Proc. Nat. Acad. Sci. USA Vol. 70, No. 11, pp. 3205-3209, November 1973

Regulatory Properties of Myocardial (ATPase activity/K +/Ca++) CLAUDIA FENNER*, DEAN T. MASONt*, ROBERT ZELISt*, AND JOAN WIKMAN-COFFELT*t * Section of Cardiovascular Medicine, * Departments of Medicine, t Biological Chemistry and t Physiology, University of California School of Medicine, Davis, California 95616 Communicated by Edwin G. Krebs, July 13, 1973

ABSTRACT The ATPase activity of purified myocardial centration of Ca++ in the presence of a low concentration of myosin was activated by either K+ or Ca++; the addition Na+; on the other hand, Ca++ depressed contraction at a of one in the presence of the other caused inhibition. According to Hill-plot analyses the K+-saturation curves high concentration of Na+. were sigmoidal (n = 2.92), while the Ca++-saturation curves were hyperbolic (n = 1.25). Ca++-saturation curves EXPERIMENTAL PROCEDURES in the presence of K+ were inhibitory with sigmoidicity (n = 4.11), while K+-saturation curves in the presence of Extraction and Purification of Myocardial Myosin (5-7, 21). Ca++ followed the Michaelis-Menten inhibition kinetics Hearts were dissected from dogs and treated as described (5). (n = 1.11). Substrate saturation curves were hyperbolic All steps were done at 40 except where indicated. The tissue for both Ca++ and K+ systems. There was no enzymatic was minced and then sheared 10 times, for 10 sec each time activity when Na+ was used as the activating metal; fur- thermore, Na+ inhibited in the presence of either K+ or (Sorvall Omni-mixer, 40,000 rpm) in 2.5 volumes of 0.05 M Ca++. Both Na+ curves of inhibition followed the Michaelis- P04 buffer (pH 6.8) containing 1 mM EDTA, 0.01 M Na Menten relationship. pyrophosphate, and 1 mM dithiothreitol. The tissue was washed three times in this low salt buffer. After each wash the Myosin has two enzymatically active heads (HMM-S) minced tissue was centrifuged for 5 min at 10,000 X g and the (1, 2), each head being able to function independently (3). pellet obtained was sheared for the same period of time in 3 HMM-S1 is obtained by papain digestion of myosin, then volumes of 0.3 M KCl-0.1 M KH2POr-0.05 M K2HPO- further purified, and shown to contain the myosin ATPase 0.01 M Na pyrophosphate-1 mM dithiothreitol-1 mM EDTA activity (4). A single head can be removed from myosin by for extraction of myosin. After it was stirred for 10 min and papain digestion in a low-salt concentration leaving the en- then centrifuged at 1300 X g for 10 min, the supernatant zyme with one head and half its original ATPase activity (1). was removed and again centrifuged at 9000 X g for 15 min. We developed procedures to obtain immunologically and elec- The twice-centrifuged supernatant was filtered through two trophoretically pure myocardial myosin (5, 6, 21)and showed layers of cheese-cloth and