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Effect of C-protein on actomyosin ATPase
141
Biochimica et Biophysica Acta, 632 (1980) 141--149 © Elsevier/North-Holland Biomedical P r e s s
BBA 29392
EFFECT OF C-PROTEIN ON ACTOMYOSIN ATPase
C. MOOS and I-NAN M. FENG
Department of Biochemistry, State University of New York, Stony Brook, N Y 11794
(U.S.A.) (Received March 3rd, 1980)
Key words: Actomyosin; A TPase; C-protein; Myosin; (Skeletal muscle)
Summary The effect of C-protein on the actin-activated ATPase of column-purified skeletal muscle myosin has been investigated at varied ionic strength. At ionic strengths below about 0.1, C-protein is a potent inhibitor. The inhibition is not reversed by increasing the actin concentration, showing that it is caused by C-protein bound to the myosin filaments. When the ionic strength is raised above about 0.12, on the other hand, the inhibition vanishes and C-protein becomes a mild activator of the actomyosin ATPase. Both effects appear rapidly upon addition of C-protein to pre-formed myosin filaments, so C-protein probably acts by binding to the surface of the filaments.
Introduction
C-protein is a component of the myofibrils in vertebrate skeletal muscle [1~2] which is structurally localized in vivo at specific positions on the thick filaments in the overlap region of the sarcomere [3,4]. It is thought to be attached to the shaft of the filament because it binds strongly in vitro not only to artificial myosin filaments but also to low ionic strength aggregates of myosin rod and light meromyosin [5]. More recently, it has also been shown to bind to F-actin, although less strongly than to myosin [6]. The interaction with actin is competitive with the binding of actin to myosin subfragment 1 (S-1) [6] and may be subject to control by the calcium-troponin system in intact filaments [7], suggesting that it might be physiologically significant. A third interesting property of C-protein is its ability to bind to the subfragment-2
Abbreviations: EGTA, ethylene glycol bis(~-aminoethyl ether)-N, N, N', N'-tetraacetate; S-1, subfrag-
ment 1 of myosin; S-2, subfragraent 2 of myosin.
142
{S-2) part of myosin, through which it could influence the movement of crossbridges in muscle [8]. The localization of C-protein on the thick filaments and its effects on the structure of myosin, myosin rod and light meromyosin aggregates, as seen in the electron microscope [5,9,10], suggested that it might play a role in thick filament architecture (see also Refs. 11 and 12), b u t its interactions with actin and S-2 raise the possibility that it might participate more actively in the contractile process. One experimental approach to this question has been to investigate the effect of C-protein on ATPase activity in various actomyosin systems. It has previously been shown that C-protein has no effect on myosin ATPase in the absence of actin [1], but that it inhibits the actin-activated ATPase of both myosin [1] and S-1 [6] at low ionic strength. In acto-S-1 the actin is the only c o m p o n e n t capable of binding C-protein (Starr and Offer [8] showed that C-protein does not bind S-1), so the ATPase inhibition probably results from competition between C-protein and S-1 for binding to actin [6]. In actomyosin, on the other hand, C-protein could bind to the myosin filaments as well as to actin. We have n o w investigated the effect of C-protein on actomyosin in detail. The inhibition at low ionic strength can reach 80% or more, and it is not reversed b y increasing the actin concentration, showing that C-protein bound to the myosin filaments is responsible. As the ionic strength is raised, however, the inhibitory effect diminishes, and in the physiological range of ionic strength, above a b o u t 0.12, C-protein activates the actomyosin ATPase. A preliminary report of some of these findings has appeared in abstract form [13]. Methods Proteins. Actin, column-purified myosin and C-protein were prepared from rabbit skeletal muscle as described previously [1,6]. A TPase measurements. The ATPase rates were measured on a pH-stat at pH 7.0 and 25°C [6,14] in reaction mixtures containing 2 mM ATP, 5 mM MgC12, 1 mM EGTA, and varying amounts of KC1. The EGTA was included to prevent unknown fluctuations in Ca 2÷ concentration, although the actin was free of tropomyosin and troponin and the experimental results were unchanged if the EGTA was omitted. We have found that actomyosin ATPase rates are sometimes significantly affected b y the manner of mixing the myosin with the actin, particularly whether or n o t an actomyosin complex is formed prior to addition of ATP. We have chosen to prepare myosin filaments separately, b y diluting a solution of myosin from 0.4 M KC1 to the ionic conditions of the particular experiment, and then to add these filaments to a reaction mixture containing the actin and ATP. In this way the myosin filaments are not subjected to ionic changes (other than addition of ATP) at the start of the ATPase reaction. C-protein, where present, was mixed with the myosin filaments before they were added to the ATPase reaction, except in Tables I and II where the sequence of addition was being studied. Variations in the time of preincubation of the C-protein and myosin from 5 min to 4 h did not affect the results. Different myosin preparations varied in their actin-activated ATPase activities under given conditions, without any accompanying differences in their Ca- or K-
