Calcium Regulation in Squid Mantle and Scallop Adductor Muscles 1

. Biochem. 89, 581-589 (1981) Calcium Regulation in Squid Mantle and Scallop Adductor Muscles 1 Kunihiko KONNO,* Ken-ichi ARM,* Mikiharu YOSHIDA,** and Shizuo WATANABE*** Department of Food Science, Faculty of Fisheries, Hokkaido University, Hakodate, Hokkaido 041, **Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo, Hokkaido 060, and ***Department of Chemistry, Faculty of Science, Tokyo Institute of Technology, Ookayama, Meguro-ku, Tokyo 152 Received for publication, July 14, 1980 Calcium binding to the regulatory light chain was studied by an equilibrium dialysis method, and it was found that calcium binding to the regulatory light chain in an isolated form was qualitatively different from that in a bound form, i.e., in myosin. This finding can acount for our previous observation (1979) that the calcium or strontium concentration required for inducing difference spectra in the regulatory light chain (in an isolated form) was higher than that required for activating ATPase or for superprecipitation of actomyosin (in a bound form). Most of the findings obtained by Asada et al. (1979) and Ashiba et al. (1980) for clam foot myosin were confirmed with squid mantle myosin and scallop adductor myosin. Therefore, the following properties are probably not confined to clam foot myosin but are common to myosins from molluscan muscles. (1) The Mg- ATPase activity of myosin alone was sensitive to calcium. (2) Removal of the regulatory light chain resulted in a reversible loss of superprecipitation ability. (3) As the ATP concentration increased, the ATPase activity of actomyosin changed in a biphasic manner, whereas that of myosin alone changed in a monophasic manner. Two of our observations appear to favor the suggestion of Asada et al. and Ashiba et al. that the primary action of calcium is to activate myosin-atpase rather than to induce actin-myosin bindings. (1) Desensitized myosin (free from the regulatory light chain) behave exactly like untreated myosin in the presence of calcium. For example, as the ATP concentration increased, the ATPase activity of myosin alone and that of actomyosin changed qualitatively and quantitatively in the same way as those of untreated myosin and acto-untreated myosin in the presence of calcium. (2) At KC1 concentrations between 0.2 M and 0.4 M, actin-activation of myosin (in the presence of calcium) was absent whereas calcium sensitivity of myosin-atpase was still detectable. Downloaded from http:jb.oxfordjournals.org at Penn State University (Paterno Lib) on May 9, 2016 1 This investigation was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. Presented at the "Kumagai-Natori" conference, held in October, 1979 at Lake Kawaguchi, Japan. Abbreviation: EGTA, ethyleneglycol bis(^-aminoethylether)a r,'-tctraacetate. Vol. 89, No. 2, 1981 581

582 K. KONNO, K. ARAI, M. YOSHIDA, and S. WATANABE In the early 1970's, Kendrick-Jones, Szent-Gyogyi, and their associates (7, 2) showed that the component responsible for calcium regulation in molluscan muscles is myosin-linked, and that it is in fact one of the two types of light chain subunits of myosin. In particular, they showed that regulatory light chains of scallop adductor myosin are released on EDTA treatment, and that removal of regulatory light chains results in loss of calcium sensitivity of actomyosin-atpase (desensitized). Recently, Asada et al. (3), studying clam foot myosin, found that removal of regulatory light chains results in uncoupling of the actomyosin- ATPase reaction from the actomyosin-superprecipitation reaction. Meanwhile, studying calcium regulation in squid mantle muscle, we were able to isolate both troponin (4) and regulatory light chains (5) in a pure form. We showed that myosin-linked regulation rather than actin-linked regulation is dominantly operating in calcium regulation of squid mantle myosin B, and that calcium and strontium ions induce difference spectra in the UV absorption of regulatory light chains (squid LC-2 and scallop EDTA-LQ but not in that of the second type of light chains (squid LC-1 and scallop SH-LQ. Calcium-induced formation of difference spectra was also reported by Nishita et al. (6) to occur with regulatory light chains isolated from Akazara adductor myosin. MATERIALS AND METHODS Squid mantle and scallop striated adductor myosins, and their regulatory light chains (LC-2 and EDTA-LQ were prepared as described in our previous papers (4, 5). Rabbit skeletal actin was obtained by the method of Spudich and Watt (7), and pyruvate kinase was prepared by the method of Tietz and Ochoa (<S). ATPase activity was determined by measuring either the liberated Pi (9) or the liberated pyruvate (10). Superprecipitation