then precipitated with 9 volumes of there is a difference in turnover rates of the myocardial chains water, containing 2 mM EDTA, and stirred for 30 min. After (7). We then sought to determine kinetically if there is any co- it was centrifuged at 10,000 X g for 10 min, the pellet was operative interaction between the two ATPase sites of pure suspended (2 mg of protein per ml) in 0.05 M Na pyro- myosin when no protein effectors are present, thereby pro- phosphate (pH 7.5)-i mM EDTA-1 mM dithiothreitol, and viding an indication of whether there is some interaction be- then made 2 mM in ATP. After it was stirred for 10 min, the tween myosin subunits. myosin was centrifuged at 50,000 X g for 20 min to precipi- Furthermore, this study was designed to determine if the tate contaminant proteins (5). By use of saturated (NH4)2SO4 antagonistic effects that Na+ and/or K+ have on Ca++ physi- adjusted to pH 6.8, the fraction between 35 and 42% satu- ologically are reflected in the effects of these ions on the ration with (NH4)2SO4 was collected and the pellet was dis- ATPase activity of purified myosin. Busselen and Carmeliet solved by homogenizing in 0.05 M Tris - HCl (pH 7.5)-i mM (8) showed that low concentrations of NaCl (0.03 M) elevated dithiothreitol-0.5 M KCl. The myosin was then dialyzed in the the effects of Ca++ on peak isometric tension of the hearts of same buffer overnight and centrifuged at 40,000 X g for 15 min various animals, including dogs, over a range of CaCl2 con- to precipitate the insoluble contaminants. centrations from 1-10 mM but depressed mechanical con- traction at high concentrations of NaCl (0.14 M); the time Myosin ATPase Activity. Assay A (Substrate-saturation to peak tension was greater in either at high concentrations with potassium-EDTA-activated ATPase). Potassium- of CaCl2 and the depressing effects of high NaCl concentra- EDTA-activated ATPase activity of myosin was measured tions were again proportional. According to Brustaert et al. in a mixture containing 0.1 M Tris HCl (pH 7.5), 5 mM (9), increasing CaCl2 concentrations from 2.5 to 7.5 mM in EDTA, 0.65 M KCl, and various concentrations of ATP. bathing solutions containing a low concentration of NaCl Assay B (Substrate-saturation with calcium-activated ATP- augmented the maximum unloaded shortening velocity of cat ase) (10). Calcium-activated ATPase activity was measured papillary muscle. These experiments indicated that the in 0.2 M Tris adjusted to pH 6.5 with maleic acid, 0.01 M strength of contraction was elevated by increasing the con- CaCl2, and various concentrations of ATP. Assay C (Potas- sium-EDTA--saturated ATPase). The reaction mixture con- Abbreviation: HMM, heavy meromyosin. tained 0.1 M Tris * HCl (pH 7.5), 4 mM ATP, 5 mM EDTA, 3205 Downloaded by guest on October 3, 2021 3206 Biochemistry: Fenner et al. Proc. Nat. Acad. Sci. USA 70 (1978)

