Presence of a unit for actin-myosin interaction during the superprecipitation of actomyosin.

The interaction of actin with myosin was studied in the presence of ATP at low ionic strength by means of measurements of the actin-activated ATPase activity of myosin and superprecipitation of actomyosin. At high ATP concentrations the ATPase activities of myosin, heavy meromyosin (HMM) and myosin subfragment 1 (S-1) were activated by actin in the same extent. At low ATP concentrations the myosin ATPase activity was activated about 30-fold by actin, whereas those of HMM and S-1 were stimulated only several-fold. This high actin activation of myosin ATPase was coupled with the occurrence of superprecipitation. The activation of HMM or S-1 ATPase by actin shows a simple hyperbolic dependence on actin concentration, but the myosin ATPase was maximally activated by actin at a 2:1 molar ratio of actin to myosin, and a further increase in the actin concentration had no effect on the activation. These results suggest the presence of a unit for actin-myosin interaction, composed of two actin monomers and one myosin molecule in the filaments.     

Myosin and actin from ascidian smooth muscle and their interaction

Myosin and actin were purified from ascidian smooth muscle. Ascidian myosin contained two classes of light chains and the pH dependence of Ca2+-activated ATPase and the KCl dependence of actin-activated ATPase of ascidian myosin differed from those of vertebrate skeletal myosin. Troponin-tropomyosin complex from ascidian increased the ATPase activity of ascidian reconstituted actomyosin in a Ca2+-dependent manner. Ascidian myosin provided the reconstituted actomyosin with the responsiveness to calcium ions. Two actin isoforms were present in ascidian, which were distinguished by isoelectric points.     

Actomyosin interactions in the presence of ATP and the N-terminal segment of actin.

The binding of myosin subfragment 1 (S-1) to actin in the presence of ATP and the acto-S-1 ATPase activities of acto-S-1 complexes were determined at 5 degrees C under conditions of partial saturation of actin, up to 90%, by antibodies against the first seven N-terminal residues on actin. The antibodies [Fab(1-7)] inhibited strongly the acto-S-1 ATPase and the binding of S-1 to actin in the presence of ATP at low concentrations of S-1, up to 25 microM. Further increases in S-1 concentration resulted in a partial and cooperative recovery of both the binding of S-1 to actin and the acto-S-1 ATPase while causing only limited displacement of Fab(1-7) from actin. The extent to which the binding and the ATPase activity were recovered depended on the saturation of actin by Fab(1-7). The combined amounts of S-1 and Fab binding to actin suggested that the activation of the myosin ATPase activity was due to actin free of Fab. Examination of the acto-S-1 ATPase activities as a function of S-1 bound to actin at different levels of actin saturation by Fab(1-7) revealed that the antibodies inhibited the activation of the bound myosin. Thus, the binding of antibodies to the N-terminal segment of actin can act to inhibit both the binding of S-1 to actin in the presence of ATP and a catalytic step in ATP hydrolysis by actomyosin. The implications of these results to the regulation of actomyosin interaction are discussed.     

Regulation of the adenosinetriphosphatase activity of cross-linked actin-myosin subfragment 1 by troponin-tropomyosin

Chalovich and Eisenberg [Chalovich, J. M., & Eisenberg, E. (1982) J. Biol. Chem. 257, 2432-2437] have suggested that at low ionic strength, troponin-tropomyosin regulates the actomyosin ATPase activity by inhibiting a kinetic step in the actomyosin ATPase cycle rather than by blocking the binding of myosin subfragment 1 (S-1) to actin. This leads to the prediction that troponin-tropomyosin should inhibit the ATPase activity of the complex of actin and S-1 (acto . S-1) even when S-1 is cross-linked to actin. We now find that the ATPase activity of cross-linked actin . S-1 prepared under milder conditions than those used by Mornet et al. [Mornet, D., Bertrand, R., Pantel, P., Audemard, E., & Kassab, R. (1981) Nature (London) 292, 301-306] is inhibited 90% by troponin-tropomyosin in the absence of Ca2+. At mu = 18 mM, 25 degrees C, the ATPase activity of this cross-linked preparation is only about 2-fold greater than the maximal actin-activated ATPase activity of S-1 obtained with regulated actin in the absence of Ca2+. At physiological ionic strength, the ATPase activity of this cross-linked actin . S-1 preparation is inhibited about 95% by troponin-tropomyosin. Since cross-linked S-1 behaves kinetically like S-1 in the presence of infinite actin concentration, it is very unlikely that inhibition of the ATPase activity of cross-linked actin . S-1 is due to blocking of the binding of S-1 to actin. Therefore, these results are in agreement with the suggestion that troponin-tropomyosin regulates primarily by inhibiting a kinetic step in the ATPase cycle.     