143
EDTA~activated ATPases at 0.5 M KCI. These variations may have been due to accidental modification of the so-called Sa thiol [15]. However, the effects of C-protein reported here were observed with all myosin preparations. C-protein binding. To determine the amount of C-protein bound to myosin filaments, samples of the myosin/C-protein mixtures used in the ATPase assays were centrifuged, at 25°C, for 30 min at 100 000 X g, and the bound C-protein was calculated from the decrease in supernatant A280 relative to a control in the absence of myosin [5]. Electron microscopy. Samples for electron microscopy were taken from the myosin filament suspensions used for ATPase measurements, or from the ATPase mixtures during the reaction, and were placed on electron microscope grids with carbon films, fixed 30 s with 2.5% (w/v) glutaraldehyde without changing the ionic milieu, then rinsed with water and stained for negative contrast with uranyl acetate [5,6]. Micrographs were taken at a magnification of 36 000× on a JEOL Model 100B microscope.
Results
Low ionic strength. The effect of C-protein on actomyosin ATPase at approximately 0.09 ionic strength is illustrated in Fig. 1. The binding of C-protein to the myosin was accompanied by a marked ATPase inhibition which reached 80% at a binding ratio of 0.6 mol C-protein/mol myosin. To ascertain whether the inhibition was indeed caused by C-protein bound to the myosin rather than by free C-protein interacting with actin, the dependence of the ATPase rate on actin concentration was investigated in the presence and absence of C-protein. Fig. 2 shows that the inhibition was not relieved by increasing the actin concentration to more than 18 times the molar concentration of C-protein or myosin. If C-protein were inhibiting by binding to the actin, as it does in the acto-S-1 system studied previously [6], then addition of
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Fig. 1. I n h i b i t i o n o f a c t o m y o s l n A T P a s e b y C-protein at 0.09 ionic strength. C o n d i t i o n s : 65 mM KCI, 2 mM ATP, 5 mM MgCI2, 1 mM EGTA, 12 #M actin, 0.10--0.63 /~M myosin, e, ATPase; ~, C-protein binding to myosin . Fig. 2. Effect o f actin c o n c e n t r a t i o n o n C-protein i n h i b i t i o n . C o n d i t i o n s as in Fig. 10 w i t h 0.64 ~M C-protein an d 0.64 ~tM my osin. M a x i m u m ATPase w i t h o u t C-protein was 1.2 ~ m o l / m i n per mg myosin.
144
excess actin should overcome the inhibition. It is clear, therefore, that the C-protein here is acting on the myosin filaments. There appeared to be three ways in which C-protein could bind to myosin filaments that might account for its inhibitory effect on actomyosin ATPase. First, it might cause the filaments to aggregate, thus preventing many of the myosin molecules from interacting with actin. Second, it might modify the assembly of myosin molecules within the filament shaft [5,10] and, consequently, indirectly alter the steric constraints on myosin-actin interactions; and third, C-protein might bind to the surface of the 'filaments and directly influence the interaction of myosin crossbridges with actin through its interaction either with actin or with myosin S-2. The first alternative, aggregation of myosin filaments, is unlikely a priori because the ATPase of myosin filaments in the absence of actin is unaffected by C-protein, but it was tested directly by electron microscopic examination of samples of control and inhibited filaments and of the ATPase reaction mixtures themselves. As illustrated in Fig. 3, there was no evidence of filament aggregation in the presence of C-protein. These micrographs also failed to reveal noticeable changes in filament structure, but stronger evidence against the second mode of action of C-protein arose from a study of the timing of the effect. Table I shows that C-protein causes comparable inhibition whether it is present during formation of the myosin filaments or is added to preformed filaments either before or during the ATPase reac-
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Fig. 3. Myosin filaments w i t h and w i t h o u t C-protein, from the e x p e r i m e n t s h o w n in Fig. I. a, Myosin filam e n t s w i t h o u t C-protein; b, m y o s i n filaments with 2.06 m o l added C-protein per mol myosin; c, sample o f ATPase reaction m i x t u r e w i t h o u t C-protein; d, sample o f A T P a s e reaction m i x t u r e with 2.06 mol C-protein per m o l myosin. Bar is 1 # m . Fig. 4. Onset of ATPase inhibition u p o n addition of C-protein during a reaction at 0.09 ionic strength, traced f r o m an actual pH-stat record u n d e r the c o n d i t i o n s o f Table I. C-protein added at arrow; b r o k e n line s h o w s progress of a control reaction w i t h o u t C-protein.
