Regulatory Proteins from Dorsal Muscle of the Carp

Transcription

1 J. Biochent. 82, (1977) Regulatory Proteins from Dorsal Muscle of the Carp Kunihiko KONNO,* Ken-ichi ARAI,* and Shizuo WATANABE** *The Department of Food Science, Faculty of Fisheries, Hokkaido University, Hakodate, Hokkaido 041, and **the Department of Chemistry, Faculty of Science, Tokyo Institute of Technology, Ookayama, Meguro-ku, Tokyo 152 Received for publication, February 28, 1977 It was found that myosin B of carp dorsal muscle is identical with that of rabbit skeletal muscle as regards both calcium and strontium sensitivities. Carp dorsal myosin B is therefore dif ferent from bovine cardiac myosin B, the strontium sensitivity of which is known to be higher than that of rabbit skeletal myosin B. The sensitivities were estimated by measuring the Mg-ATPase activity in the presence of various concentrations of calcium or strontium ions. Half-maximal activation of the ATPase activity was obtained with either 1 ƒêm free calcium ions or 20 ƒêm free strontium ions. Relaxing protein (also called "native" tropomyosin) and troponin were prepared from carp dorsal muscle, and were shown to be as effective as those prepared from rabbit skeletal muscle in inhibiting Mg-ATPase of either rabbit or carp "desensitized" myosin B and in conferring calcium sensitivity on either of them. It was, however, observed that when desensitized myosin B is prepared from "frozen" instead of "fresh" rabbit skeletal muscles, Mg-ATPase of the desensitized myosin B of frozen muscle is only weakly inhibited by carp relaxing protein or carp troponin (plus rabbit tropo myosin), whereas it is fully inhibited by rabbit relaxing protein or rabbit troponin plus rabbit tropomyosin. It was then found that the desensitized myosin B prepared from frozen rabbit skeletal muscle can be made equally responsive to carp and rabbit relaxing proteins (or troponin plus tropomyosin) either by heating the desensitized myosin B in M KCl at 30 to 40 Ž for 10 to 30 min or by treating it with 1 to 1.5 M urea at 25 Ž for 20 min. It is thus suggested that freezing rabbit muscle caused a minor and reversible change in the actomyosin con formation. The observations described above were also reproduced using "reconstituted" actomyosin in place of "desensitized" myosin B. For example, it was observed that Mg-ATPase of rabbit actomyosin reconstituted from actin of frozen muscle and myosin of fresh muscle is only weakly inhibited by carp relaxing protein. It was, however, found that carp relaxing protein is as effective as rabbit relaxing protein if it is added to actin of frozen muscle before actin and myosin are mixed to reconstitute actomyosin. Abbreviations: EGTA, ethyleneglycol-bis(a-aminoethylether)-n, N Œ-tetraacetate; SDS, sodium dodecyl sulfate. Vol. 82, No. 4,

2 932 K. KONNO, K. ARAI, and S. WATANABE In a previous report (1, 2) from our laboratory, was shown that actin and myosin prepared from carp dorsal muscle were nearly identical, in bio chemical functions, with those of rabbit muscle. In the present report it is shown that relaxing protein ("native" tropomyosin) and troponin prepared from carp dorsal muscle are also essential ly identical, in biochemical functions, with those of rabbit skeletal muscle. However, it has been observed that Mg-ATPase of "desensitized" myosin B (3) prepared from "frozen" rabbit skeletal muscle is not inhibited by carp relaxing protein, whereas it is fully inhibited by rabbit relaxing protein. The reason for the effect of freezing muscle