The aggregation characteristics of column-purified rabbit skeletal myosin in the presence and absence of C-protein at pH 7.0.

The aggregation properties of column-purified rabbit skeletal myosin at pH 7.0 were investigated as functions of ionic strength, protein concentration, and time. Filaments prepared by dialysis exhibited the same average length and population distribution at 0.10 and 0.15 M KCl at protein concentrations greater than 0.10 mg/ml; similar results were obtained at .0.20 M KCl, although average filament length was approximately 0.5 micrometer shorter. Once formed, these length distributions remained virtually unchanged over an 8-d period. At and below 0.10 mg/ml, average filament length decreased as a function of protein concentration; filaments prepared from an initial concentration of 0.02 mg/ml were half the length of those prepared at 0.2 mg/ml. Filaments prepared by dilution exhibited a sharp increase in average length as the time-course increased up to 40 s, then altered only slightly over a further period of 4 min. Addition of C-protein in a molar ratio of 1-3.3 myosin molecules affected most of these results. Average filament length was affected neither by ionic strength nor by initial protein concentration down to 0.04 mg/ml or over an 8-d period. Filaments formed by dilution in the presence of C-protein exhibited a constant average length and hypersharp length distribution over variable time courses up to 7 min. It is possible that C-protein acts to stabilize the antiparallel intermediate during filamentogenesis, and may also affect subunit addition to this nucleus.     

Hydrostatic pressure studies of native and synthetic thick filaments: in vitro myosin aggregates at pH 7.0 with and without C-protein

Column-purified myosin at pH 7.0 will reproducibly aggregate into filaments of known average length and structure when dialyzed against a low ionic strength medium under controlled conditions. When exposed to increased hydrostatic pressure, followed by quick return to atmospheric pressure, the original filaments shorten linearly with increasing pressure; in addition, a second population of filaments is seen, presumably the result of reaggregation of myosin after release of pressure. This second population is about 0.5 microns long, bipolar, and about half the diameter of the original filaments. The number of these filaments, but not their physical characteristics, is a function of the shortening of the original filament population. Both the remnants of the original population and the new aggregates, once formed, are stable over time and at room temperature. The addition of C-protein to myosin solutions before filament preparation results in a filament population of slightly shorter length. When these filaments are exposed to increased hydrostatic pressure, they are more resistant to disaggregation than myosin filaments without C-protein. However, like the filaments prepared in the absence of C-protein, a second population of shorter, thinner filaments is visible after exposure to pressure.     

Developmental relationship of myosin binding proteins (myomesin, connectin and C-protein) to myosin in chicken somites as studied by immunofluorescence microscopy

The developmental relationship of myosin binding proteins (myomesin, connectin and C-protein) to myosin was studied in chicken cervical somites by immunofluorescence microscopy. Muscle and non-muscle myosins initially appeared as slender rods at the same sites, and then, fused to form non-striated fibrils. As muscle myosin formed striated structures (A bands), non-muscle myosin disappeared from this structure. Myomesin (reactive with monoclonal antibodies MyB4 and MyBB78) and connectin (carboxy terminal region, reactive with monoclonal antibody T51) were seen as dots in the center of these myosin rods. These proteins then formed characteristic mature striations on non-striated fibrils of myosin. Earlier alignment of these myosin binding proteins rather than myosin indicates that the correct assembly of these proteins seems to be related to the formation of initial myosin rods as well as subsequent linear and periodic alignment of myosin molecules to form early A bands. Connectin spots reactive with 9D10 were scattered around myosin rods/myomesin dots/connectin T51 dots. These spots may represent radiating connectin filaments from these rods/dots to link myosin rods to the I-Z-I structures of myofibrils to be incorporated. Since the slow isoform of C-protein formed its characteristic bands ("doublets") prior to H zone formation within A bands by myosin, this isoform may help to precisely align myosin filaments within the A band region. The presence of the slow, then the slow and the cardiac, and finally the co-existence of the slow and the fast isoforms of C-protein may interfere with the incorporation and co-polymerization of non-adult isoforms into myofibrils.     