145 TABLE I EFFECT OF SEQUENCE OF ADDITION ON ATPase INHIBITION BY C-PROTEIN C o n d i t i o n s : 0 . 2 1 / ~ M m y o s i n , 0 . 2 1 / ~ M C - p r o t e i n , 1 2 ~M a c t i n , 6 5 m M KCI. C-protein addition
ATPase (/~mol/min per mg myosin)
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0 . 7 5 3 +- 0 . 0 4 0 0 . 1 8 1 -+ 0 . 0 1 6 0 . 1 9 0 -+ 0 . 0 0 6 0 . 8 8 -+ 0 . 1 9 **
* M y o s i n in 0 . 5 M KCI m i x e d w i t h C - p r o t e i n in 6 5 m M K C I , giving a f i n a l KCI c o n c e n t r a t i o n o f 0 . 2 4 M, t h e n d i l u t e d t o 6 5 m M KCI. * * Rates before and after C-protein addition. -l/ c
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Fig. 5. E f f e c t o f i o n i c s t r e n g t h t o ATPase i n the absence (o) o r presence ( e ) o f C - p r o t e i n a d d e d t o m y o s i n f i l a m e n t s b e f o r e u s e . T o t a l i o n i c s t r e n g t h is 0 . 0 2 6 p l u s KCI c o n c e n t r a t i o n . F i n a l K C I e o n c e n i z a t l o n w a s adjusted in each reaction mixture, a, myosin filaments were formed by dilution to 50 mM KCI; reaction m i x t u r e s c o n t a i n e d 0 . 2 1 /AM m y o s i n , 0 . 2 1 # M C - p r o t e i n a n d 1 2 /~M a c t i n , b , myozfln f i l a m e n t s w e r e f o r m e d a t 1 0 0 m M K C I ; r e a c t i o n m i x t u r e s c o n t a i n e d 0 . 2 1 - - 2 . 1 /~M m y o s i n . 1 . 7 m o l C - p r o t e i n p e r m o l myosin, and 9.5/~M aetin.
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Fig. 7. Onset o f A T P a s e activation u p o n a d d i t i o n o f C-protein duzing a re a c t i on at 0.125 i o n i c strength. An actual pH-stat record u n d e r c o n d i t i o n s o f Table II; C-protein added at arrow. Fig. 8. E f f e c t o f actin c o n c e n t r a t i o n o n ATPase activation b y C-protein at 0.125 ionic strength. Myosin, 0.5--1 /~M. • 1.0 m o l C-protein/mol m y o s i n ; 4 no C-protein.