on the response of rabbit desensitized myosin B to carp relaxing protein is investigated in this work. EXPERIMENTAL All the muscular proteins we used were prepared from rabbit skeletal and carp dorsal muscles, fresh or stored at -20 Ž for a week. Myosin B was prepared from rabbit skeletal muscle by a routine method, extracting with Weber-Edsall solution, and also from carp dorsal muscle, extracting with 0.6 M KCl buffered with 40 mm sodium phosphate (ph 7.5) (4). "Desensitized" myosin B was obtained by the procedure described by Schaub et al. (3). Myosin was prepared by the method of Connell (5), extracting with 0.45 M KCl, 5 mm ATP-Mg, and 50 mm sodium phosphate (ph 6.4). Actin free from relaxing protein was prepared by the method of Spudich and Watt (6). Recon stituted actomyosin was prepared by mixing myosin and actin in a weight ratio of 3 : 1. Rabbit and carp relaxing proteins were obtained by the method originally reported for the preparation from rabbit skeletal muscle (7, 8). Troponin was also prepared from both carp dorsal and rabbit skeletal muscles by the method of Ebashi et al. (9). Tropomyosin was prepared only from rabbit skeletal muscle by the method of Bailey (10). ƒ - Actinin was prepared according to the method of Ebashi et al. (11). The Mg-ATPase reaction was carried out at 25 Ž in a medium containing 30 mm KCl, 20 mm Tris-maleate buffer (ph 7.5), 2 mm MgCl2, 2 mm ATP, and an appropriate concentration (0.1 mg/ml) of myosin B, "desensitized" myosin B or recon it stituted actomyosin. The reaction was stopped by adding perchioric acid (to 5%), and the in organic orthophosphate (Pi) liberated was de termined according to Fiske and Subbarow (12), though p-methylaminophenol was used in place of 1, 2, 4-aminonaphthol sulfonic acid as the reducing reagent. The initial rate of phosphate liberation in the steady state (ƒêmol Pi per min per mg of protein) was used to express the Mg-ATPase ac tivity. Ca sensitivity was defined by the following relationship: "Superprecipitation" of "desensitized" myosin B was also studied by measuring absorbance at 550 nm (13). The temperature was maintained at 20 Ž. SDS-gel electrophoresis was carried out ac cording to Weber and Osborn (14). A gel rod of 10 % polyacrylamide was used, and Coomassie Blue was used to stain the protein bands. The protein concentration was determined by the biuret method. RESULTS AND DISCUSSION The Calcium and Strontium Sensitivities of Myosin B-The Mg-ATPase activity of myosin B in a low salt medium was measured with various concentrations of calcium or strontium ions. The concentration of free ions was calculated by assum ing that the stability constants under the experi mental conditions employed are 3 x 105 M-1 for Ca-EGTA complex and 4 x 102 M-1 for Sr-EGTA complex (15). In Fig. 1, the ATPase activity is plotted as a function of pca or psr (negative logarithms of the free calcium or strontium concen trations). It is clear (Fig. 1) that carp dorsal myosin B is essentially identical with rabbit skeletal myosin B in both calcium and strontium sensitivi ties. The half-maximal activation of ATPase occurred with either I ƒêm free calcium ions or 20 ƒêm free strontium ions. It is known (15, 16) that bovine cardiac myosin B has a higher strontium sensitivity than rabbit skeletal myosin B. Carp myosin B is thus different in strontium sensitivity from bovine cardiac myosin B. Relaxing Protein (or "Native" Tropomyosin) "Desensitized" myosin B's and relaxing proteins were prepared from rabbit skeletal and carp dorsal J. Biochem.