E 0.4 -

Z 0.3- E

0.2-

0.1

0.2 0.4 0.6 0.8 1.0 mg Protein FIG. 1. Comparative release of Pi with increasing concentra- tions of the , myosin ATPase, for both the K+ (EDTA) and Ca++ reactions. Assay conditions were those of assay A for K + (EDTA) activation and assay B for the Ca++ activation. Saturating concentrations of ATP were used (4 mM). (0) 5 mM EDTA + 0.65 M KCl; (A) 10 mM CaCl2.

and various concentrations of KCl. Assay D (Calcium-satu- rated ATPase). ATPase was measured in 0.2 M Tris * maleate (pH 6.5), 4 mM ATP, and various concentrations of CaCl2. Assay E (Potassium-EDTA-inhibited-ATPase in the presence of CaCl2). Potassium inhibition was measured in 0.2 M Tris- maleate (pH 6.5), 4 mM ATP, 0.01 M CaCl2, and various con- ATM, mM centrations of KCl. Assay F (Calcium-inhibited ATPase in FIG. 2. Dependence of myosin ATPase activity on the the presence of KCl). Calcium inhibition was measured in concentration of substrate, ATP, in the presence of activators. 0.1 M Tris HCl (pH 7.5), 4 mM ATP, 2 mM EDTA, 0.65 M (A) With KCI, as in assay A. Vmax = 0.56 AM Pi/mg-min; KCl, and various concentrations of CaCl2. Assay C was used n = 1.17; S0.5 = 0.48 mM. (B) With CaCI2 as in assay B. Vmax = for Na+ saturation curves, except similar concentrations of 0.36,M Pi/mg-min; n = 1.12; SO.5 = 0.29 mM. Inset is a plot for KCl. Assays E and F were used of log [v/Vmax - v] against the ATP concentration as plotted NaCl were substituted semilogarithmically. Vmax was obtained from reciprocal plots of for inhibition curves of Na+ except NaCl was substituted for v against substrate concentration. SO.5 is the concentration of KCl in Assay E and NaCl was substituted for CaCl2 in Assay substrate giving 50% of the maximal activity. F. All assays were done in a total volume of 2 ml. Assays A, C, and F were incubated at 250 for 5 min, while assays B, D, assay buffer to be used for enzymatic analyses. Protein con- and E were incubated at 300 for 5 min. The reactions were centrations were determined according to Lowry et al. (12). stopped by addition of 1 ml of 20% trichloroacetic acid; 1-ml RESULTS aliquots were assayed for Pi by the method of Fiske and Sub- All was washed in phosphate-free de- Fig. 1 is a correlation between increasing enzyme concentra- baRow (11). glassware K+ tergent. All myosin solutions were dialyzed against the specific tion and the velocity of myosin ATPase activity where served as a metal, compared to the Ca++ enzyme. Incubation mixtures were chosen for the two metals such that myosin TABLE 1. Summary of differences between would remain in solution. For 5-min incubations, 250 was the K+ and Ca++ ATPase optimal temperature for monovalent cation activation of myosin while 30° was optimal for divalent cation activation. K+ enzyme Ca++ enzyme We showed that the pH optimum for monovalent cation acti- vation of myosin was pH 7.5, while for the divalent cations it ATP saturation Hyperbolic; n 1 Hyperbolic; n _ 1 Rate against K+ Sigmoidal curves of Michaelis-Menten was 5.5 (Wikman-Coffelt, J., Zelis, R., Fenner, C. & Mason, concentration activation; n > 2 curves for inhibi- D. T., Nature New Biol., submitted). Myosin, prepared by tion; n 1 the procedures presented here, was shown in other studies Rate against Ca++ Sigmoidal curves of Activation curves to be electrophoretically and immunologically pure myosin concentration inhibition; n > 2 are essentially (5-7,21) and gave similar enzymatic activity compared to hyperbolic; that which we obtained by a more lengthy procedure (5). n. 1 Fig. 2 is the substrate saturation curves for these two en- Rate against Na+ Michaelis-Menten Michaelis-Menten zyme reaction systems. To determine if there are two inter- concentration curves for in- curves for in- for _ _ acting substrate-binding sites, we fitted the kinetic data hibition; n 1 hibition; n 1 ATP hydrolysis to the empirical Hill equation. The kinetics Downloaded by guest on October 3, 2021 Proc. Nat. Acad. Sci. USA 70 (1973) Myocardial Myosin 3207 of activation by substrate gave no evidence for interaction between ATP sites: the n value for the Hill equation was close to 1 for either Ca++ or K+ and the curves were essen- tially hyperbolic for both. High concentrations of ATP had some inhibitory effects on the K+ enzyme but no effects were noted for the Ca++ enzyme. The Vmax (0.56MuM Pi/mg -min) E bi 0.5 -+1.5 for K+ activation curves was higher than that for Ca++ E o7i +1.0 activation curves (0.36 AM Pi/mg. min). The amount of ATP 0.4_ needed to reach half-maximal saturation (S0.5) was 0.48 mM E5> +0.5- E Ef 0 for the K+-activated enzyme, somewhat higher than the 0.29 C~~~~~~~~~KI-0.5 mM needed for half-maximal saturation of the Ca++ enzyme. 