preformed filaments. Table II shows that the activation b y C-protein was similar whether the C-protein was added to the myosin before or after filament formation, or was added to the ATPase reaction mixture before the myosin. Likewise, varying the time of preincubation of the myosin filaments with C-protein from 5 min to 4.5 h did n o t change the activating effect of the C-protein. Addition of C-protein during the actomyosin ATPase reaction caused a rapid increase in rate (Fig. 7). Therefore, the ATPase activation, like the inhibition at lower ionic strength, is unlikely to depend on an internal restructuring of the myosin filaments. The effect of actin concentration is shown in Fig. 8. Although the activation is small, the proportional activation, a b o u t 25% in this case, is consistently found to be roughly constant for actin concentrations up to 1.5 mg/ml, the highest concentration tested. Since the ATPase rates do n o t approach actin saturation under these conditions, it cannot be determined from such experiments whether the activation represents an increase in the absolute ATPase of each activated myosin molecule or an increase in the fraction of myosin molecules activated, i.e., an increase in the effective affinity of the actin-myosin interaction. Discussion We have shown here that C-protein significantly alters the actin-activated ATPase of artificial filaments of purified myosin. At low ionic strength, C-protein is a p o t e n t inhibitor, whereas when the ionic strength is raised above a b o u t 0.12 the effect of C-protein becomes a less dramatic but, nevertheless, reproducible activation. Because the inhibition at low salt is n o t overcome by increasing the actin concentration, it is clear that C-protein b o u n d to the myosin filaments is responsible for the effect. Although a similar kinetic argument
148 cannot be made in the case of the activation at higher salt, it is likely that this effect too is caused by C-protein on the myosin filaments because more than half the added C-protein is bound to myosin and the remaining free C-protein, less than 0.05 mg/ml in Fig. 8, is unlikely to bind significantly to actin at this ionic strength [6]. The a m o u n t of C-protein normally present in myosin would have a noticeable but small effect in both cases. However, bound C-protein at a mole ratio of 1 : 3, which is approximately the proportion present in the C-protein-containing part of the thick filaments in muscle [1], would cause at least 50% inhibition at low ionic strength and about half-maximal activation under the conditions of Fig. 6. The effects of C-protein on actomyosin ATPase are, therefore, large enough to be significant in vivo. The rapid onset of both the inhibition and the activation indicates that the C-protein acts by binding to the surface of the filaments, which corresponds to the way it is attached to the thick filaments in muscle [4]. Beyond this, however, it is not clear how it produces either effect. One can consider three general hypotheses, none of which is ruled out by evidence now available. The first is a direct action on the myosin. A gross effect such as filament aggregation has been rejected, and if C-protein blocked access to myosin heads simply by burying them, its effect should be inhibitory even at high ionic strength. It is conceivable, however, that it alters the angles or distances at which the myosin heads project from the filament in a subtle, salt
149 no longer rate-limiting. Under these conditions, the ATPase rate might be limited by the mechanical restriction on the movement of attached crossbridges [18] imposed by constraints on the relative motions of the actin and myosin filaments. Interfilament C-protein bridges could then inhibit the ATPase by virtue of the additional impediment they would place on filament motion. In considering the implications of these results for intact muscle, a question that arises immediately is how C-protein influences actomyosin in which the actin filaments contain tropomyosin and troponin. Calcium-sensitive actomyosin systems are currently under investigation, and although ~ve are not yet in a position to present detailed results, it appears that the effects of C-protein reported here do occur in regulated actomyosin as well. That is, C-protein inhibits the ATPase at low ionic strength in the presence of calcium (but not in EGTA), but does not inhibit, and may activate slightly, at ionic strengths above about 0.12. In addition, at either ionic strength, it appears that the Ca2+ concentration required for half-maximal ATPase activation may be decreased when C-protein is added to the myosin filaments. In other words, the presence of C-protein seems to enhance Ca 2÷ binding. This would be consistent with the calcium-enhancement of C-protein binding to the thin filaments of myofibrils [7] if C-protein on the myosin filaments in actomyosin does indeed interact with the actin filaments as well.
Acknowledgements This work was supported by a grant from the Muscular Dystrophy Association and by U.S. National Science Foundation Grant PCM 77-26785.