3 REGULATORY PROTEINS FROM CARP MUSCLE 933 fresh muscles. SDS-gel electrophoretic patterns of the proteins thus obtained are shown in Fig. 2. The effect of relaxing protein on the Mg-ATPase activity of desensitized myosin B was studied, and the results obtained are summarized in Table I. Carp relaxing protein appears to be as effective as rabbit relaxing protein in inhibiting Mg-ATPase of either carp or rabbit desensitized myosin B and in conferring calcium sensitivity on desensitized myosin B. Rabbit Desensitized Myosin B of Frozen Muscle-It was, however, found (Fig. 3) that rabbit desensitized myosin B prepared from "frozen" muscle (a week at -20 Ž) behaves differently from that prepared from "fresh" muscle, whereas carp desensitized myosin B obtained from frozen muscle behaves in the same way as that prepared from fresh muscle. When rabbit de sensitized myosin B prepared from frozen muscle was used, its Mg-ATPase activity was not inhibited by carp relaxing protein, but it was strongly in hibited by rabbit relaxing protein. On the other Fig. 1. Activation effect of calcium and strontium ions on the Mg-ATPase activity of carp dorsal and rabbit skeletal myosin B's. (A): The EGTA concentration was kept at 0.5 mm, and the concentration of SrCl2 was varied. 143 ƒêg/ml of carp myosin B ( ü) or 110 ƒêg/ml of rabbit myosin B ( ) was ued. (B): The CaCl2 concentration was kept at I mm, and the concentration of EGTA was varied. 143 ƒêg/ml of carp myosin B ( ü) or 107 ƒêg/ml of rabbit myosin B ( ) was used. ATPase assay was carried out at 25 Ž in a medium containing 2 mm MgCl2, 2 mm ATP, 30 mm KCl, 20 mm Tris maleate (ph 7.0 in A, and ph 6.7 in B). hand, when carp desensitized myosin B prepared from frozen muscle was used, both carp and rabbit relaxing proteins were equally effective in inhibiting Mg-ATPase and accordingly in conferring calcium sensitivity. Table II shows that the strontium sensitivity was also low with a combination of carp relaxing protein and rabbit desensitized myosin B from frozen muscle. The difference between rabbit and carp relaxing proteins was also observed when superprecipitation instead of Mg-ATPase ac tivity was studied (Fig. 4). Superprecipitation of Fig. 2. SDS-gel electrophoresis of myosin, actin, desensitized myosin B, troponin, tropomyosin, and relaxing protein obtained from fresh and frozen muscles of carp and rabbit. SDS gels (10% polyacrylamide) of 10 cm length and 0.5 cm diameter were pre pared according to Weber and Osborn. Electrophoresis was carried out in 0.1 M sodium phosphate (ph 7.0) containing 0.1 % SDS at a constant current of 8 ma per gel for 4 h. Approximately 50 tug of myosin and desensitized myosin B (d-myosin B), 20ƒÊg of actin and relaxing protein (RP), and 10 ƒêg of troponin (TN) and tropomyosin (TM) were applied to each gel rod. The proteins were obtained from rabbit fresh skeletal (R), rabbit "frozen" skeletal (R Œ), carp fresh dorsal (C), and carp "frozen" dorsal (C Œ) muscles. Vol. 82, No. 4, 1977

4 934 K. KONNO, K. ARAI, and S. WATANABE TABLE I. Effect of relaxing protein on the Mg-ATPase activity of desensitized myosin B prepared from "fresh" muscles of carp and rabbit. (A) ƒêg/ml of rabbit desensitized myosin B and 37.0 ƒêg/ml of rabbit relaxing protein or 47.4 ƒêg/ml of carp relaxing protein were used. (B) ƒêg/ml of carp desensitized myosin B and 30.0 ƒê g/ml of rabbit relaxing protein or 29.0 ƒêg/ml of carp relaxing protein were used. The Mg-ATPase assay was carried out at 25 C in a medium containing 20 mm Tris-maleate (ph 7.5), 30 mm KCl, 2 mm MgCl2, and 0.1 mm CaCl2 (+Ca) or 0.5 mm EGTA (-Ca). Ca sensitivity was calculated as described in "EXPERIMENTAL." RP and d-myosin B denote relaxing protein and desensitized myosin B, respectively. The activity is expressed in ƒêmol Pi/min/mg