was studied by measuring changes in absorbance at 550 nm (see Ref. 5). In the study of the ATP-concentration dependence, an ATP-regenerating system of pyruvate kinase and phosphoenol pyruvate was used to maintain the ATP concentration. Calcium bindings to myosin and to its regulatory light chains were measured by an equilibrium dialysis method, using "Ca (11). The equilibrium dialysis was conducted at 20 C in 0.1 M KC1 and 20 mm histidine buffer (ph 6.8), or in 0.3 M KC1 and 20 mm histidine buffer (ph 6.8). The free Ca 1+ concentration was set by using Ca-EGTA buffer (12). RESULTS Calcium Binding Previously (5), we studied the effects of calcium and strontium ions on the UV absorption spectra of light chain subunits isolated from squid myosin and from scallop adductor myosin. We then found (a) that the divalent cations induced difference spectra in regulatory light chains (squid LC-2 and scallop EDTA-LQ but not in the second type of light chains (LC-1 and SH-LQ, and (b) that the strontium and calcium concentrations required for inducing difference spectra were much higher than those required for activation of the Mg-ATPase of myosin B. In other words, the results obtained were qualitatively in favor of, but quantitatively inconsistent with, the possibility that the divalent cation-induced change in the regulatory light chain conformation may be the first step in a series of reactions, which lead to "contraction" of actomyosin. In the present study, we measured calcium binding with regulatory light chains in an isolated form and with myosins (light chains in a bound form). Isolated regulatory light chains were purified by Sephadex G-75 gel filtration. The molecular size of scallop EDTA-LC estimated by the gel filtration was about 5x10* daltons and that of squid LC-2 was about 2x10* daltons (data not shown). Scallop EDTA-LC was therefore suggested to be in an aggregated form. Calcium binding to regulatory light chain was then measured by using "Ca in equilibrium dialysis (11). As shown in Fig. 1, isolated light chains had two types of calcium binding sites: calcium binding with the first type of sites occurred at about 1-10 fim free calcium whereas that with the second type occurred at about 100-1,000 M free calcium. The amount of calcium bound to the first type of sites was small: approximately 0.4 mol of calcium per mol of squid LC-2 (1.5x10* daltons), and 0.15 mol of calcium per mol of scallop EDTA-LC (1.7x10* daltons). Moreover, the amount of calcium bound was greatly reduced when 2.5 mm Downloaded from http:jb.oxfordjournals.org at Penn State University (Paterno Lib) on May 9, 2016. Biochem.

Ca REGULATION IN SQUID AND SCALLOP 583 MgCl, was present. Lower calcium binding ability of isolated scallop EDTA-light chain was also reported by Szent-Gyorgyi et al. (2). In the present study, we found that squid regulatory light chain retained its calcium binding ability even if it was released from heavy chain. In this respect, squid regulatory light chain is clearly different from EDTAlight chain of scallop. On the other hand, as shown in Fig. 2, calcium binding with myosins was very different from 1.0.3 0 (A) 0, y O. (B) 3 7 pca Fig. 1. Calcium binding to isolated regulatory light chains from squid and from scallop muscles. Calcium binding was measured at 20 C by using "Ca in equilibrium dialysis conducted in a medium containing 0.1 M KC1, 20 mm histidine (ph 6.8), and no (O) or 2.5 min MgCli (). Free calcium concentration was set by using a Ca-EGTA buffer system. (A) Squid LC-2, (B) scallop EDTA-LC. j o that with isolated light chains. The amount of calcium bound with myosin was about 2 mol per mol of myosin (4.8 x 10 5 daltons), which is equivalent to 1 mol of calcium per mol of light chain (in a bound form). Only one type of binding site was detectable, and the half-maximal binding occurred at about 10~ s - 7 M free calcium with squid myosin, and at about 10~ 6-1 M free calcium with scallop myosin. These concentrations of free calcium ions were about equal to those required Fig. 2. Calcium binding to myosins from squid and scallop muscles. Calcium binding was measured under the conditions described in Fig. 1, except that 0.3 M KC1 was used instead of 0.1 M KC1. The same symbols as in Fig. 1 are used. (A) Squid myosin. (B) Scallop myosin. Downloaded from http:jb.oxfordjournals.org at Penn State University (Paterno Lib) on May 9, 2016 Fig. 3. Calcium binding to desensitized and nsensitized scallop myosins. Calcium binding was measured under the same conditions as in Fig. 1. Symbols are the same as in Fig. 2. (A) Desensitized scallop myosin, (B) resensitized scallop myosin with squid LC-2, and (Q resensitized scallop myosin with scallop EDTA-LC. Vol. 89, No. 2, 1981