References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Offer, G., Moos, C. and Start, R. (1973) J. Mol. Biol. 74,653---676 Morimoto, K. and Harrington, W.F. (1973) J. MoL Biol. 77, 165--175 Pepe, F.A. and Drucker, B. (1975) J, MoL Biol. 99, 609--617 Craig, R. and Offer, G. (1976) Proc. Roy. Soc., Set. B 192,451--461 Moos, C., Offer, G., Start, R. and Bennett, P. (1975) J. MoL Biol. 97, 1---9 Moos, C., Mason, C.M., Besterman, J.M., Feng, I.M. and Dubin, J.H. (1978) J. Mol. Biol. 124, 571-586 Moos, C. (197.9) Biophys. J. 2 5 , 2 4 4 a Start, R. and Offer, G. (1978) Biochem. J. 171,813---816 Chowrashi, P.K. and Pepe, F.A. (1977) J. Cell Biol. 74,136--152 Koretz, J.F. (1979) Biophys. J. 2 7 , 4 3 3 - - 4 4 6 Offer, G. (1972) Cold Spring Harbor Syrup. Quant. Biol. 37, 87--93 Sj~str6m, M. and Squire, J.M. (1977) J. Mol. Biol. 109, 49--68 Moos, C. and Feng, I.M. (1979) Fed, Proc. 38, 339 FAsenberg, E. and Moos, C. (1967) J. Biol. Chem. 242, 2945--2951 Horigome, T. and Yamashita, T. (1979) J. Biochem. 85,229--237 Maruyama, K. and Gergely, J. (1962) J. BioL Chem. 237, 1095--1099 Maruyama, K. and Gergely, J. (1962) J. Biol. Chem. 237, 1100--1106 Eisenberg, E. and Hill, T.L. (1978) Prog. Biophys. Mol. Biol. 33, 55--62
Biochimica et Biophysica Acta, 632 (1980) 141--149 © Elsevier/North-Holland Biomedical P r e s s
BBA 29392
EFFECT OF C-PROTEIN ON ACTOMYOSIN ATPase
C. MOOS and I-NAN M. FENG
Department of Biochemistry, State University of New York, Stony Brook, N Y 11794
(U.S.A.) (Received March 3rd, 1980)
Key words: Actomyosin; A TPase; C-protein; Myosin; (Skeletal muscle)
Summary The effect of C-protein on the actin-activated ATPase of column-purified skeletal muscle myosin has been investigated at varied ionic strength. At ionic strengths below about 0.1, C-protein is a potent inhibitor. The inhibition is not reversed by increasing the actin concentration, showing that it is caused by C-protein bound to the myosin filaments. When the ionic strength is raised above about 0.12, on the other hand, the inhibition vanishes and C-protein becomes a mild activator of the actomyosin ATPase. Both effects appear rapidly upon addition of C-protein to pre-formed myosin filaments, so C-protein probably acts by binding to the surface of the filaments.
Introduction
C-protein is a component of the myofibrils in vertebrate skeletal muscle [1~2] which is structurally localized in vivo at specific positions on the thick filaments in the overlap region of the sarcomere [3,4]. It is thought to be attached to the shaft of the filament because it binds strongly in vitro not only to artificial myosin filaments but also to low ionic strength aggregates of myosin rod and light meromyosin [5]. More recently, it has also been shown to bind to F-actin, although less strongly than to myosin [6]. The interaction with actin is competitive with the binding of actin to myosin subfragment 1 (S-1) [6] and may be subject to control by the calcium-troponin system in intact filaments [7], suggesting that it might be physiologically significant. A third interesting property of C-protein is its ability to bind to the subfragment-2
Abbreviations: EGTA, ethylene glycol bis(~-aminoethyl ether)-N, N, N', N'-tetraacetate; S-1, subfrag-
ment 1 of myosin; S-2, subfragraent 2 of myosin.
142
{S-2) part of myosin, through which it could influence the movement of crossbridges in muscle [8]. The localization of C-protein on the thick filaments and its effects on the structure of myosin, myosin rod and light meromyosin aggregates, as seen in the electron microscope [5,9,10], suggested that it might play a role in thick filament architecture (see also Refs. 11 and 12), b u t its interactions with actin and S-2 raise the possibility that it might participate more actively in the contractile process. One experimental approach to this question has been to investigate the effect of C-protein on ATPase activity in various actomyosin systems. It has previously been shown that C-protein has no effect on myosin ATPase in the absence of actin [1], but that it inhibits the actin-activated ATPase of both myosin [1] and S-1 [6] at low ionic strength. In acto-S-1 the actin is the only c o m p o n e n t capable of binding C-protein (Starr and Offer [8] showed that C-protein does not bind S-1), so the ATPase inhibition probably results from competition between C-protein and S-1 for binding to actin [6]. In actomyosin, on the other hand, C-protein could bind to the myosin filaments as well as to actin. We have n o w investigated