of myosin B. arp and rabbit relaxing proteins prepared from frozen muscles were as effective those as prepared from fresh muscles in inhibitin Mg-ATPase and superprecipitation of desensitized myosin B of fresh muscles. We investigated the possibility that rabbit desensitized myosin B from frozen muscle is in capable of binding carp relaxing protein. De sensitized myosin B and relaxing protein were homogenized in 0.1 M KCl (ph 7.5), and centri fuged at 20,000 x g. The sediment was then analyzed by SDS-gel electrophoresis. The results Fig. 3. Effect of relaxing protein on the Mg-ATPase activity of desensitized myosin B obtained from "frozen" muscle of carp and rabbit. (A) 112 ƒêg/ml of rabbit desensitized myosin B, and carp ( œ, ü) or rabbit (, ) relaxing protein. (B) 97 ƒêg/ml of carp desensitized myosin B, and carp ( œ, ü) or rabbit (, ) relaxing protein. œ +Ca; ü, -Ca. Conditions for the ATPase assay were as in Table I. rabbit desensitized myosin B from "frozen" muscle is only weakly inhibited by carp relaxing protein, whereas it is strongly inhibited by rabbit relaxing protein (Fig. 4A and 4B). On the other hand, when carp desensitized myosin B prepared from frozen muscle was used, both carp and rabbit relaxing proteins were equally effective in inhibiting superprecipitation (Fig. 4C and 4D). It should also be mentioned (though the results are not pre (though not presented) indicated that rabbit de sensitized myosin B from frozen muscle can bind carp relaxing protein as strongly as it binds rabbit relaxing protein. Troponin-The observations described above suggest that relaxing protein can be replaced by troponin plus tropomyosin. We were able to prepare troponin from carp dorsal muscle using the same procedures reported for the preparation of rabbit skeletal troponin (9). The patterns obtained on SDS-gel electrophoresis of carp and rabbit tro ponins are shown in Fig. 2. In agreement with the results of other investigators (9), rabbit troponin showed three protein bands of 37,000, 24,000, and 19,000 daltons. Carp troponin also showed three protein bands, though they corresponded to 30,000, 21,000, and 19,000 daltons. The electrophoretic patterns of tropomyosin and relaxing proteins obtained are also shown in Fig. 2. J. Biochem.

5 REGULATORY PROTEINS FROM CARP MUSCLE 935 TABLE II. Effect of relaxing protein (RP) on the Mg-ATPase activity of desensitized myosin B from "frozen" muscles of carp and rabbit: strontium sensitivity. (A) 75.2 ƒêg/ml of rabbit desensitized myosin B and 37.9 ƒêg/ml of rabbit relaxing protein or 44.8 beg/ml of carp relaxing protein were used. (B) ƒêg/ml of carp desensitized myosin B and 37.9 ƒêg/ml of rabbit relaxing protein or 44.5 ƒêg/ml of carp relaxing protein were used. The Mg ATPase assay was carried out at 25 Ž in a medium containing 20 mm Tris-maleate (ph 7.5), 30 mm KCl, 2 mm ATP, 12 mm MgCl2, and 0.5 mm EGTA plus 5 x 10-" m SrCl2 (+Sr) or 0.5 mm EGTA (-Sr). The activity was expressed in ƒêmol P, min/mg of myosin B. Sr sensitivity is calculated using the relationship: When desensitized myosin B obtained from frozen rabbit muscle was used (Fig. 5A), its Mg ATPase was only weakly inhibited by carp troponin (plus rabbit tropomyosin) but was strongly inhibited by rabbit troponin (plus rabbit tropomyosin). When desensitized myosin B obtained from frozen carp muscle was employed (Fig. 5B), its Mg- ATPase was strongly inhibited by carp troponin as well as by rabbit troponin. Rabbit desensitized Fig. 4. Effect of relaxing protein on superprecipitation of desensitized myosin B obtained from frozen muscles of carp and rabbit. Superprecipitation of 308 ƒêg/ml of rabbit (A, B) or 320 beg/ml of carp (C, D) desensitized myosin B was followed by measuring the absorbance at 550 nm. The reaction was carried out at 20 