0.2 -1.0 There was no enzymatic activity when Na+ was substituted for K+ in assay A or when Na+ was substituted for Ca++ in 0.100t~0.01 0.1 [KCIJ 1.0 10.0 assay B. 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 Fig. 3 shows the saturation curve for KCl and CaCl2. KCI, M Either alone activate and are essential for enzymatic activity. B Fig. 3 shows that 50% of the maximal activation (Ao.5) af- 0.4- fected by KCl occurs at a concentration of 0.45 M. Since the Vmax for KCl was large and out of physiological range, 0.65 M KCl was used in assays A and F so as not to deviate too 0.3- far from cellular conditions. The n value (Hill coefficient) for the K+-activation curve was larger than 2 (Fig. 3A), thus showing strong sigmoidicity. There was little enzymatic acti- -1.5- vation at low K+ concentrations. E +1.~~~~~0. 1. 1. The concentrations of CaCl2 required for half-maximal > activation (Ao.5) was 1.9 mM (Fig. 3B), indicating a greater affinity of myosin for Ca++ compared to its affinity for K+ where the Ao.5 for KCl was 0.45 M. A Hill plot of the data gave an n value of 1.25 for CaCl2 saturation curves (Fig. 3B, inset); the curves were essentially hyperbolic. The Hill equation can be applied to the kinetics of inhibi- FIG. 3. Activation of myocardial myosin ATPase activity. tion (13). A plot of enzyme activity as a function of increasing (A)ByKC asinassayC.n = 2.92;Ao. = 0.45M. (B) By CaCis concentrations of inhibitor at a saturating concentration of as in assay D. n = 1.25; A0.5 = 1.9 mM. A0.5 is the concentration substrate (4 mM ATP) gave a straight line. In Fig. 4A, with of salt required for 50% of maximal stimulation, and n is the 10 mM CaCl2 and assay E, the concentration of KCl at which Hill interaction coefficient. Inset is a Hill plot of the data, where half-maximal inhibition by K+ occurred was 1.52 M v is the velocity at a given concentration of calcium or potassium; (Io.5) change in rate is the increase in velocity due to saturating con- KCl. The kinetics of inhibition by K+ in the presence of Ca++ centrations of (A) potassium and (B) calcium. Avv is the increase gave an n value of about 1, which was quite different from in velocity due to the addition of activator, i.e., the velocity the sigmoidal activation curves with K+ where no other obtained upon addition of a certain amount of activator to the metal was present (assay C). This same type of inhibition was reaction mixture minus the velocity of the reaction mixture noted when Na+ (Fig. 5A) was substituted for K+ using containing no activator. Vmax was obtained from double reciprocal assay E; the Io.5 for NaCl was 1.05 M and the n value was 1.16. plots of v against activator concentration. [The Hill plot (inset) in The inhibition curve for NaCl followed a Michaelis-Menten B is the inhibition data in the presence of 0.65 M KCl.J relationship. The kinetics of inhibition by Ca++ in the presence of K+ did not follow the classical Michaelis-Menten relationship A summary of the kinetics data, as presented here, is shown (Fig. 4B). The curves were sigmoidal and the n value was 4.11 in Table 1. (Fig. 4B, inset). Thus in the presence of 0.65 M KCl, low concentrations of Ca++ had little effect on the enzyme whereas DISCUSSION concentrations larger than 2 mM became inhibitory. The con- Having developed a procedure for purification of myocardial centration of CaCl2 needed to reach half-maximal inhibition myosin which, according to electrophoresis and immunology, (Io.5) was 6.1 mM CaCl2, smaller than the 10.5 for KCl. At contained no major contaminants (5-7,21), we could examine lower concentrations of activator (0.3 M KCl), the inhibition the kinetics of myosin, as presented here, without the inter- curves for CaCl2 were again sigmoidal and the n value was the ference of inhibitors or activators (14, 15). It has been pro- same as that described for CaCl2 inhibition in the presence of posed that myosin has two independent sites which bind ATP 0.65 M KCl. Inhibition was again noted when Na+ was sub- (2). According to our Hill-plot analyses for the ATP satura- stituted for Ca++ in the presence of K+ (Fig. 5B). However, tion curves, there was no cooperative interaction between the inhibition curves for Na+ were not sigmoidal as in Ca++ these sites, with either the K+ or theCaau enyzme; the n inhibition. The n value for Na+ inhibition curves was 1.16; value was about 1 for both and the curves were hyperbolic. the curves followed a typical Michaelis-Menten relationship. In the two activation curves with ion saturation, the Kin The Io.5 for NaCl was 0.11 M, one-tenth that in the presence saturation curve was highly sigmoidal, giving Hill constants of CaCl2. greater than 2; there was little activation at low concentra- Downloaded by guest on October 3, 2021 3208 Biochemistry: Fenner et al. Proc. Nat. Acad. Sci. USA 70 (1973)