the effect of C-protein on actomyosin in detail. The inhibition at low ionic strength can reach 80% or more, and it is not reversed b y increasing the actin concentration, showing that C-protein bound to the myosin filaments is responsible. As the ionic strength is raised, however, the inhibitory effect diminishes, and in the physiological range of ionic strength, above a b o u t 0.12, C-protein activates the actomyosin ATPase. A preliminary report of some of these findings has appeared in abstract form [13]. Methods Proteins. Actin, column-purified myosin and C-protein were prepared from rabbit skeletal muscle as described previously [1,6]. A TPase measurements. The ATPase rates were measured on a pH-stat at pH 7.0 and 25°C [6,14] in reaction mixtures containing 2 mM ATP, 5 mM MgC12, 1 mM EGTA, and varying amounts of KC1. The EGTA was included to prevent unknown fluctuations in Ca 2÷ concentration, although the actin was free of tropomyosin and troponin and the experimental results were unchanged if the EGTA was omitted. We have found that actomyosin ATPase rates are sometimes significantly affected b y the manner of mixing the myosin with the actin, particularly whether or n o t an actomyosin complex is formed prior to addition of ATP. We have chosen to prepare myosin filaments separately, b y diluting a solution of myosin from 0.4 M KC1 to the ionic conditions of the particular experiment, and then to add these filaments to a reaction mixture containing the actin and ATP. In this way the myosin filaments are not subjected to ionic changes (other than addition of ATP) at the start of the ATPase reaction. C-protein, where present, was mixed with the myosin filaments before they were added to the ATPase reaction, except in Tables I and II where the sequence of addition was being studied. Variations in the time of preincubation of the C-protein and myosin from 5 min to 4 h did not affect the results. Different myosin preparations varied in their actin-activated ATPase activities under given conditions, without any accompanying differences in their Ca- or K-
143
EDTA~activated ATPases at 0.5 M KCI. These variations may have been due to accidental modification of the so-called Sa thiol [15]. However, the effects of C-protein reported here were observed with all myosin preparations. C-protein binding. To determine the amount of C-protein bound to myosin filaments, samples of the myosin/C-protein mixtures used in the ATPase assays were centrifuged, at 25°C, for 30 min at 100 000 X g, and the bound C-protein was calculated from the decrease in supernatant A280 relative to a control in the absence of myosin [5]. Electron microscopy. Samples for electron microscopy were taken from the myosin filament suspensions used for ATPase measurements, or from the ATPase mixtures during the reaction, and were placed on electron microscope grids with carbon films, fixed 30 s with 2.5% (w/v) glutaraldehyde without changing the ionic milieu, then rinsed with water and stained for negative contrast with uranyl acetate [5,6]. Micrographs were taken at a magnification of 36 000× on a JEOL Model 100B microscope.
Results
Low ionic strength. The effect of C-protein on actomyosin ATPase at approximately 0.09 ionic strength is illustrated in Fig. 1. The binding of C-protein to the myosin was accompanied by a marked ATPase inhibition which reached 80% at a binding ratio of 0.6 mol C-protein/mol myosin. To ascertain whether the inhibition was indeed caused by C-protein bound to the myosin rather than by free C-protein interacting with actin, the dependence of the ATPase rate on actin concentration was investigated in the presence and absence of C-protein. Fig. 2 shows that the inhibition was not relieved by increasing the actin concentration to more than 18 times the molar concentration of C-protein or myosin. If C-protein were inhibiting by binding to the actin, as it does in the acto-S-1 system studied previously [6], then addition of
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Fig. 1. I n h i b i t i o n o f a c t o m y o s l n A T P a s e b y C-protein at 0.09 ionic strength. C o n d i t i o n s : 65 mM KCI, 2 mM ATP, 5 mM MgCI2, 1 mM EGTA, 12 #M actin, 0.10--0.63 /~M myosin, e, ATPase; ~, C-protein binding to myosin . Fig. 2. Effect o f actin c o n c e n t r a t i o n o n C-protein i n h i b i t i o n . C o n d i t i o n s as in Fig. 10 w i t h 0.64 ~M C-protein an d 0.64 ~tM my osin. M a x i m u m ATPase w i t h o u t C-protein was 1.2 ~ m o l / m i n per mg myosin.