Ž in a medium containing 2 mm MgCl2, 0.5 mm ATP, 60 mm KCl, 20 mm Tris-maleate (ph 7.0), and 0.5 mm EGTA (open symbols) or 0.1 mm CaCl2 (filled symbols). The arrow on the curve with open symbols indicates addition of 1.7 mm CaCl2. Circles, carp relaxing protein; squares rabbit relaxing protein; triangles, control (with no relaxing protein). Fig. 5. Effect of troponin (plus tropomyosin) on the Mg-ATPase activity of desensitized myosin B obtained from frozen muscle of carp and rabbit. (A) 91.9 ƒêg/ml of rabbit desensitized myosin B. (B) 120 ƒêg/ml of carp desensitized myosin B. Circles ( œ, ü), carp troponin; squares (, ), rabbit troponin. Rabbit skeletal tropo myosin was always added at a concentration equal to the concentration of troponin added. Conditions for the ATPase assay were as in Table I. Vol. 82, No. 4, 1977

6 936 K. KONNO, K. ARAI, and S. WATANABE myosin B obtained from frozen muscle was thus different from that obtained from fresh muscle as regards response to carp relaxing protein or carp troponin, although they showed no difference in shows that the effect of heat treatment requires KCl at a concentration higher than 0.4 M. Effects very similar to those of heat treatment were observed when rabbit desensitized myosin B SDS-gel electrophoretic pattern (Fig. 2). Treatment with Heat or Urea-It was found, however, that rabbit desensitized myosin B prepared from frozen muscle can be made as responsive to carp relaxing protein as that prepared from fresh muscle by treating the desensitized myosin B with heat or urea. Figure 6 shows that the Mg-ATPase activities in the presence and absence of calcium ions are both reduced by heat treatment, but that the activity is more strongly reduced in the absence of calcium ions than in its presence, thus resulting in an increase in the calcium ions than in its pres ence, thus resulting in an increase in the calcium sensitivity of the heat-treated desensitized myosin B (30 to 40 Ž). The effect of heat treatment at 35 Ž was studied as a function of time. Figure 7 shows that rabbit desensitized myosin B of frozen muscle acquires calcium sensitivity in 10 to 15 min, while that obtained from fresh muscle is not affected in any way by the same heat treatment. Figure 8 Fig. 7. Heat treatment of desensitized myosin B's of rabbit fresh and frozen muscles, and resensitization to Cal+ by carp relaxing protein. (A) ƒêg/ml of desensitized myosin B of fresh rabbit muscle and 44.2 ƒê g/ml of carp relaxing protein. (B) leg/ml of desensitized myosin B of frozen rabbit muscle and 44.6 ƒêg/ml of carp relaxing protein. Desensitized myo sion B was incubated at 35 Ž for various times. Con ditions for the ATPase assay were as in Table I. The symbols used are the same as in Fig. 6. Fig. 6. Heat treatment of desensitized myosin B of rabbit frozen muscle, and resensitization to Cal+ by carp relaxing protein. Rabbit desensitized myosin B (4 mg/ml) in 0.6 M KCl-20 mm Tris-maleate, ph 7.5, was incubated at various temperatures for 30 min. After cooling to 0 Ž, the Mg-ATPase activity was assayed with 98.4 ƒêg/ml of rabbit desensitized myosin B and 46.5 leg/ml of carp relaxing protein ( œ -I-Ca; ü, -Ca). The Ca sensitivity ( ) was calculated as described in " EXPERIMENTAL." The Mg-ATPase activity of desensitized myosin B alone was also assayed (x). Conditions for the ATPase assay were as in Table T. Fig. 8. KCl concentration and heat treatment of rabbit desensitized myosin B of rabbit frozen muscle. Rabbit desensitized myosin B (4 mg/ml) was treated at 35 Ž for 30 min at various KCl concentrations. 105 leg/ml of rabbit desensitized myosin B and 50.3 leg/ml of carp relaxing protein were used for the Mg-ATPase assay. Conditions for the ATPase assay were as in Table I. The symbols used are the same as in Fig. 6. J. Biochem.