A

0.4 +1.5 100, + 1.0 _\O ~i> +0.5 0.3 > 0 c n -0.5 E ° 0 0 1.0 (2 0E ~~~~~~~- 0.2 -1.5 E 0.1 1.0 10.0 100.0 [KCI] 0.1

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 KCI, M B NaCI, M 0.4 +1.5 100l +1.0 0.3 E 0E I 0 0 E 0.2 -0.5 >( -1.0 ._ -1.5 0.1 1.0 10 [Ca 102 103

2 4 6 8 10 12 14 16 18 20 CaCI2, mM FIG. 4. Inhibition of myosin ATPase activity. (A) By KCl in presence as assay n = 1.11; = the of 10 mM of CaCl2 in E. 1o.5 NaCI, M 1.52 M. (B) By CaCl2 in the presence of 0.65 M KCl (A) as in assay F and (0) 0.30 M KCl. n = 4.11; Io.5 = 6.1 mM. Inset: (A) Increasing concentrations of KCl with respect to log[v/Vo - FIG. 5. Inhibition of myosin ATPase activity by NaCl in the v] where V0 is the amount of Pi released in 1 min in the absence presence of (A) 10 mM CaCl2 as in assay E (n = 1.16; Io.5 = of inhibitor, KCl, and v is the observed velocity at a given con- 1.05 M), and (B) 0.65 M KCl as in assay F (n = 1.16; Io.s = centration of inhibitor; (B) Increasing concentrations of CaCl2 0.11 M). Inhibition kinetics are as those described in Fig. 4. with respect to log[v/Vo - v] where Vo is the amount of Pi released in 1 min in the absence of inhibitor, CaCl2. Io.5 is the concentration of inhibitor giving 50% inhibition under the condi- tions of the experiment. yses, this inhibition was sigmoidal (n = 4.11). The sensitivity of the enzyme to inhibition was not modulated by activator concentrations. Thus, at a lower concentration of KCl (0.3 tions of KCl. Bowen and Kerwin (16), working with skeletal M), Ca++ was still inhibitory, giving the same sigmoidal muscle myosin, have demonstrated that the EDTA-ATPase curve but with lower maximum activity (Vo). Using various activity is greatly accelerated by increasing the KCl concen- conditions, with or without EDTA, we did not find an acti- tration. The saturation curve for CaCl2 was hyperbolic and vating effect of Ca++ in the presence of K+ as reported by the n value was about one. The Ao.5 for KCl was 0.41 M while other investigators (18). We attribute our results, partly, to that for CaCl2 was only about 1.9 mM, showing a greater ap- a highly purified enzyme with good specific activity (5, 6, 21). parent affinity of the enzyme for Ca++. There was no myosin Increasing concentrations of KCl also had inhibitory effects ATPase activity when Na+ was substituted as the activating on myosin ATPase activity in the presence of CaCl2 (10 mM), ion. Other investigators have shown that NaCl has no acti- similar to that noted by Trayer and Perry for vating effects on the EDTA-ATPase of skeletal muscle myo- myosin (19). The inhibitory effect with K+ was not sigmoidal sin (16,17). In the presence of either Ca++ or K+, Na+ was (n - 1) in contrast to the allosteric kinetics obtained with K+ inhibitory; the 1o.5 for NaCl in the presence of Ca++ was 1.05 myosin in the absence of Ca++ (n > 2). M, 10-fold higher than its Io.5 in the presence of K+, which It has been proposed that muscle contraction is directly re- was 0.11 M. lated to the enzymatic activity of myosin (20). Thus, the alter- It was reported that increasing concentrations of Ca++ ations in mechanical contraction observed with various concen- activated myocardial myosin ATPase activity in the presence trations of Ca++ and Na+ or Ca++ and K+ (8, 9) may be a di- of 0.5 M KCl (18). In contrast, we found that low concentra- rect result of the influence of these ions on myosin ATPase ac- tions of Ca++ in the presence of 0.5 M KCl had no effect on tivity. The concentration of Na+ in the presence of Ca++, the enzymatic activity while higher concentrations of CaC12 and vice versa, which activated or depressed mechanical con- (2-10 mMI) in the presence of KCl inhibited the myosin traction (8, 9), had also similar results on myosin ATPase ATPase activity. Furthermore, according to Hill-plot anal- activity as described in these studies. Downloaded by guest on October 3, 2021 Proc. Nat. Acad. Sci. USA 70 (1973) Myocardial Myosin 3209