Effects of phosphorylated and unphosphorylated C-protein on cardiac actomyosin ATPase

C-protein, a component of the thick filaments of striated muscles, is reversibly phosphorylated and dephosphorylated in heart. It has been hypothesized that C-protein may be involved in regulating contraction, because the extent of C-protein phosphorylation correlates with the rate of cardiac relaxation. To test this hypothesis, the effects of phosphorylated and unphosphorylated C-protein on the actin-activated ATPase activity of myosin filaments prepared from DEAE-Sephadex-purified myosin were examined. Unphosphorylated C-protein (0.1 microM to 1.5 microM) stimulated actin-activated myosin ATPase activity in a dose-dependent manner. With a myosin: C-protein molar ratio of approximately 1, actin-activated myosin ATPase activity was elevated up to 3.2 times that of the control. Phosphorylated C-protein (2.5 mol PO4/mol C-protein) stimulated the activity somewhat less (2.5 times that of control). The stimulation of ATPase activity by C-protein was due to an increase in the Vmax value (from 0.25/second to 0.62/second) and a decrease in the Km value (from 11.9 microM to 6.7 microM). The addition of C-protein to actomyosin solutions produced an increase in the light-scattering of the actomyosin solution and a distinct precipitation of the actomyosin with time. Phosphorylated C-protein had a smaller effect on light-scattering than dephosphorylated C-protein. C-protein had a negligible effect on Ca-ATPase, EDTA-K-ATPase, or Mg-ATPase activities in the absence of actin. C-protein had only small effects on the actin-activated ATPase of heavy meromyosin. These results suggest that C-protein stimulates actin-activated myosin ATPase activity by enhancing the formation of stable aggregates between actin and myosin filaments.     

Effect of C-protein on actomyosin ATPase

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.     

Myosin thick filaments from adult rabbit skeletal muscles

Myosin subfragment 1 (S1) forms dimers in the presence of Mg(2+) or MgADP or MgATP. The entire myosin molecule forms head-head dimers in the presence of MgATP. The angle between the two subunits in the S1 dimer is 95 degrees. Assuming that the length of the globular part of S1 is approximately 12 nm and that the S1/S2 joint (lever arm approximately 7 nm) is clearly bent, the cylinder tangent to this dimer should have a diameter of approximately 18 nm, close to the approximately 16-20 nm suggested by many studies for the diameter of thick filaments in situ. These conclusions led us to re-examine our previous model, according to which two heads from two opposite myosin molecules are inserted into the filament core and interact as dimers. We studied synthetic filaments by electron microscopy, enzyme activity assays, controlled digestion and filament-filament interaction analysis. Synthetic filaments formed by rapid dilution in the presence of 1 mM EDTA at room temperature ( approximately 22 degrees C) had all their myosin heads outside the backbone. These filaments are called superfilaments (SF). Synthetic filaments formed by slow dilution, in the presence of either 2 mM Mg(2+) or 0.5 mM MgATP and at low temperature ( approximately 0 degrees C) had one myosin head outside the backbone and one head inside. These filaments are called filaments (F). Synthetic filaments formed by slow dilution, in the presence of 4 mM MgATP at low temperature ( approximately 0 degrees C) had most of their heads inserted in the filament core. These filaments are called antifilaments (AF). These experimental results provide important new information about myosin synthetic filaments. In particular, we found that myosin heads were involved in filament assembly and that filament-filament interactions can occur via the external heads. Native filaments (NF) from rabbit psoas muscle were also studied by enzyme assays. Their structure depended on the age of the rabbit. NF from 4-month-old rabbits were three-stranded, i.e. six myosin heads per crown, two of which were inside the core and four outside. NF from 18-month-old rabbits were two-stranded (similar to F).     

Effects of C-protein on synthetic myosin filament structure.

In the absence of C-protein, synthetic filaments prepared from column-purified myosin exhibit the following features: individual filament diameters are uniform over a long length, but a wide distribution of diameters is apparent over the population; approximately 25% of the filaments have a frayed appearance and take up stain poorly, whereas the remaining 75% are well-stained; optical diffraction of well-stained filaments reveals a 14.3-nm subunit period and a 43-nm axial period (Koretz, 1978; Koretz, 1979). Addition of C-protein to myosin before filament formation affects all of these features in a manner related to C-protein concentration. At the physiological ratio of C-protein to myosin in the banded region of the natural thick filament, synthetic aggregates are uniform in diameter over the population and show less than 10% frays. Whereas the subunit period remains unchanged, the axial period has increased to 114.4 nm, or eight times the subunit repeat. Above and below the physiological ratio, disorder of a specific nature is apparent. Addition of C-protein after filament formation appears to coat the aggregates so that elements of backbone ultrastructure are obscured, and some evidence of axial period change is visible in diffraction patterns. A model is presented for the binding of C-protein to myosin, and its observed effects on filament structure are explained in terms of this model.     