7 REGULATORY PROTEINS FROM CARP MUSCLE 937 of frozen muscle was incubated with urea at 25 Ž for 20 min. The Mg-ATPase activity was then measured in the presence of carp relaxing protein. Figure 9 shows that 1 to 1.5 M urea can make the desensitized myosin B responsive to the addition of carp relaxing protein. Since heat and urea treatments are generally thought to cause some change in the protein conformation, it is suggested that rabbit desensitized myosin B obtained from frozen muscle is different in conformation from that obtained from fresh muscle. On the other hand, it was reported by Fukazawa et al. (17) that freezing of rabbit skeletal or chicken breast muscle makes the Z-line structure more fragile. It is therefore possible that rabbit desensitized myosin B from frozen muscle is contaminated by a-actinin (11), one of the constituents of the Z-line structure - However, (though the result is not shown) addition of ƒ -actinin obtained from rabbit skeletal muscle to desensitized myosin B obtained from fresh rabbit muscle did not affect the Mg-ATPase activity of the latter. "Reconstituted" Actomyosin -We next in vestigated whether actin or myosin in the de Fig. 9. Urea treatment of desensitized myosin B of frozen muscle. Rabbit desensitized myosin B (2 mg/ml) in 0.6 M KCl-20 mm Tris-maleate (ph 7.5) was treated with various concentrations of urea at 25 Ž for 20 min. The treatment was halted by adding eight volumes of distilled water containing other components for the ATPase assay ƒêg/ml of rabbit desensitized myosin B and 50.3 ƒêg/ml of carp relaxing protein were used for the ATPase assay. Conditions for the ATPase assay were as in Table I. The symbols used are the same as Fig. 6. sensitized myosin B is changed, probably in conformation, upon freezing rabbit skeletal muscle. Myosins and actins were prepared from both frozen and fresh muscles of rabbit and carp. In the SDSgel electrophoretic patterns (Fig. 2), actins and myosins prepared from frozen muscles were not distinguishable from those obtained from fresh muscles. However, when the Mg-ATPase activity of "reconstituted" actomyosin was measured (sixteen different combinations), it was found that TABLE III. Resensitization effect of relaxing protein on the Mg-ATPase activity of "reconstituted" actomyosin. Myosins were always prepared from fresh muscles of carp (C) and rabbit (R). Actins were prepared from both fresh and frozen muscles. Relaxing protein (40% relative to actomyosin) was added to reconstituted actomyosin (1 part of actin and 3 parts of myosin by weight). Conditions for the ATPase assay were as described in Table I. The activity is expressed in ƒêmol Pi/min/mg of actomyosin. Vol. 82, No. 4, 1977

8 938 K. KONNO, K. ARAI, and S. WATANABE rabbit actin of frozen muscle behaves differently from the other three actins. Table III summarizes the results obtained for eight combinations with fresh myosins. Although omitted from the table, essentially the same results were obtained for eight other combinations with frozen muscle myosins. In other words, freezing of muscles caused no detectable change in carp and rabbit myosins. On the other hand, as shown in Table III, whenever rabbit actin of frozen muscle was used to reconsti tute actomyosin, the calcium sensitivity (or the relaxing protein-induced inhibition) was low. Freezing rabbit muscle must, therefore, have caused a definite change in rabbit actin, whereas it did not seem to cause any appreciable change in carp actin. It should, however, be noted that the effect of freezing rabbit skeletal muscle on the properties of the actin preparation does not fully account