This paper was supported by Research Program Project 9. Brustaert, D. L., Claes, V. A. & Goethals, M. A. (1973) Grant HL-14780 and Health Sciences Advancement Award "Effects of calcium on force-velocity-length relations of RR-06138 from the National Institutes of Health, and a Grant heart muscle of the cat," Circ. Res. 32, 385-392. from the Sacramento-Yolo-Sierra Heart Association. 10. Luchi, R. J., Kritcher, E. M. & Conn, H. L. (1965) "Mo- lecular characteristics of canine cardiac myosin," Circ. Res. 1. Margossian, S. S. & Lowey, S. (1973) "Substructure of 16, 74-82. myosin molecule III. Preparation of single-headed deriva- 11. Fiske, C. H. & SubbaRow, Y. (1925) "Estimation of in- tives of myosin," J. Mol. Biol. 74, 301-311. organic phosphate," J. Biol. Chem. 66, 375-380. 12. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, 2. Maruyama, K. & Weber, A. (1972) "Binding of adenosine R. J. (1951) "Protein measurement with the Folin phenol triphosphate to myofibrils during contraction and relaxa- reagent," J. Biol. Chem. 193, 265-275. tion, " Biochemistry 11, 2990-2998. 13. Changeau, J. P. (1963) "Allosteric interaction on bio- 3. Margossian, S. S. & Lowey, S. (1973) "Substructure of synthetic L-threonine deaminase from E. coli K12," Cold myosin molecule IV. Interaction of myosin and its sub- Spring HarborSymp. Quant. Biol. 28, 497-504. fragments with adenosine triphosphate and F-actin," 14. Luchi, R. J. & Kritcher, E. M. (1966) "Cardiac myosin J. Mol. Biol. 74, 313-330. ATPase activity" Circ. Res. 19, 283-294. 15. Spudich, J. A. & Watt, S. (1971) "The regulation of rabbit 4. Mueller, H. & Perry, S. V. (1962) "The degradation of skeletal muscle contraction," J. Biol. Chem. 246, 4866- heavy meromyosin by trypsin," Biochem. J. 85, 431-437. 4871. 5. Wikman-Coffelt, J., Zelis, R., Fenner, C. & Mason, D. T. 16. Bowen, W. J. & Kerwin, T. D. (1954) "A study of the (1973) "Myosin chains of myocardial tissue I. Purification effects of ethylenediaminetetraacetic acid in myosin adeno- and immunological properties of myosin heavy chains," sinetriphosphatase," J. Biol. Chem. 211, 237-243. Biochem. Biophys. Res. Commun. 51, 1097-1104. 17. Kielley, W. W., Kalckar, H. M. & Bradley, L. B. (1956) 6. Wikman-Coffelt, J., Zelis, R., Fenner, C. & Mason, D. T. "The hydrolysis of purine and pyrimidine nucleoside (1973) "Studies on the synthesis and degradation of light triphosphates by myosin," J. Biol. Chem. 219, 95-103. and heavy chains of cardiac myosin," J. Biol. Chem. 248, 18. Brahms, J. & Kay, C. M. (1963) "Molecular and enzymatic 5206-5207. properties of cardiac myosin A as compared with those of skeletal myosin A," J. Biol. Chem. 238, 198-205. 7. Wikman-Coffelt, J., Zelis, R., Fenner, C. & Mason, D. T. 19. Trayer, I. P. & Perry, S. V. (1966) "The myosin of develop- (1973) "Purification and immunological properties of ing skeletal muscle," Biochem. Z. 345, 87-100. myocardial myosin light chains," J. Biol. Chem. in press. 20. Barany, M. (1967) "ATPase activity of myosin correlated 8. Busselen, P. & Carmeliet, E. (1973) "Protagonistic effects with speed, muscle shortening," J. Gen. Physiol. 50, 6 Pt, of Na+ and Ca++ on tension development in cardiac muscle 197. at low extracellular Na+ concentrations," Nature New Biol. 21. Wikman-Coffelt, J., Nelis, R., Fenner, C. & Mason, D. T., 243, 57-59. Prep. Biochem. in press. Downloaded by guest on October 3, 2021