Effect of ethylene glycol on the interaction of different myosin subfragment 1.nucleotide complexes with actin

To elucidate the structure of the cross-bridge intermediates in the actomyosin ATPase cycle, several laboratories have added both ethylene glycol and AMP-PNP to muscle fibers. These studies suggested that ethylene glycol shifts the structure of myosin.AMP-PNP toward the weak-binding conformation, i.e., toward the structure of myosin.ATP. Since only the weak-binding conformation of myosin subfragment 1 (S-1) binds with no apparent cooperativity to the troponin-tropomyosin-actin complex (regulated actin), we used this as a probe to examine the conformation of various S-1.nucleotide complexes in ethylene glycol. Our results show that ethylene glycol markedly weakens the binding strength of S-1, S-1.ADP, and S-1.AMP-PNP to actin but has almost no effect on the binding strength of S-1.ATP. As in muscle fibers, at 40% ethylene glycol, the binding strength of S-1.AMP-PNP to actin becomes very similar to the binding strength of S-1.ATP. In the presence of troponin-tropomyosin, the binding of S-1.AMP-PNP to actin shows no apparent cooperativity in 40% ethylene glycol. Therefore, our results confirm that ethylene glycol shifts the structure of the myosin.AMP-PNP toward the weak-binding conformation. However, our results also suggest that ethylene glycol has a direct effect on the regulated actin complex. This is shown by the fact that ethylene glycol markedly increases the cooperative binding of S-1.ADP to regulated actin both in the presence and in the absence of Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxygen exchange between phosphate and water accompanies calcium-regulated ATPase activity of skinned fibers from rabbit skeletal muscle

The extent of oxygen exchange between phosphate and water has been measured for the calcium-regulated magnesium-dependent ATPase activity of chemically skinned fibers from rabbit skeletal muscle. The oxygen exchange was determined for isometrically held fibers by measuring with a mass spectrometer the distribution of 18O atoms in the product inorganic phosphate when ATP hydrolysis was carried out in H2(18)O. The extent of exchange was much greater in relaxed muscle (free Ca2+ less than 10(-8) M) than in calcium-activated muscle (free Ca2+ approximately equal to 3 X 10(-5) M). Activated fibers had an ATPase activity at least 30-fold greater than the relaxed fibers. These results correlate well with the extents of oxygen exchange accompanying magnesium-dependent myosin and unregulated actomyosin ATPase activities, respectively. In relaxed fibers, comparison of the amount of exchange with the ATPase activity suggests that the rate constant for the reformation of myosin-bound ATP from the myosin products complex is about 10 s-1 at 20 degrees C and pH 7.1. In each experiment the distribution of 18O in the Pi formed was incompatible with a single pathway for ATP hydrolysis. In the case of the calcium-activated fibers, the multiple pathways for ATP hydrolysis appeared to be an intrinsic property of the actomyosin ATPase in the fiber. These results indicate that in muscle fibers, as in isolated actomyosin, cleavage of protein-bound ATP is readily reversible and that association of the myosin products complex with actin promotes Pi release.     

Biochemical kinetics of skeletal actosubfragment-1 at high subfragment-1 concentrations.

The actomyosin ATPase activity of skeletal myosin subfragment-1 (S-1) is typically studied by keeping the S-1 concentration low and varying the actin concentration. General agreement exists over the kinetic data observed. Another way of studying the ATPase activity is to keep the actin concentration low and vary the S-1 concentration. The picture that has emerged is that the maximal ATPase rate (per micromolar actin), Vamax, is several fold greater than the Vsmax measured at fixed S-1. Likewise, the apparent activation constant Kam is several fold weaker than KATPase. In addition it is found that Kam, henceforth Kam(At), varies with the total actin concentration At, but controversy continues over the actin dependence of Vamax. Of particular interest is the fact that the Lymn-Taylor and refractory state models could not account for the data. Here we have repeated studies on the ATPase activity at fixed actin concentration in an attempt to determine if the current models for the actin activated myosin ATPase activity can account for both the constant actin and constant S-1 data simultaneously, or if these data imply that new kinetic models need be postulated. We conclude that the current kinetic models can account for the data.     