for that on the properties of desensitized myosin B preparation. The effect (for example, the inhibi tory effect) of carp relaxing protein observed here with actomyosin reconstituted by using rabbit actin of frozen muscle is stronger (50 or 55 in Table III) than that observed with rabbit desensitized myosin B of frozen muscle (10 or 16 in Table II). It was, moreover, observed (though not shown in the table) that even after heat or urea treatment of rabbit actin of frozen muscle, the inhibitory effect of carp relaxing protein on the reconstituted actomyosin was not enhanced: 59 before and 65 after urea treatment, and 59 before and 60 after heat treat ment. On the other hand, it was found that when rabbit actomyosin reconstituted from rabbit actin and myosin of fresh muscle was kept frozen at -20 Ž for 7-10 days, it behaved exactly like rabbit desensitized myosin B of frozen muscle. In other words, Mg-ATPase of the reconstituted actomyosin was only weakly inhibited (10-15 instead of 50-59) by carp troponin (plus tropomyosin), and was fully inhibited by carp troponin (plus tropomyosin) after urea or heat treatment. It may be reasonable to assume that rabbit actin (in the filament form) of frozen muscle assumes an irregular conformation, relative to a regular conformation assumed by rabbit actin obtained from fresh muscle, and also that actin in the irregular conformation binds irregularly with myosin. If so, the observations described above suggest that (a) Carp troponin is different from combined with tropomyosin) can normalize the irregular binding between actin and myosin. (b) The irregular binding in reconstituted acto myosin is different from that in desensitized myosin B of frozen rabbit muscle. In view of the following findings, the differences seem to be of a subtle nature. When carp relaxing protein was added to rabbit actin of frozen muscle prior to reconstituting actomyosin, it was found to be as effective as rabbit relaxing protein in inhibiting Mg-ATPase of the reconstituted actomyosin. In all other experiments presented in this report, relaxing protein was added after actin and myosin had been mixed. It is therefore suggested that the binding of relaxing protein to rabbit actin of frozen muscle restored the conformation of the latter to the regular one, which is in turn capable of binding in the regular way with the subsequently added myosin. REFERENCES 1. Takashi, R. (1973) Bull. Japanese Soc. Sci. Fisheries (Nihon Suisan Gakkai Shi) (Abstract in English) 39, Seki, N., Kitao, M., Konno, K., & Arai, K. (1973) Bull. Japanese Soc. Sci. Fisheries (Nihon Suisan Gakkai Shi) (Abstract in English) 39, Schaub, M.C., Hartshorne, D.J., & Perry, S.V. (1964) Biochem. J. 104, Takashi, R., Arai, K., & Saito, T. (1970) Bull. Japanese Soc. Sci. Fisheries (Nihon Suisan Gakkai Shi) (Abstract in English) 36, Connell, J.J. (1954) Biochem. J. 58, Spudich, J.A. & Watt, S. (1971) J. Biol. Chem. 246, Wilkinson, T.M., Perry, S.V., Cole, H.A., & Trayer, I.P. (1972) Biochem. J. 127, Arai, K. & Watanabe, S. (1968) J. Biochem. 64, Ebashi, S., Wakabayashi, T., & Ebashi, F. (1971) J. Biochem. 69, Bailey, K. (1948) Biochem. J. 43, Ebashi, S. & Ebashi, F. (1965) J. Biochem. 58, Fiske, C.H. & Subbarow, Y. (1925) J. Biol. Chem. 66, Yasui, T. & Watanabe, S. (1965) J. Biochern. 240, Weber, K. & Osborn, M. (1969) J. Biol. Chem. 244, Ebashi, S., Kodama, A., & Ebashi, F. (1968) J. Biochem. 64, Staprans, I., Takahashi, H., Russell, M.P., & Watanabe, S. (1972) J. Biochem. 72, Fukazawa, T. Hashimoto, Y., & Tonomura, Y. (1963) Biochim. Biophys. Ada 75, rabbit troponin in that only the latter (when J. Biochem.