144
excess actin should overcome the inhibition. It is clear, therefore, that the C-protein here is acting on the myosin filaments. There appeared to be three ways in which C-protein could bind to myosin filaments that might account for its inhibitory effect on actomyosin ATPase. First, it might cause the filaments to aggregate, thus preventing many of the myosin molecules from interacting with actin. Second, it might modify the assembly of myosin molecules within the filament shaft [5,10] and, consequently, indirectly alter the steric constraints on myosin-actin interactions; and third, C-protein might bind to the surface of the 'filaments and directly influence the interaction of myosin crossbridges with actin through its interaction either with actin or with myosin S-2. The first alternative, aggregation of myosin filaments, is unlikely a priori because the ATPase of myosin filaments in the absence of actin is unaffected by C-protein, but it was tested directly by electron microscopic examination of samples of control and inhibited filaments and of the ATPase reaction mixtures themselves. As illustrated in Fig. 3, there was no evidence of filament aggregation in the presence of C-protein. These micrographs also failed to reveal noticeable changes in filament structure, but stronger evidence against the second mode of action of C-protein arose from a study of the timing of the effect. Table I shows that C-protein causes comparable inhibition whether it is present during formation of the myosin filaments or is added to preformed filaments either before or during the ATPase reac-
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Fig. 3. Myosin filaments w i t h and w i t h o u t C-protein, from the e x p e r i m e n t s h o w n in Fig. I. a, Myosin filam e n t s w i t h o u t C-protein; b, m y o s i n filaments with 2.06 m o l added C-protein per mol myosin; c, sample o f ATPase reaction m i x t u r e w i t h o u t C-protein; d, sample o f A T P a s e reaction m i x t u r e with 2.06 mol C-protein per m o l myosin. Bar is 1 # m . Fig. 4. Onset of ATPase inhibition u p o n addition of C-protein during a reaction at 0.09 ionic strength, traced f r o m an actual pH-stat record u n d e r the c o n d i t i o n s o f Table I. C-protein added at arrow; b r o k e n line s h o w s progress of a control reaction w i t h o u t C-protein.
145 TABLE I EFFECT OF SEQUENCE OF ADDITION ON ATPase INHIBITION BY C-PROTEIN C o n d i t i o n s : 0 . 2 1 / ~ M m y o s i n , 0 . 2 1 / ~ M C - p r o t e i n , 1 2 ~M a c t i n , 6 5 m M KCI. C-protein addition
ATPase (/~mol/min per mg myosin)
Control: no C-protein A d d e d t o m y o s i n f i l a m e n t s a s in M e t h o d s Present during myosin filament formation * A d d e d d t t r i n g r e a c t i o n ( f r o m F i g . 4)
0 . 7 5 3 +- 0 . 0 4 0 0 . 1 8 1 -+ 0 . 0 1 6 0 . 1 9 0 -+ 0 . 0 0 6 0 . 8 8 -+ 0 . 1 9 **
* M y o s i n in 0 . 5 M KCI m i x e d w i t h C - p r o t e i n in 6 5 m M K C I , giving a f i n a l KCI c o n c e n t r a t i o n o f 0 . 2 4 M, t h e n d i l u t e d t o 6 5 m M KCI. * * Rates before and after C-protein addition. -l/ c
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Fig. 5. E f f e c t o f i o n i c s t r e n g t h t o ATPase i n the absence (o) o r presence ( e ) o f C - p r o t e i n a d d e d t o m y o s i n f i l a m e n t s b e f o r e u s e . T o t a l i o n i c s t r e n g t h is 0 . 0 2 6 p l u s KCI c o n c e n t r a t i o n . F i n a l K C I e o n c e n i z a t l o n w a s adjusted in each reaction mixture, a, myosin filaments were formed by dilution to 50 mM KCI; reaction m i x t u r e s c o n t a i n e d 0 . 2 1 /AM m y o s i n , 0 . 2 1 # M C - p r o t e i n a n d 1 2 /~M a c t i n , b , myozfln f i l a m e n t s w e r e f o r m e d a t 1 0 0 m M K C I ; r e a c t i o n m i x t u r e s c o n t a i n e d 0 . 2 1 - - 2 . 1 /~M m y o s i n . 1 . 7 m o l C - p r o t e i n p e r m o l myosin, and 9.5/~M aetin.