Structural evidence for the interaction of C-protein (MyBP-C) with actin and sequence identification of a possible actin-binding domain

C-protein (MyBP-C) is a myosin-binding protein that is usually seen in two sets of seven to nine positions in the C-zones in each half of the vertebrate striated muscle A-band. Skeletal muscle C-protein is a modular structure containing ten sub-domains (C1 to C10) of which seven are immunoglobulin-type domains and three (C6, C7 and C9) are fibronectin-like domains. Cardiac muscle C-protein has an extra N-terminal domain (C0) and also some sequence insertions, one of which provides phosphorylation sites. It is conceivable that C-protein has both a structural and regulatory role within the sarcomere. The precise mode of binding of C-protein to the myosin filament has not been determined. However, detailed ultrastructural studies have suggested that C-protein, which binds to myosin, can give rise to a longer periodicity (about 435A) than the intrinsic myosin filament repeat of 429A. The reason for this has remained a puzzle for over 25 years. Here we show by modelling and computation that the presence of this longer periodicity could be explained if the myosin-binding part of C-protein binds to myosin with the expected 429A repeat, but if there are systematic interactions of the N-terminal end of C-protein with the neighbouring actin filaments in the hexagonal lattice of filaments in the A-band. We also show that if they occur these interactions would probably only arise in defined muscle states. Further analysis of the MyBP-C sequence identifies a possible actin-binding domain in the Pro-Ala-rich sequence found at the N terminus of skeletal MyBP-C and between domains C0 and C1 in the cardiac sequence.     

Effects of BTS (N-benzyl-p-toluene sulphonamide), an inhibitor for myosin-actin interaction, on myofibrillogenesis in skeletal muscle cells in culture

Actin filaments align around myosin filaments in the correct polarity and in a hexagonal arrangement to form cross-striated structures. It has been postulated that this myosin-actin interaction is important in the initial phase of myofibrillogenesis. It was previously demonstrated that an inhibitor of actin-myosin interaction, BDM (2,3-butanedione monoxime), suppresses myofibril formation in muscle cells in culture. However, further study showed that BDM also exerts several additional effects on living cells. In this study, we further examined the role of actin-myosin interaction in myofibril assembly in primary cultures of chick embryonic skeletal muscle by applying a more specific inhibitor, BTS (N-benzyl-p-toluene sulphonamide), of myosin ATPase and actin-myosin interaction. The assembly of sarcomeric structures from myofibrillar proteins was examined by immunocytochemical methods with the application of BTS to myotubes just after fusion. Addition of BTS (10-50 microM) significantly suppressed the organization of actin and myosin into cross-striated structures. BTS also interfered in the organization of alpha-actinin, C-protein (or MyBP-C), and connectin (or titin) into ordered striated structures, though the sensitivity was less. Moreover, when myotubes cultured in the presence of BTS were transferred to a control medium, sarcomeric structures were formed in 2-3 days, indicating that the inhibitory effect of BTS on myotubes is reversible. These results show that actin-myosin interaction plays a critical role in the process of myofibrillogenesis.     

Immunohistochemical analysis of C-protein isoforms in cardiac and skeletal muscle of the axolotl,Ambystoma mexicanum

Of the several proteins located within sarcomeric A-bands, C-protein, a myosin binding protein (MyBP) is thought to regulate and stabilize thick filaments during assembly. This paper reports the characterization of C-protein isoforms in juvenile and adult axolotls, Ambystoma mexicanum, by means of immunofluorescent microscopy and Western blot analyses. C-protein and myosin are found specifically within the A-bands, whereas tropomyosin and -actin are detected in the I-bands of axolotl myofibrils. The MF1 antibody prepared against the fast skeletal muscle isoform of chicken C-protein specifically recognizes a cardiac isoform (Axcard1) in juvenile and adult axolotls but does not label axolotl skeletal muscle. The ALD66 antibody, which reacts with the C-protein slow isoform in chicken, localizes only in skeletal muscle of the axolotl. This slow axolotl isoform (Ax_slow) displays a heterogeneous distribution in fibers of dorsalis trunci skeletal muscle. The C₃₁₅ antibody against the chicken C-protein cardiac isoform identifies a second axolotl cardiac isoform (Ax_card2), which is present also in axolotl skeletal muscle. No C-protein was detected in smooth muscle of the juvenile and adult axolotl with these antibodies.This work was supported by NIH grants HL-32184 and HL-37702 and a grant-in-aid from the American Heart Association to L.F.L.     