584 K. KONNO, K. ARAI, M. YOSHIDA, and S. WATANABE for activation of the ATPase of acto-squid myosin and for that of acto-scallop myosin (see Figs. 4 and 5A). Therefore, the difference between the calcium concentration required for activating myosin B-ATPase and that required for inducing difference spectra of isolated light chains may be understood as the difference in calcium binding between a bound form of light chains and an isolated form of light chains (Figs. 1 and 2). Moreover, we were also able to show (Fig. 3) that the regulatory light chain is responsible for the calcium binding described above. We studied the effect of desensitization and resensitization of myosin on its calcium binding ability. Figure 3 shows that "desensitized" scallop myosin, which was completely free from EDTA-LC, lost its calcium binding ability (A), and that the lost ability was fully recovered by adding either scallop EDTA-LC (Q or squid LC-2 (B) to desensitized scallop myosin (approximately 2 mol of light chain per mol of myosin). It should be pointed out that calcium binding to "resensitized" myosin, like that to untreated myosin (see Fig. 2), was unaffected by the addition of 2.5 mm MgCl 2. We found that scallop myosin and scallop resensitized myosin were capable of binding approximately 2 mol of calcium per mol of myosin.6 to I c I S.2 (A) 0- ' 1 o o'' 0 (B) o o-o-o o- pca Fig. 4. Calcium requirement for activation of Mg- ATPase of squid myosin and for activation of that of acto-squid myosin. Mg-ATPase of myosin alone (O) and of actomyosin () were assayed in a medium containing 30 mm KC1, 20 mm Tris-maleate (ph 6.8), 2 mm MgCl,, and 1 mm ATP. Rabbit skeletal actin (0.09 mgml) was combined with squid mantle myosin (0.19 mgml) to obtain actomyosin. Free calcium concentration was set by using a Ca-EGTA buffer system. (C) f - (D) 7 o-o.o' 0^ 5 pca Fig. 5. Calcium requirement for the activation of Mg-ATPases of scallop myosins and of rabbit acto-scallop myosins. ATPase was assayed under the conditions described in Fig. 4, and the same symbols as in Fig. 4 are used. (A) Scallop untreated myosin, (B) scallop desensitized myosin, (Q scallop resensitized myosin with squid LC-2, and (D) scallop resensitized myosin with scallop EDTA-LC. * Downloaded from http:jb.oxfordjournals.org at Penn State University (Paterno Lib) on May 9, 2016. Biochem.

Ca REGULATION IN SQUID AND SCALLOP 585 even if MgCli was absent. Moreover, EDTAlight chain in an isolated form was found to have little calcium binding ability at around 10 m calcium ions (see Fig. 1). Therefore, these results rather suggest that EDTA-light chain was present in a form bound to myosin even if MgCl s was absent. This view was further supported by the recent finding of Ojima et al. (personal communication) that the release of EDTA-light chain from Akazara scallop myosin was prevented by the presence of small amounts of calcium even if MgCl t was absent. Calcium Sensitivity As shown in Figs. 4 and 5A, Mg-ATPase of squid myosin as well as that of scallop myosin was activated by the same concentration (1-100 ftm) of free calcium as those of rabbit acto-squid myosin and of rabbit acto-scallop myosin were. However, it should be noted that scallop myosin showed a higher ATPase activity than squid myosin did in the absence of calcium. We then studied, using scallop desensitized myosin, the role of regulatory light chain in the calcium sensitivity of myosin alone. Figure 5 shows that the Mg-ATPase activity of "desensitized" scallop myosin as well as that of actodesensitized scallop myosin lost calcium sensitivity. The ATPase activity of scallop myosin in the calcium medium (lo-'-lo" 6 M free calcium) was unaffected by EDTA washing, whereas the activity in the absence of calcium (10~ 8-10~ 7 M) was greatly increased by the same washing procedure, thus resulting in loss of calcium sensitivity. The calcium sensitivity of the ATPase activity of actoscallop myosin was lost upon EDTA washing, but the ATPase activity in the presence of calcium was also reduced by the same washing procedure (B). Calcium sensitivity of myosin ATPase and that of actomyosin ATPase were both recovered on addition of either squid LC-2 (Q or scallop EDTA-LC (D). Moreover, the reduced ATPase activity of acto-desensitized myosin (B) was also recovered on addition of regulatory light chains. Similar results were also obtained by measuring superprecipitation activity instead of ATPase activity. Figure 6A shows that acto-scallop myosin lost its superprecipitation ability as EDTA washing of scallop myosin progressed, even in the presence of calcium. Figure 6 (B and Q shows that the lost ability was recovered on addition of either squid LC-2 or scallop EDTA-LC. The two o E I"' o.2 (B) (C) 4 6 8 10 TIME (min) Fig. 6. Effects