The phosphorylated L2 light chain of skeletal myosin is a modifier of the actomyosin ATPase

Incubation of rabbit skeletal myosin with an extract of light chain kinase plus ATP phosphorylated the L2 light chain and modified the steady state kinetics of the actomyosin ATPase. With regulated actin, the ATPase activity of phosphorylated myosin (P-myosin) was 35 to 181% greater than that of unphosphorylated myosin when assayed with 0.05 to 5 micro M Ca2+. Phosphorylation had no effect on the Ca2+ concentration required for half-maximal activity, but it did increase the ATPase activity at low Ca2+. With pure actin, the percentage of increase in the actomyosin ATPase activity correlated with the percentage of phosphorylation of myosin. Steady state kinetic analyses of the actomyosin system indicated that 50 to 82% phosphorylation of myosin decreased significantly the Kapp of actin for myosin with no significant effect on the Vmax. Phosphorylaton of heavy meromyosin similarly modified the steady state kinetics of the acto-heavy meromyosin system. Both the K+/EDTA- and Mg-ATPase activities of P-myosin and phosphorylated heavy meromyosin were within normal limits indicating that phosphorylaiion had not altered significantly the hydrolytic site. Phosphatase treatment of P-myosin decreased both the level of phosphorylation of L2 and the actomyosin ATPase activity to control levels for unphosphorylated myosin. It is concluded levels for unphosphorylated myosin. It is concluded from these results that the ability of P-myosin to modify the steady state kinetics of the actomyosin ATPase was: 1) specific for phosphorylation; 2) independent of the thin filament regulatory proteins.     

Interactions between actin, myosin, and an actin-binding protein from rabbit alveolar macrophages. Alveolar macrophage myosin Mg-2+-adenosine triphosphatase requires a cofactor for activation by actin.

The interactions were analyzed between actin, myosin, and a recently discovered high molecular weight actin-binding protein (Hartwig, J. H., and Stossel, T. P. (1975) J. Biol Chem.250,5696-5705) of rabbit alveolar macrophages. Purified rabbit alveolar macrophage or rabbit skeletal muscle F-actins did not activate the Mg2+ATPase activity of purified rabbit alveolar macrophage myosin unless an additional cofactor, partially purified from macrophage extracts, was added. The Mg2+ATPase activity of cofactor-activated macrophage actomyosin was as high as 0.6 mumol of Pi/mg of myosin protein/min at 37 degrees. The macrophage cofactor increased the Mg2+ATPase activity of rabbit skeletal muscle actomyosin, and calcium regulated the Mg2+ATPase activity of cofactor-activited muscle actomyosin in the presence of muscle troponins and tropomyosin. However, the Mg2+ATPase activity of macrophage actomyosin in the presence of the cofactor was inhibited by muscle control proteins, both in the presence and absence of calcium. The Mg2+ATPase activity of the macrophage actomyosin plus cofactor, whether assembled from purified components or studied in a complex collected from crude macrophage extracts, was not influenced by the presence of absence of calcium ions. Therefore, as described for Acanthamoeba castellanii myosin (Pollard, T. D., and Korn, E. D. (1973) J. Biol. Chem. 248, 4691-4697), rabbit alveolar macrophage myosin requires a cofactor for activation of its Mg2+ATPase activity by F-actin; and no evidence was found for participation of calcium ions in the regulation of this activity.In macrophage extracts containing 0.34 M sucrose, 0.5 mM ATP, and 0.05 M KCl at pH 7.0,the actin-binding protein bound F-actin into bundles with interconnecting bridges. Purified macrophage actin-binding protein in 0.1 M KCl at pH 7.0 also bound purified macrophage F-actin into filament bundles. Macrophage myosin bound to F-actin in the absence but not the presence of Mg2+ATP, but the actin-binding protein did not bind to macrophage myosin in either the presence or absence of Mg2+ATP.     