Immunochemical analysis of porcine cardiac C-protein

C-protein has been isolated from pig heart and its immunochemical properties studied. It is extracted with myosin, and separated from the myosin on a DEAE-Sephadex column. The amount of C-protein recovered from crude myosin is approx. 3.5%. The molecular weight of C-protein is 150,000. Anti-C-protein serum reacts with crude myosin and purified C-protein but not with purified myosin in immunodiffusion plates. Cardiac C-protein does not react with anti-skeletal white muscle C-protein serum. Immunoblotting experiments show that anti-cardiac C-protein serum reacts with a Mr = 150,000 component in myofibrils or crude myosin. C-protein is located in the A-band, except the M-line region, of the myofibrils. These results indicate that C-protein is an intrinsic component of the thick filaments in pig heart myofibrils.     

Control of filament length by the regulatory light chains in skeletal and cardiac myosins

The possible role of the regulatory light chains (LC2) in in vitro assembly of rabbit skeletal and dog cardiac myosins was examined by formation of minifilaments and synthetic thick filaments. After LC2 was removed, the resulting myosin preparations exhibited little aggregation in 0.5 M KCl and 0.05 M potassium phosphate (pH 6.5). Minifilaments migrated as a single, hypersharp peak during sedimentation velocity, but electron microscopic analysis revealed a more destabilized structure for LC2-deficient minifilaments. Thick filaments were formed in buffers containing 0.15 M KCl and the following: 20 mM imidazole; 20 mM imidazole, 5 mM ATP; or 20 mM imidazole, 5 mM ATP, and 5 mM MgCl2, all at pH 7.0. Skeletal and cardiac myosin filaments formed in imidazole buffer alone were bipolar, tapered at both ends, and about 1.6 micron long. Removal of LC2 resulted in the formation of shorter thick filaments (1.2 micron long). This effect could be reversed by reassociation with LC2. Inclusion of ATP in the buffer disrupted the filament structure, resulting in irregular, short filaments (less than 0.6 micron); addition of both ATP and MgCl2 largely reversed the effects of ATP alone. In cardiac myosin filaments, the bare zone diameter increased from 16 nm as measured in control and LC2-recombined samples to 20 nm in LC2-deficient myosin assemblies. These results implicate LC2 in an active role in controlling synthetic thick filament length in both skeletal and cardiac muscles.     

The mechanochemical basis of amoeboid movement: II. Cytoplasmic filament stability at low divalent cation concentrations

The role played by Ca²⁺ in the stability of cytoplasmic actin and myosin filaments was investigated ultrastructurally with negatively stained isolated cytoplasm from Chaos carolinensis. Cytoplasm was incubated in solutions containing 5, 10, 15 and 25 mM EGTA for periods of time varying from 2 to 20 min. As either the EGTA concentration or duration of incubation was increased, the extent of myosin and actin filament depolymerization increased. The actin filaments depolymerized except where they were stabilized by interaction with myosin. With longer incubation times or higher EGTA concentrations complete depolymerization of the actin filaments could be accomplished. Myosin aggregates also disassembled and became shorter, while monomeric myosin labelled adjacent thin filaments to form arrowhead complexes resembling myosin enriched actomyosin [1]. These actomyosin complexes were relatively stable at low Ca²⁺ concentrations. In addition, the complexes showed a characteristic 35 nm periodicity and were dissociable in the presence of Mg²⁺-ATP. The actin containing filaments were more labile at low Ca²⁺ concentrations than the myosin aggregates. These results suggest that in cells capable of regulating their Ca²⁺ concentrations efficiently, filament polymerization-depolymerization could play a role in the control of cytoplasmic streaming.     