of desensitization and of resensitization on the superprecipitation of rabbit acto-scallop myosin. Superprecipitation was measured in a medium containing 30 mm KC1, 20 mm Tris-maleate (ph6.8), 2 nun MgCI,, 0.1 mm CaCl,, and 0.5 mm ATP. The turbidity (absorbance) change of actomyosin suspension after addition of ATP was followed at 550 nm. (A) Actodesensitized scallop myosin; numbers in the figure denote the numbers of repetitions of EDTA-washing. (B and C) Acto-resensitized scallop myosin; numbers in the figure denote the amounts of squid LC-2 (B) and of scallop EDTA-LC (C) added to desensitized scallop myosin (%, ww). regulatory light chains were equally effective in the recovery of superprecipitation ability. The recovery was saturated when the amount of regulatory light chains added reached about 7% of that of desensitized scallop myosin, which is approximately equivalent to a molar ratio of 11 for light chainheavy chain. This value (7 %) was also equal to that required for recovery of calcium sensitivity in the ATPase activity (5). ATP-Concentration Dependence We studied the calcium sensitivities of myosin alone, of myosin B and of actomyosin from squid and scallop muscles. The results thus obtained showed some qualitative similarities to and some quantitative variations from the results reported by Ashiba et al. (13), who studied clam foot myosin. Figure Downloaded from http:jb.oxfordjournals.org at Penn State University (Paterno Lib) on May 9, 2016 Vol. 89, No. 2, 1981

586 K. KONNO, K. ARAI, M. YOSHIDA, and S. WATANABE 7 shows the ATP-concentration dependence of the superprecipitation activity of myosin B's from squid and scallop muscles. Superprecipitation occurred in the absence of calcium when the ATP concentration was very low (1-10 JM ATP). Moreover, superprecipitation of both myosin B's 10 (A) A (B) A? -Log [ATP) (M) Fig. 7. ATP-concentration dependence of the superprecipitation activities of myosin B's from squid and scallop muscles. Superprecipitation was measured in a medium containing 30 mm KC1, 20 HIM Tris-maleate (ph 6.8), 2 mm MgCl,, either 0.1 mm CaCl, (), or 0.5 mm EGTA (O) and various concentrations of ATP (1 im-1 mm). Pyruvate kinase (0.3 mgm]) and phosphoenol pyruvate (1 mm) were added to maintain the ATP concentration. Superprecipitation activity was expressed in terms of AAxlJT U t, where A A is the maximal increase in turbidity, and rut is th e me rfrquired to reach the half-maximal increase. (A) Squid t' myosin B. (B) Scallop myosin B. was practically insensitive to calcium at 1-10 JUM ATP. The superprecipitation activity of both myosin B's, either in the absence or presence of calcium, responded in a biphasic manner to change in the ATP concentration, and the maximal activity was obtained at about 3-10 [in ATP in the absence of calcium and at about 100-300 (IM ATP in the presence of calcium. Figure 8 shows that the ATPase activities of scallop and squid myosin B's responded essentially in the same manner as their superprecipitation activities did (Fig. 7). Figure 9 shows that Mg-ATPase activity of squid myosin and that of scallop myosin responded in a monophasic manner to change in the ATP concentration. We also observed (see also Fig. 5) that the Mg-ATPase activity of scallop myosin was higher than that of squid myosin in the absence of calcium at all the ATP concentrations tested, and accordingly that calcium sensitivity at 1 fita ATP was more obvious with squid myosin than with scallop myosin. In order to study the effects of regulatory light chain on the ATP concentration dependence of myosin alone and on that of actomyosin, we examined the ATP-concentration dependence of "desensitized" scallop myosin and that of actodesensitized scallop myosin as well as those of "resensitized" scallop myosin and of acto-resensitized myosin. As shown in Fig. 10, the essential features of the ATP-concentration dependence of both myosin-atpase and actomyosin-atpase were unaffected by desensitization and by resensitization: for example, as the ATP concentration increased, myosin-atpase increased in a monophasic Downloaded from http:jb.oxfordjournals.org at Penn State University (Paterno Lib) on May 9, 2016 3 2 6 3 -Log [ATP] (M) Fig. 8. ATP-concentration dependence of Mg-ATPases of myosin B's from squid and scallop muscles. The ATPase activity of myosin B was assayed under the conditions described in Fig. 7, and the same symbols as in Fig. 7 are used. (A) Squid myosin B, and (B) scallop myosin B. 3 6 9 -Log IATP1 Fig. 9. ATP-concentration dependence of Mg-ATPases of myosins from squid and scallop muscles. The ATPase activity was assayed under the conditions described in Fig. 7, and the same symbols as in Fig. 7 are used. (A) Squid myosin, and (B) scallop myosin. J. Biochem.