Catalytic cooperativity induced by SH1 labeling of myosin filaments

Modifications of SH1 groups on isolated myosin subfragment 1 (S-1) and myosin in muscle fibers affect differently the acto-S-1 ATPase and the fiber properties. Consistent with the findings of earlier work on fibers, the modification of SH1 groups in relaxed myofibrils with phenylmaleimide caused a loss of their shortening. This loss paralleled the decrease in the Vmax of extracted myosin but was not linear with the extent of SH1 labeling. Strikingly, the decrease in Vmax of S-1 prepared from the modified myofibrils was directly proportional to the extent of SH1 labeling. The specificity of SH1 labeling in myofibrils was verified by ATPase activities, thiol titrations, radiolabeling experiments, and comparisons to myosin labeled on SH1 in solution. To test for intermolecular interactions in the myosin filaments and their contribution to the differences between S-1 and myosin, the catalytic properties of copolymers of myosin were examined. Copolymers of myosin and rod minifilaments were formed in 5 mM citrate-Tris (pH 8.0) buffer, and their homogeneity was verified by sedimentation velocity analysis. The inhibition of actomyosin ATPase by rod particles was related to the decrease in the Km value. When rod particles were replaced in these minifilaments by SH1-modified myosin, the ATPase of the copolymers was increased over that of the combined ATPases of the individual filaments. The actomyosin ATP turnover rates on the unmodified heads were increased severalfold by the modified heads.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of the reaction of N-ethylmaleimide with myosin and heavy meromyosin subfragment 1

Myosin reacted at low ionic strength with NEM forms an actomyosin which is Ca⁺⁺ insensitive. With HMM S-1 the reaction with NEM causes a marked loss of the actin activated ATPase activity and the Ca⁺⁺ sensitivity is reduced but not eliminated. The presence of actin during the sulfhydryl reaction does not significantly alter this result. HMM S-1 prepared from myosin previously desensitized by NEM regains Ca⁺⁺ sensitivity. These results indicate that the conformations of myosin and HMM S-1 are different and could reflect a difference between insoluble (filamentous) myosin and myosin, or its fragments, in solution.     

Antibody against the amino terminus of alpha-actin inhibits actomyosin interactions in the presence of ATP

Actomyosin interactions in the presence of ATP were examined by using site-specific antibodies directed against the first seven N-terminal residues on skeletal alpha-actin. Fab fragments of these antibodies (S alpha N Fab) inhibited effectively the actin-activated ATPase of myosin subfragment 1 (S-1) at both 5 and 25 degrees C. Binding experiments carried out in the presence of ATP at 5 degrees C revealed that the catalytic inhibition was related to the inhibition of S-1 binding to actin by Fab. At equimolar ratios of Fab to actin, the binding of S-1 to actin and the activated ATPase were inhibited by 75 and 82%, respectively. These results, when contrasted with the small effect of Fab on rigor actomyosin binding, suggest ATP-induced changes at the interface of actin and myosin.     

Synthetic peptide of the sequence 632-642 on myosin subfragment 1 inhibits actomyosin ATPase activity.

A synthetic peptide corresponding to a sequence 632-642 (S632-642) on the myosin subfragment 1 (S-1) heavy chain and spanning the 50/20 kDa junction of S-1 binds to actin in the presence and absence of S-1. The binding of 1.0 mole of peptide per actin causes almost complete inhibition of actomyosin ATPase activity and only partial inhibition of S-1 binding to actin. The binding of S632-642 to the N-terminal segment of actin is supported by competitive carbodiimide cross-linking of S-1 and S632-642 to actin and the catalytic properties of cross-linked acto-S-1 and actin-peptide complexes. These results show that the sequence 632-642 on S-1 is an autonomous binding site for actin and confirm the catalytic importance of its interactions with the N-terminal segment of actin.     

A 72,000-mol-wt protein from tomato inhibits rabbit acto-S-1 ATPase activity

Tomato activation inhibiting protein (AIP) is a molecule of an apparent molecular weight of 72,000 that co-purifies with tomato actin. In an assay system containing rabbit skeletal muscle F-actin and rabbit skeletal muscle myosin subfragment-1 (myosin S-1), tomato AIP dissociated the acto-S-1 complex in the absence of Mg+2ATP and inhibited the ability of F-actin to activate the low ionic strength Mg+2ATPase activity of myosin S-1. At a molar ratio of 5 actin to 1 AIP, a 50% inhibition of the actin-activated Mg+2ATPase activity of myosin S-1 was observed. The inhibition can be reversed by raising the calcium ion concentration to 1 X 10(-5) M. The AIP had no effect on the basal low ionic strength Mg+2ATPase activity of myosin S-1 in the absence of actin. The protein did not bind directly to actin nor did it cause depolymerization or aggregation of F-actin but appeared, instead, to interact with the actin binding site on myosin S-1. Since AIP is a potent, reversible inhibitor of the rabbit acto-S-1 ATPase activity, it is postulated that it may be responsible for the low levels of actin activation exhibited by tomato F-actin fractions containing the AIP.     