Heat-induced Gelation of Myosin from Leg and Breast Muscles of Chicken

Heat-induced gelation of myosin from leg and breast muscles of chicken was studied in 0.6 m KC1. Gel strength of breast myosin was higher than that of leg myosin between pH 5.2 and 6.0. Turbidity of breast myosin increased below pH 6.0 but that of leg myosin did not increase at pH 5.7. Turbidity of leg myosin was higher than that of breast myosin below pH 5.6. Viscosity of breast myosin increased between pH 5.5 and 6.0 as the pH decreases, although that of leg myosin decreased. The breast myosin assembled to form long filaments at pH 5.7, but leg myosin failed to form long filaments. At pH 5.4, breast myosin filaments became longer and leg myosin assembled into filaments though they were shorter than breast myosin filaments. The strength of heat-induced gel formed from the filamentous leg and breast myosins at acidic region was not influenced by F-actin. These results indicate that the strength of heat-induced gel of both myosins is closely related to their morphological properties.     

Dense precipitate of brain tubulin with skeletal muscle myosin

Purified tubulin from porcine brain formed a dense precipitate at 37 degrees C with muscle myosin filaments from rabbit skeletal muscle; this effect was greater than that with partially purified tubulin. ATP or GTP, which prevented the myosin filaments from precipitating, inhibited the formation of the dense precipitate, but did not dissociate the dense precipitate once formed. The dense precipitate was found by thin-section electron microscopy to be composed to side-by-side aggregates of myosin filaments whose projections might be decorated by tubulin. The decoration was also seen by negative-stain electron microscopy. The binding of tubulin to myosin filaments decreased the Mg2+- and Ca2+-GTPase activity of the myosin by about half, but did not affect either Mg2+- or Ca2+-ATPase activity. The binding ratio of tubulin to myosin in the presence of 5 mM MgCl2 was 2.2 mol/mol using purified tubulin and 1.8 mol/mol using partially purified tubulin. Five mM ATP and GTP in the presence of 5 mM MgCl2 decreased the tubulin binding by 1.6-2.0 and 1.1-1.3 mol/mol, respectively, when added before an encounter of tubulin with myosin filaments, but did not cause any decrease when added after such an encounter.     

Alterations in Ca2+ sensitive tension due to partial extraction of C- protein from rat skinned cardiac myocytes and rabbit skeletal muscle fibers

C-protein, a substantial component of muscle thick filaments, has been postulated to have various functions, based mainly on results from biochemical studies. In the present study, effects on Ca(2+)-activated tension due to partial removal of C-protein were investigated in skinned single myocytes from rat ventricle and rabbit psoas muscle. Isometric tension was measured at pCa values of 7.0 to 4.5: (a) in untreated myocytes, (b) in the same myocytes after partial extraction of C-protein, and (c) in some myocytes, after readdition of C-protein. The solution for extracting C-protein contained 10 mM EDTA, 31 mM Na2HPO2, 124 mM NaH2PO4, pH 5.9 (Offer et al., 1973; Hartzell and Glass, 1984). In addition, the extracting solution contained 0.2 mg/ml troponin and, for skeletal muscle, 0.2 mg/ml myosin light chain-2 in order to minimize loss of these proteins during the extraction procedure. Between 60 and 70% of endogenous C-protein was extracted from cardiac myocytes by a 1-h soak in extracting solution at 20-23 degrees C; a similar amount was extracted from psoas fibers during a 3-h soak at 25 degrees C. For both cardiac myocytes and skeletal muscle fibers, partial extraction of C-protein resulted in increased active tension at submaximal concentrations of Ca2+, but had little effect upon maximum tension. C-protein extraction also reduced the slope of the tension-pCa relationships, suggesting that the cooperativity of Ca2+ activation of tension was decreased. Readdition of C-protein to previously extracted myocytes resulted in recovery of both tension and slope to near their control values. The effects on tension did not appear to be due to disruption of cooperative activation of the thin filament, since C-protein extraction from cardiac myocytes that were 40-60% troponin-C (TnC) deficient produced effects similar to those observed in cells that were TnC replete. Measurements of the tension-pCa relationship in skeletal muscle fibers were also made at a sarcomere length of 3.5 microns which, because of the distribution of C-protein on the thick filament, should eliminate any interaction between C-protein and actin. The effects of C-protein extraction were similar at long and short sarcomere lengths. These data are consistent with a model in which C-protein modulates the range of movement of myosin, such that the probability of myosin binding to actin is increased after its extraction.     