Ca REGULATION IN SQUID AND SCALLOP 587 6 -Log [ATPJ Fig. 10. Effects of desensitization and of resensitization on the ATP-concentration dependence of Mg-ATPases of myosin and of actomyosin from scallop muscle. The ATPase activity was assayed under the conditions described in Fig. 7. (A,«) 0.1 mm CaCl,, (A,O) 0.5 mm EGTA, and (0,0) myosin alone, (A,A) actomyosin. (A) Desensitized scallop myosin, (B) resensitized with squid LC-2, and (C) resensitized with scallop EDTA-LC. manner whereas actomyosin-atpase did so in a biphasic manner. It should be pointed out that resensitized myosin with squid LC-2 showed much lower ATPase activity than did that with scallop EDTA-LC, in the absence of calcium. These differences would arise not from differences in the properties of heavy chains but from differences in the properties of squid LC-2 and scallop EDTA- LC. In accord with the observations of Ashiba et al. (13) on clam foot myosin, we observed that calcium sensitivity of squid myosin-mg-atpase was detectable even if the ph of the ATPase assay was changed from 6.0 to 9.5 (data not shown). Moreover, we observed that change in the temperature of the ATPase assay from 5 C to 30 C also did not affect the calcium sensitivity of squid myosin (data not shown). As shown in Fig. 11 A, calcium sensitivity was observed in the ATPase activity of squid myosin alone when the KCl concentration was lower than 0.4 M, whereas it was not observed in high-salt media (higher than 0.4 M KCl). On the other hand, calcium-modulated Mg-ATPase activity of acto-squid myosin was higher than that of squid myosin alone when the KCl concentration was lower than 0.2 M; in other words, actin-activation was observed only when the KCl concentration was lower than 0.2 M. Figure 11B shows that in Fig. 11. KCl-concentration dependence of Mg-ATPase and that of superprecipitation of acto-squid myosin. (A) The ATPase activity of myosin (, O) and that of acto-squid myosin (A, A) were assayed at 25 C in a medium containing 20 mm Tris-maleate (ph 6.8), 2 mm MgClt, 1 mm ATP, either 0.1 HIM CaCl, (closed) or 0.5 mm EGTA (open), and various concentrations of KCl (from 0.03 M to 0.5 M). (B) The superprecipitation activity of acto-squid myosin was assayed under the conditions used for the ATPase activity (A), but in the presence of 0.1 mm CaCl,. The activity was expressed as described in Fig. 7..3 Downloaded from http:jb.oxfordjournals.org at Penn State University (Paterno Lib) on May 9, 2016 Vol. 89, No. 2, 1981

588 K. KONNO, K. ARAI, M. YOSHTDA, and S. WATANABE accordance with the latter observation in Fig. 11 A, superprecipitation of acto-squid myosin occurred only when the KCl concentration was lower than 0.2 M. We were thus able to distinguish the effect of KCl concentration on the actin-activation from that on calcium sensitivity of myosin alone. It is therefore suggested that binding of the light chain to myosin heavy chain may not be directly related to the binding of actin to heavy chain. Further work on this point is now in progress. DISCUSSION Asada et al. (3) and Ashiba et al. (13) studied clam foot myosin and reported four findings: (a) Mg-ATPase of myosin alone was as sensitive to calcium or strontium as that of rabbit acto-clam myosin. (b) When the regulatory light chain was removed from clam foot myosin, the superprecipitation ability of rabbit acto-clam myosin in the presence of calcium was reversibly lost, whereas its ATPase activity remained unaffected in the presence of calcium and increased in the absence of calcium, (c) As the ATP concentration increased, the ATPase activity of actomyosin (or that of myosin B) in the presence of calcium changed in a biphasic manner, whereas that of myosin alone in the presence of calcium increased in a monophasic manner, (d) Calcium sensitivity of myosin ATPase was detectable even when the KC1 concentration was sufficiently high to dissociate actin from myosin. We were able to confirm all