Identification of Three Major Components in Fish Sarcoplasmic Proteins

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1 Nippon Suisan Gakkaishi 54(6), (1988) Identification of Three Major Components in Fish Sarcoplasmic Proteins Takayuki Nakagawa,*1 Shugo Watabe,*2 and Kanehisa Hashimoto*2 (Received November 6, 1987) Attempts were made to identify the native proteins ("43K","40K" and "35K" components) whose subunits appeared as three bands in SDS-gel electrophoretograms of sarcoplasmic proteins from each of red sea bream, Pacific mackerel and carp ordinary muscle. Judging from gel filtration behaviors on Sephadex G-150, along with subunit molecular weights and other properties, "43K","40K" and "35K" components were identified as creatine kinase, aldolase and glyceraldehyde-3-phosphate dehydrogenase, respectively. Specific activity of creatine kinase ranged from units/mg through the three fish species, whereas those of aldolase and glyceraldehyde-3-phosphate dehydrogenase were more species-specific, ranging from and units/mg, respectively. Molecular weights of isolated creatine kinase, aldolase and glyceraldehyde-3-phosphate dehydrogenase were determined to be 86,000-88,000, 160,000 and 136, ,000, respectively, regardless of fish species. Major sarcoplasmic proteins of fish are glycolytic enzymes as are those of mammals.1,2) In general, however, specific activity of glycolytic enzyme widely differs depending upon species. For example, regulatory kinases such as hexokinase, phosphofructokinase and pyruvate kinase in carp muscle show lower activities than those in rat muscle.2) Several glycolytic enzymes of migrating fish such as salmon and mackerel exhibit higher activities than those of bottom fish such as cod.3) On the other hand, levels of sarcoplasmic enzymes in fish muscle have systematically not been examined as yet. In a previous paper,4) we reported that fish ordinary muscle was generally rich in three sarcoplasmic proteins, tentatively designated "43K","40K" and "35K" components whose subunit molecular weights were 43,000, 40,000 and 35,000, as determined by sodium dodecyl sulfate (SDS)-gel electrophoresis. In addition, a quantitative estimation of the three bands allowed us to categorize the fish tested into three groups: marine white-fleshed, marine redfleshed and freshwater fish groups. This situation aroused us to identify the three major sarcoplasmic proteins which may closely be involved in energy metabolism in fish muscle. The present paper deals with the results obtained with three species of fish, red sea bream, Pacific mackerel and carp. Materials and Methods Fish The ordinary muscle was excised from live specimens of the red sea bream Pagrus major and carp Cyprinus carpio, and from Pacific mackerel Scomber japonicus specimens which were stored frozen at -80 Ž immediately after catch. Estimation and Determination of Molecular Weight Thirty grams of ordinary muscle of each species was minced, homogenized with 5 volumes of 0.1M Tris-HCl buffer (ph 7.6) containing 0.2mM EDTA (buffer A), and was centrifuged at 15,000 ~g for 30min. A portion (5ml) of the supernatant was applied to a Sephadex G-150 column (2.6 ~70cm) equilibrated with buffer A and developed with the same buffer at a flow rate of 20ml/h. Four milliliter fractions were collected. The calibration curve between the molecular weight of protein and elution volume was obtained with the following standard proteins (Boehringer Mannheim Biochemicals); catalase (molecular weight, 240,000), aldolase (158,000), bovine serum albumin (68,000), ovalbumin (45,000), chymotrypsinogen A (25,000) and cytochrome c (12,500). Some selected fractions were analyzed for protein composition by SDS-gel electrophoresis5)