147 0.6 0.4
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Fig. 7. Onset o f A T P a s e activation u p o n a d d i t i o n o f C-protein duzing a re a c t i on at 0.125 i o n i c strength. An actual pH-stat record u n d e r c o n d i t i o n s o f Table II; C-protein added at arrow. Fig. 8. E f f e c t o f actin c o n c e n t r a t i o n o n ATPase activation b y C-protein at 0.125 ionic strength. Myosin, 0.5--1 /~M. • 1.0 m o l C-protein/mol m y o s i n ; 4 no C-protein.
preformed filaments. Table II shows that the activation b y C-protein was similar whether the C-protein was added to the myosin before or after filament formation, or was added to the ATPase reaction mixture before the myosin. Likewise, varying the time of preincubation of the myosin filaments with C-protein from 5 min to 4.5 h did n o t change the activating effect of the C-protein. Addition of C-protein during the actomyosin ATPase reaction caused a rapid increase in rate (Fig. 7). Therefore, the ATPase activation, like the inhibition at lower ionic strength, is unlikely to depend on an internal restructuring of the myosin filaments. The effect of actin concentration is shown in Fig. 8. Although the activation is small, the proportional activation, a b o u t 25% in this case, is consistently found to be roughly constant for actin concentrations up to 1.5 mg/ml, the highest concentration tested. Since the ATPase rates do n o t approach actin saturation under these conditions, it cannot be determined from such experiments whether the activation represents an increase in the absolute ATPase of each activated myosin molecule or an increase in the fraction of myosin molecules activated, i.e., an increase in the effective affinity of the actin-myosin interaction. Discussion We have shown here that C-protein significantly alters the actin-activated ATPase of artificial filaments of purified myosin. At low ionic strength, C-protein is a p o t e n t inhibitor, whereas when the ionic strength is raised above a b o u t 0.12 the effect of C-protein becomes a less dramatic but, nevertheless, reproducible activation. Because the inhibition at low salt is n o t overcome by increasing the actin concentration, it is clear that C-protein b o u n d to the myosin filaments is responsible for the effect. Although a similar kinetic argument
148 cannot be made in the case of the activation at higher salt, it is likely that this effect too is caused by C-protein on the myosin filaments because more than half the added C-protein is bound to myosin and the remaining free C-protein, less than 0.05 mg/ml in Fig. 8, is unlikely to bind significantly to actin at this ionic strength [6]. The a m o u n t of C-protein normally present in myosin would have a noticeable but small effect in both cases. However, bound C-protein at a mole ratio of 1 : 3, which is approximately the proportion present in the C-protein-containing part of the thick filaments in muscle [1], would cause at least 50% inhibition at low ionic strength and about half-maximal activation under the conditions of Fig. 6. The effects of C-protein on actomyosin ATPase are, therefore, large enough to be significant in vivo. The rapid onset of both the inhibition and the activation indicates that the C-protein acts by binding to the surface of the filaments, which corresponds to the way it is attached to the thick filaments in muscle [4]. Beyond this, however, it is not clear how it produces either effect. One can consider three general hypotheses, none of which is ruled out by evidence now available. The first is a direct action on the myosin. A gross effect such as filament aggregation has been rejected, and if C-protein blocked access to myosin heads simply by burying them, its effect should be inhibitory even at high ionic strength. It is conceivable, however, that it alters the angles or distances at which the myosin heads project from the filament in a subtle, salt
149 no longer rate-limiting. Under these conditions, the ATPase rate might be limited by the mechanical restriction on the movement of attached crossbridges [18] imposed by constraints on the relative motions of the actin and myosin filaments. Interfilament C-protein bridges could then inhibit the ATPase by virtue of the additional impediment they would place on filament motion. In considering the implications of these results for intact muscle, a question that arises immediately is how C-protein influences actomyosin in which the actin filaments contain tropomyosin and troponin. Calcium-sensitive actomyosin systems are currently under investigation, and although ~ve are not yet in a position to present detailed results, it appears that the effects of C-protein reported here do occur in regulated actomyosin as well. That is, C-protein inhibits the ATPase at low ionic strength in the presence of calcium (but not in EGTA), but does not inhibit, and may activate slightly, at ionic strengths above about 0.12. In addition, at either ionic strength, it appears that the Ca2+ concentration required for half-maximal ATPase activation may be decreased when C-protein is added to the myosin filaments. In other words, the presence of C-protein seems to enhance Ca 2÷ binding. This would be consistent with the calcium-enhancement of C-protein binding to the thin filaments of myofibrils [7] if C-protein on the myosin filaments in actomyosin does indeed interact with the actin filaments as well.
Acknowledgements This work was supported by a grant from the Muscular Dystrophy Association and by U.S. National Science Foundation Grant PCM 77-26785.
References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
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