four findings, by using squid mantle myosin and scallop adductor myosin in place of clam foot myosin. It is therefore suggested that the four properties of myosin and of actomyosin are probably common to myosins and actomyosins from molluscan muscles. Moreover we added two new findings to observations (c) and (d). As shown in Fig. 10, desensitized myosin and acto-desensitized myosin behaved in essentially the same manner as undesensitized myosin and acto-undesensitized myosin in the presence of calcium; that is, when the ATP concentration was increased above 0.3 mm, the ATPase activity of actomyosin decreased, presumably due to dissociation of actin from myosin. On the assumption that the decrease in the actomyosin- ATPase activity is due to dissociation of actin from myosin, the ATP concentration required for dissociation of actomyosin was the same, regardless of whether the regulatory light chain was present or absent, and in spite of the fact that the amount of calcium bound to myosin was supposed to be sufficient to keep myosin-atpase fully activated. Similarly, as shown in Fig. 11, at KCl concentrations between 0.2 M and 0.4 M, no actin-activation of myosin-atpase, and no superprecipitation of actomyosin occurred, though the amount of calcium bound to myosin was supposed to be sufficient to keep myosin-atpase activated. Therefore, the two observations described (see Figs. 10 and 11) seem to favor the suggestion of Asada et al. and Ashiba et al. that the primary role of the regulatory light chain is to inhibit myosin-atpase rather than to inhibit actin-myosin bindings, and accordingly that the primary action of calcium is to activate (or to remove the inhibition of) myosin-atpase rather than to induce (or to remove the inhibition of) actin-myosin bindings. It can be seen in Fig. 5B that upon removal of regulatory light chains, the activity level of actomyosin-atpase was lowered whereas that of myosin-atpase stayed high. This decrease in actomyosin-atpase activity may correspond to the decrease in the superprecipitation ability of acto-desensitized myosin (Fig. 6). However, removal of regulatory light chain did not always result in decrease in the actomyosin-atpase activity (5). Moreover, Asada et al. (personal communication) observed that removal of regulatory light chains invariably resulted in loss of the superprecipitation ability but not always in decrease in the actomyosin-atpase activity. It is therefore suggested that removal of regulatory light chains is followed by a subsequent change, perhaps in the myosin conformation, which may result in a change in the actin binding site. The authors are grateful to Professor K. Yagi of Hokkaido University for valuable discussions and encouragement. REFERENCES 1. Kendrick-Jones, J., Lehman, W., & Szent-Gydrgyi, A.G. (1970) J. Mol. Biol. 54, 313-326 2. Szent-Gydrgyi, A.G., Szentkiralyi, E.M., & Kendrick-Jones, J. (1973). Mol. Biol. 74, 170-203 Downloaded from http:jb.oxfordjournals.org at Penn State University (Paterno Lib) on May 9, 2016. Biochem.

Ca REGULATION IN SQUID AND SCALLOP 589 3. Asada, T., Ashiba, G., & Watanabe, S. (1979) 9. Fiske, C.H. & Subbarow, Y. (1962). Biol. Chem. J. Biochem. 85, 1543-1546 66, 375-^00 4. Konno, K. (1978). Biochem. 84, 1431-1440 10. Reynard, A.M., Haas, L.F., Jacobsen, D.D., & 5. Konno, K., Arai, K., & Watanabe, S. (1979) J. Boyer, P.D. (1961). Biol. Chem. 236, 2277-2291 Biochem. 86, 1939-1950 11. Kuwayama, H. & Yagi, K. (1979). Biochem. 85, 6. Nishita, K., Ojima, T., & Watanabe, S. (1979). 1245-1255 Biochem. 86, 663-673 12. Ebashi, S., Kodama, A., & Ebashi, F. (1968) 7. Spudich, J.A. & Watt, S. (1971). Biol. Chem. 246,. Biochem. 64, 465^77 604-613 13. Ashiba, G., Asada, T., & Watanabe, S. (1980) 8. Tietz, A. & Ochoa, S. (1958) Arch. Biochem. Biophys. J. Biochem. 88, 837-846 78, 477^93 Downloaded from http:jb.oxfordjournals.org at Penn State University (Paterno Lib) on May 9, 2016 Vol. 89, No. 2, 1981