2 using 10% polyacrylamide gels. For determination of molecular weights of purified enzymes, a Sephacryl S-200 column (1.9 ~ 95cm), Sephadex G-200 column (2.2 ~45cm) or Sephadex G-150 column (2.6 ~70cm) was used after equilibration with 0.1M Tris-HCl buffer (ph 7.5 or 8.0), essentially by the same procedures as above. Flow rate was 20ml/h. Purification Procedures of "43K","40K" and "35K" Components As described below,"43k","40 K" and "35 K" components were assumed to be creatine kinease (EC ), aldolase (EC ) and glyceraldehyde-3-phosphate dehydrogenase (EC ), respectively, by intact and subunit molecular weight measurements.6) For further characterization, attempts were made to isolate those enzymes as follows. All the operations were carried out in a cold room at 2-4 Ž. The ordinary muscle (30-50g) of each species was excised and homogenized with 5 volumes of 10mM Tris-HCl buffer (ph 8.0) containing 0.15M KCl and 5mM 2-mercaptoethanol for "43K" and "35K" components, or homogenized with 5 volumes of 10mM Tris-HCl buffer (ph 7.5) containing 0.2mM EDTA for "40K" component. Each homogenate was centrifuged at 20,000 ~g for 30 min. Ammonium sulfate fractionation was performed by adding solid ammonium sulfate to the resulting supernatant. The fractionation range adopted was 40-70% saturation for "43K" component, 30-60% saturation for "40K" component, and % saturation for "35K" component.7,8) The mixture was allowed to stand for 30 min after each addition of ammonium sulfate and the precipitate formed was collected by centrifugation. The pellet obtained was dissolved in and dialyzed against an appropriate buffer overnight, then applied to a CM-Sepharose CL-6B column (2.6 ~ 40cm) for "43K" component, to a DEAEcellulose column (2.0 ~40cm) for "40K" component, and to a DEAE-Sepharose CL-6B column (2.6 ~40cm) for "35K" component. Proteins were eluted with a linear gradient of KCl from 0 to 0.5M, in a total volume of 1,000ml of 10mM Tris-HCl buffer (ph 8.0) containing 5mM 2- mercaptoethanol for "43K" and "35K" components, or 1,000ml of 10mM Tris-HCl buffer (ph 7.5) containing 0.2mM EDTA for "40K" component. Enzymatically active fractions were collected and further purified by a DEAE-cellulose column (2.2 ~40cm) chromatography, followed by gel filtration on a Sephadex G-150 column (2.6 ~70 cm) or a Sephacryl S-200 column (1.9 ~95cm) as described in the legend for Fig. 3 in detail. Activity Assay Creatine kinase activity was measured spectrophotometrically by the method of Forster et al.,9) whereas aldolase and glyceraldehyde-3-phosphate dehydrogenase activities were measured spectrophotometrically by the methods of Scopes.7) Through the three enzymes, one activity unit was defined as the amount of enzyme which catalizes the reduction of 1 ƒêmol NADP+ or the oxidation of 1 ƒêmol NADH per min at 25 Ž. Protein concentration was determined by the micro-biuret method10) using bovine serum albumin as a standard. Results and Discussion For the estimation of intact molecular weights of "43K","40K" and "35K" components, the ordinary muscle extract of Pacific mackerel was applied to Sephadex G-150 gel filtration. As shown in Fig. 1, three peaks A-C appeared. From the position of elution, peak A was judged to be composed of high molecular weight proteins. Peak B contained most of the sarcoplasmic proteins, and peak C was considered to consist of peptides and free amino acids. From the calibration curve (Fig. 2), intact molecular weights of "43K","40K" and "35K" components were estimated to be 80,000, 160,000 and 130,000, respectively. Judging from intact and subunit molecular weights, "43K","40K" and "35K" components were assumed to be creatine kinase, aldolase and glyceraldehyde-3-phosphate dehydrogenase, respectively, by reference to the data of those enzymes isolated from other sources.6,11) Attempts were then made to isolate the three components by the method essentially consisting of two to three types of chromatography, using each enzyme activity as parameter. Elution patterns at the final purification step for Pacific mackerel enzymes are shown in Fig. 3. Activity and protein peaks coincided with each other, irrespective of enzyme. Essentially so was the case with the other two fishes (data not shown). Relative mobilities of the subunits derived from the three enzymes, regardless of fish species, agreed well with those of the three major bands in the