In situ reconstitution of myosin filaments within the myosin-extracted myofibril in cultured skeletal muscle cells

We studied the in situ reconstitution of myosin filaments within the myosin-extracted myofibrils in cultured chick embryo skeletal muscle cells using the electron microscope and polarization microscope. Myosin was first extracted from the myofibrils in glycerinated muscle cells with a high-salt solution containing 0.6 M KCl. When rabbit skeletal muscle myosin was added to the myosin-extracted cells in the high-salt solution, thin filaments in the ghost myofibrils were bound with myosin to form arrowhead complexes. Subsequent dilution of KCl in the myosin solution to 0.1 M resulted in the formation of thick myosin filaments within the myofibrils, increasing the birefringence of the myofibrils. When Mg-ATP was added such myosin-reassembled myofibrils were induced either to form supercontraction bands or to restore the sarcomeric arrangement of thick and thin filaments. Under the polarization microscope, vibrational movement of the myofibrils was seen transiently upon addition of Mg-ATP, often resulting in a regular arrangement of myofibrils in register. These myofibrils, with reconstituted myosin filaments, structurally and functionally resembled the native myofibrils. The findings are discussed with special reference to the myofibril formation in developing muscle cells.     

The myosin filament. VI. Myosin content.

There has been some disagreement about the number of myosin molecules in vertebrate skeletal myosin filaments calculated from the myosin to actin weight ratio determined by quantitative sodium dodecyl sulfate/polyacrylamide gel electrophoresis (Tregear &; Squire, 1973; Potter, 1974; Morimoto &; Harrington, 1974). In this work it was found that (1) thoroughly washed fibrils are required to obtain the true value for the myosin to actin weight ratio. (2) Neither actin nor myosin is extracted preferentially during the required washing procedure. (3) There are four myosin molecules per 14.3 nm interval along the myosin filament or about 400 myosin molecules per filament.From published estimates of the number of molecules of C-protein per myosin filament (Offer et al., 1973; Morimoto &; Harrington, 1974) and the findings in this work, we conclude that there are four molecules of C-protein at each of the 14 C-protein binding positions along the filament, i.e. one C-protein molecule for each of the four myosin molecules contributing to the cross-bridges at each position.     

Electric and flow birefringence properties of myosin in aqueous urea and sodium pyrophosphate.

The electric dipole moment of rabbit skeletal myosin was estimated from the electric and flow birefringence properties. Myosin formed small polydisperse aggregates (0.2-1.1 microM in length) with an apparent electric dipole moment of 5,000-20,000 Debye in aqueous urea or sodium pyrophosphate at low ionic strength. Permanent dipole moment contributed substantially to the apparent dipole moment. An anti-parallel association of myosin was suggested from the dependence of the apparent dipole moment on myosin concentration. Some interactions between myosin and C-protein were detected in 1 M urea by flow birefringence and analytical ultracentrifugation studies. The apparent dipole moment of myosin aggregates was less dependent on myosin concentration in the presence of C-protein.     

The binding of skeletal muscle C-protein to F-actin, and its relation to the interaction of actin with myosin subfragment-1.

C-protein is a component of thick filaments of skeletal muscle myofibrils. It is bound to the assembly of myosin tails that forms the filament backbone. We report here that C-protein can also bind to F-actin, with a limiting stoichiometry of approximately one C-protein molecule per 3 to 5 actin subunits and a dissociation constant in the micromolar range at ionic strength 0·07. The binding is not significantly affected by ATP, calcium ions or temperature, or by the presence of tropomyosin on the actin, but it is weakened by increasing ionic strength. Myosin subfragment-1 (S-1) competes with C-protein for binding to actin. In the absence of ATP, S-1 displaces nearly all bound C-protein from actin, while in the presence of ATP, C-protein inhibits the actin activation of S-1 ATPase. Although there is no direct evidence that interaction of C-protein with actin is physiologically significant, the lenght of the C-protein molecule is sufficient so that it could make contact with the thin filaments in muscle while remaining attached to the thick filaments.