Table II) and showed a trypsin-like substrate specificity (Table I). The absorption on Arg-Sepharose (Fig. 3) also indicates the affinity of this proteinase for the basic amino acid residue. A proteinase reported by Busconi et al. (11) and Folco et al. (12) in white croaker (Micropogon opercularis) muscle seemed to be similar to our latent proteinase in respect of enzymatic properties as a serine proteinase, induction of the activity by heating and strong MHC degrading activity, but was different from our proteinase in the inhibition by NaCl, higher molecular weight and alkaline ph dependency (optimum around ph 8.5). The molecular weight of our proteinase was estimated to be 77,000 by SDS-PAGE or 70,000 by gel filtration. This difference could be ascribed to its glycoprotein nature. It is noteworthy that the MHC and BLTR-degrading activities of this proteinase were latent and were only induced by heating in the presence of NaCl. Because little MHC-degrading activity was observed below 0.1% NaCl, it is proposed that NaCl may act to solubilize intact myofibrils and increase their digestibility by this proteinase. How ever, BLTR-degrading activity was also increased in the presence of higher concentrations of NaCl, and it is prob able that NaCl also acts directly on the proteinase molecule. HAP activity was also latent and induced by heating, but was reduced in the presence of NaCl (13). It is also interesting that SDS was ineffective for the induction of this latent proteinase activity (data not shown), although it effectively induced chymotrypsin-like activity of HAP (14, 15). No change was observed in the mobility on SDS-PAGE analysis in the presence or absence of the substrate (Fig. 9). From this result, autolytic activation seemed unlikely. Induction of this latent proteinase was probably due simply to a slight conformational change caused by heating, which was not detected as a change in migration on SDS-PAGE. Because the proteinase once heated at 60 C without sub strate and then cooled to 4 C did not show the activity around the physiological temperature range and again required heating for the induction of the activity (data not shown), the inducing process by heating is supposed to be reversible. It is generally accepted that neutral proteinase activity is strictly regulated in the cells by the presence of endogenous inhibitor or by the requirement of some other factor to avoid unwanted proteolysis. For example, calpain, cytosolic Ca"-dependent neutral proteinase, is known to co-exist with an endogenous inhibitor, calpastatin, and to show an absolute requirement of Ca" for the activity (16, 17). As to our novel latent proteinase, its activity seems to be in latent form under physiological conditions and may be induced as required by some unknown mechanism. The existence of endogenous inhibitor is also probable, because strong trypsin inhibitory activity was detected in fish muscle (11, 12, 17, 18). On the other hand, the biological function of this novel latent proteinase still remains obscure. We are now examining the seasonal change of this proteinase activity to obtain a clue to its function. REFERENCES 1. Toyohara, H. & Shimizu, Y. (1988) Agric. Biol. Chem. 52, Toyohara, H., Kinoshita, M., & Shimizu, Y. (1990) J. Food Sci. 55, Dahlmann, B., Kuehn, L., Ishiura, S., Tsukahara, T., Sugita, H., Tanaka, K., Rivett, J., Hough, F.R., Rechsteiner, M., Mykles, L.D., Fagan, M.J., Waxman, L., Ishii, S., Sasaki, M., Kloetzel, M.P., Harris, H., Ray, K., Behal, J.F., Demartino, N.G., and McGuire, J.M. (1988) Biochem. J. Lett. 255, Makinodan, Y., Yamamoto, M., & Simidu, W. (1963) Nippon Suissan Gakkaishi (in Japanese) 29, Makinodan, Y. & Ikeda, S. (1969) Nippon Suissan Gakkaishi 35, Makinodan, Y., Toyohara, H., & Niwa, E. (1985) J. Food Sci. (in Japanese) 50, Makinodan, Y., Kitagawa, T., Toyohara, H., & Shimizu, Y. (1987) Nippon Suisan Gakkaishi (in Japanese) 53, Davis, B.J. (1964) Ann. N.Y. Acad. Sci. 121, Laemmli, U.K. (1970) Nature 447, Barrett, A.J. (1977) Proteinases in Mammalian Cells and Tissues, pp. 1-55, North-Holland Publishing, Amsterdam 11. Busconi, L., Folco, E.J., Martone, C., Trucco, R.E., & Sanchez, J.J. (1984) FEBS Lett. 176, Folco, E.J., Busconi, L., Martone, C., Trucco, R.E., & Sanchez, J.J. (1984) FEBS Lett. 176, Toyohara, H., Kinoshita, M., Makinodan, Y., & Shimizu, Y. (1988) Comp. Biochem. Physiol. 92, Toyohara, H., Kinoshita, M., Yokoyama, Y., Shimizu, Y., Makinodan, Y., & Fukui, I. (1986) Seikagaku (in Japanese) 58, Folco, E.J., Busconi, L., Mar-tone, C.B., & Sanchez, J.J. (1988) Arch. Biochem. Biophys. 267, Murachi, T. (1989) Biochem. Int. 18, Toyohara, H., Makinodan, Y., Tanaka, K., & Ikeda, S. (1983) Agric. Biol. Chem. 47, Toyohara, H., Makinodan, Y., & Ikeda, S. (1984) Comp. Bio chem. Physiol. 80, Vol. 107, No. 4, 1990