3 Fig. 1. Gel filtration of the extract of Pacific mackerel on a Sephadex G-150 column. The figure inserted shows SDS-gel electrophoretic patterns of the crude extract and some selected fractions. Fig. 2. Molecular weight estimation of "43K", "40K" and "35K" components in sarcoplasmic proteins from Pacific mackerel ordinary muscle by Sephadex G-150 gel filtration. Standard proteins used were: CAT, catalase; ALD, aldolase; BSA, bovine serum albumin; OVA, o valbumin; CHY, chymotrypsinogen A; CYT, cytochrome c. crude extract (Fig. 4), supporting the assumpiont that "43K","40K" and "35K" components were creatine kinase, aldolase and glyceraldehyde- 3-phosphate dehydrogenase, respectively. As summarized in Table 1, the specific activity of each enzyme differed depending upon fish species. Specific activity of creatine kinase from red sea bream (32.8units/mg) was comparable to that from carp (33.3), whereas it was slightly higher than that from Pacific mackerel (25.9). Specific activity of aldolase widely differed among the three species, ranging between units/mg. The activity of glyceraldehyde-3- phosphate dehydrogenase ranged from units/mg. Czok and Bucher12) proposed an estimation method for an enzyme protein in muscle extract, by use of "relative specific activity", which was defined as the ratio of the specific activity of an enzyme in the extract, against that of the purified enzyme. They demonstrated that one can approximate the percentage of a given enzyme in muscle extract by relative specific activity of the enzyme. Therefore, an attempt was made to approximate the three enzymes in muscle extract. Results showed that the relative specific activity of creatine kinase ranged from 39.0 to 42.6% through the three fish species, which were more than two times as high as the relative amounts of this enzyme reported previously ( %).4) These differences seem to indicate the presence of some activator (s) in the extract. However, the reason

4 Fig. 3. Elution patterns of creatine kinase (CK; Sephacryl S-200), aldolase (ALD; DEAEcellulose) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Sephadex G-150) from the ordinary muscle of Pacific mackerel. Experimental conditions used were as follows: For CK, fractions eluted from a CM- Sepharose CL-6B column (2.6 ~40cm) were combined, concentrated, and applied to a Sephacryl S-200 column (1.9 ~95cm) equilibrated with 0.1M Tris-HCl buffer (ph 8.0). Proteins were eluted with the same buffer. Fractions of 3.4ml were collected. For ALD, fractions eluted from a CM-Sepharose CL-6B column (2.6 ~35cm) were combined, dialyzed against 10mM Tris-HCl buffer (ph 7.5) containing 0.2mM EDTA, and applied to a DEAE-cellulose column (2.2 ~40cm) equilibrated with the same buffer. Proteins were eluted with a linear gradient of KCl from 0-0.5M, in a total volume of 1,000ml of the same buffer. Fractions of 5ml were collected. For GAPDH, fractions eluted from a DEAE-Sepharose CL-6B column (2.6 ~32cm) were combined and dialyzed against an ammonium sulfate-saturated 0.1M Tris-HCl buffer (ph 8.0) containing 5mM 2-mercaptoethanol. The precipitate formed was collected by centrifugation and dissolved in a small volume of the above buffer. The enzyme solution thus obtained was applied to a Sephadex G-150 column (2.6 ~70cm) equilibrated with the same buffer. Fractions of 2.6ml were collected. Pooled fractions were indicated by both arrows.

5 Fig. 4. SDS-gel electrophoretic patterns of purified preparations (P) of creatine kinase (CK), aldolase (ALD) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), along with the crude extract (C), from the ordinary muscle of red sea bream, Pacific mackerel and carp. remains to be explained. Relative specific activities of this enzyme in green sunfish13) and dogfish14) were calculated to be 24.4 and 23.9%, respectively. Relative specific activity of aldolase was 19.2% for red sea bream, 14.4% for Pacific mackerel and 13.7% for carp. The value from carp was comparable to its relative amounts, whereas those from red sea bream and Pacific mackerel differed from the relative amounts (Table 1). The relative specific activity of aldolase in cod was calculated to be 12.3%.15) Relative specific activities of glyceraldehyde-3- phosphate dehydrogenase in carp was also close to its relative amount. This was not the case with red sea bream and Pacific mackerel, which showed clearly higher values than the relative amounts.4) Relative amounts of glyceraldehyde- 3-phosphate dehydrogenase in total extracted proteins of rabbit16) and kokanee salmon17) were calculated to be 14 and 13%, respectively. As shown in Table 2, intact molecular weights of creatine kinase, aldolase and glyceraldehyde-3- phosphate dehydrogenase were determined to be 86,000-88,000, 160,000 and 136, ,000, respectively. These values were in a good agreement with those reported from other sources including fishes; 82,700-85, ) for creatine kinase; 155, ,00015,24-27) for aldolase; 140, , ) for glyceraldehyde-3-phosphate dehydrogenase. Molecular weights of purified creatine kinase and glyceraldehyde-3-phosphate dehydrogenase were about 6,000 and 10,000 larger than those estimated with the crude extract. These differences may have been due to some retardation in elution of the enzyme, caused by the presence of high molecular weight proteins. The subunit molecular weights of fish creatine kinase, aldolase and glyceraldehyde-3-phosphate dehydrogenase were estimated to be about 43,000-44,000, 40,000 and 36,000-36,500, respectively (Table 2). These values agreed well with those reported from other

6 Enzymology" Vol. 90, (ed. by W. A. Wood), Academic Press, New York, 1982, pp ) G. Forster, E. Rernt, and H.-U. Bergmeyer: in "Methods in Enzymatic Analysis" (ed. by H.-U. Bergmeyer), Academic Press, New York, 1963, pp ) R. F. Itzhaki and D. M. Gill: Anal. Biochem., 9, (1964). 11) R. C. Ruth and F. Wold: Comp. Biochem. Physiol., 54B, 1-6 (1976). 12) R. Czok and T. Bucher: Adv. Prot. Chem., 15, (1960). 13) S. E. Fischer and G. S. Whitt: Anal. Biochem., 94, (1979). 14) B. Simonarson and D. C. Watts: Biochem. J., sources; 42,000-44, ) for creatine kinase, 40,000-45, ) for aldolase and 35,000-37,00028,30-32) for glyceraldehyde-3-phosphate dehydrogenase. From both intact and subunit molecular weights, it was found that creatine kinase, aldolase and glyceraldehyde-3-phosphate dehydrogenase of the three fish species were composed of 2, 4 and 4 identical subunits as in the case of rabbit.19,24,28) Enzymatic properties of these enzymes will be published elsewhere. Acknowledgements The expenses of the present study were defrayed in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture. References 1) R. K. Scopes: Biochem. J., 107, (1968). 2) P. M. F. Avelar, D. Giacometti, and M. Bacila: Comp. Biochem. Physiol., 60B, (1978). 3) T. Shibata: Mem. Fac. Fish., Hokkaido Univ., 24, 1-80 (1977). 4) T. Nakagawa, S. Watabe, and K. Hashimoto: Nippon Suisan Gakkaishi, 54, (1988). 5) U. K. Laemmli: Nature, 227, (1970). 6) D. W. Darnall and I. M. Klotz: Arch. Biochem. Biophys., 166, (1975). 7) R. K. Scopes: Biochem. J., 161, (1971). 8) R. K. Scopes and A. Stoter: in "Methods in 128, (1972). 15) C. Y. Lai and C. Chen: Arch. Biochem. Biophys., 144, (1971). 16) R. E. Amelunxen and D. O. Carr: Biochim. Biophys. Acta, 132, (1967). 17) T. Nakai, Y. Miyamoto, and T. Shibata: Bull. Fac. Fish., Hokkaido Univ., 26, (1975). 18) C. Gosselin-Rey and C. Gerday: Biochim. Biophys. Acta, 221, (1970). 19) R. H. Yue, R. H. Palmieri, O. E. Olson, and S. A. Kuby: Biochemistry, 6, (1967). 20) T. Takasawa, K. Fukushi, and H. Shiokawa: J. Biochem., 89, (1981). 21) T. Takasawa and H. Shiokawa: J. Biochem., 90, (1981). 22) D. M. Dawson, H. M. Eppenberger, and N. O. Kaplan: J. Biol. Chem., 242, (1967). 23) I. Kumudavalli, B. H. Morerand, and D. C. Watts: Biochem. J., 117, (1970). 24) K. Kawahara and C. Tanford: Biochemistry, 5, (1966). 25) S. Tazeen-Pasha and A. Salahuddin: Biochim. Biophys. Acta, 483, (1977). 26) E. Nagahisa and Y. Tsuchiya: Tohoku J. Agric. Res., 22, (1971). 27) S. K. Komatsu and R. E. Feeney: Biochim. Biophys. Acta, 206, (1970). 28) V. D. Hoagland, Jr. and D. C. Teller: Biochemistry, 8, (1969). 29) M. M. Vieila, L. A. Veiga, and M. Nakano: Comp. Biochem. Physiol., 74B, (1983). 30) L. D. Byers: in "Methods in Enzymology" Vol. 89, (ed. by W. A. Wood), Academic Press, New York, 1982, pp ) R. E. Amelunxen: in "Methods in Enzymology" Vol. 41, (ed. by W. A. Wood), Academic Press, New York, 1975, pp ) K. Suzuki, M. Watanabe, and K. Imahori: J. Biochem., 77, (1975). 33) F. C. Greene and R. E. Feeney: Biochim. Biophys. Acta, 220, (1970). Nippon Suisan Gakkaishi: Formerly Bull. Japan. Soc. Sci. Fish.