Measurement of ATPase activity of immobilized myosin heads

Myosin, heavy meromyosin, and myosin subfragment-1 (S-1) were immobilized on the inner surfaces of glass capillary tubes, the inside walls of which had been coated with nitrocellulose. The ATPase activities of the immobilized proteins were measured using radiolabeled ATP and electrophoretic separation of the reaction products. The activity was proportional to the amount of immobilized protein. Activation by actin of the ATPase was also observed.     

A solid-liquid biphasic model for characterization of properties of muscle and platelet contractile proteins.

Actomyosin, myosin, and actin from different sources are adsorbed, apparently as a monolayer, by polystyrene particles teins for 1 mg of Lytron were about 10-7 liters mol-1, while heterogeneity indices (alpha) varied from 0.70 to 1.0 presumably as a function of spontaneous aggregation in the liquid phase. Adsorption was irreversible. Orientation of absorbed molecules permitted association of bound muscle actin with platelet or muscle myosin. The association constant of the former reaction was 2.78 times 10-6 liters mol-1. Enzymatic properties of adsorbed actomyosin, Mg2+ATPase activity was abolished, but association of myosin with bound actin, or association of actin with bound myosin was accompanied by restoration of Mg2+ATPase activity. Every subunit of F-actin strands, unless F-actin had been fully depolymerized to G-actin, could bind myosin and activate Mg2+ATPase activity. Immunogenic characteristics of muscle myosin were enhanced by Lytron adsorption. Elicited antibodies showed selective specificity for an antigenic determinant located near or at the actin combining site of muscle myosin. Antibodies did not react with actomyosin. Antibodies prevented association of actin with muscle myosin because they inhibited both superprecipitation and development of Mg2+ATPase activity.     

Caldesmon freezes the structure of actin filaments during the actomyosin ATPase cycle

Hybrid contractile apparatus was reconstituted in skeletal muscle ghost fibers by incorporation of skeletal muscle myosin subfragment 1 (S1), smooth muscle tropomyosin and caldesmon. The spatial orientation of FITC-phalloidin-labeled actin and IAEDANS-labeled S1 during sequential steps of the acto-S1 ATPase cycle was studied by measurement of polarized fluorescence in the absence or presence of nucleotides conditioning the binding affinity of both proteins. In the fibers devoid of caldesmon addition of nucleotides evoked unidirectional synchronous changes in the orientation of the fluorescent probes attached to F-actin or S1. The results support the suggestion on the multistep rotation of the cross-bridge (myosin head and actin monomers) during the ATPase cycle. The maximal cross-bridge rotation by 7 degrees relative to the fiber axis and the increase in its rigidity by 30% were observed at transition between A**.M**.ADP.Pi (weak binding) and A--.M--.ADP (strong binding) states. When caldesmon was present in the fibers (OFF-state of the thin filament) the unidirectional changes in the orientation of actin monomers and S1 were uncoupled. The tilting of the myosin head and of the actin monomer decreased by 29% and 90%, respectively. It is suggested that in the "closed" position caldesmon "freezes" the actin filament structure and induces the transition of the intermediate state of actomyosin towards the weak-binding states, thereby inhibiting the ATPase activity of the actomyosin.     

Amphidinolide B, a powerful activator of actomyosin ATPase enhances skeletal muscle contraction

Amphidinolide B caused a concentration-dependent increase in the contractile force of skeletal muscle skinned fibers. The concentration-contractile response curve for external Ca2+ was shifted to the left in a parallel manner, suggesting an increase in Ca2+ sensitivity. Amphidinolide B stimulated the superprecipitation of natural actomyosin. The maximum response of natural actomyosin to Ca2+ in superprecipitation was enhanced by it. Amphidinolide B increased the ATPase activity of myofibrils and natural actomyosin. The ATPase activity of actomyosin reconstituted from actin and myosin was enhanced in a concentration-dependent manner in the presence or absence of troponin-tropomyosin complex. Ca2+-, K+-EDTA- or Mg2+-ATPase of myosin was not affected by amphidinolide B. These results suggest that amphidinolide B enhances an interaction of actin and myosin directly and increases Ca2+ sensitivity of the contractile apparatus mediated through troponin-tropomyosin system, resulting in an increase in the ATPase activity of actomyosin and thus enhances the contractile response of myofilament.     

Effects of actin filament cross-linking and filament length on actin- myosin interaction

We have used two actin-binding proteins of the intestinal brush border, TW 260/240 and villin, to examine the effects of filament cross-linking and filament length on myosin-actin interactions. TW 260/240 is a nonerythroid spectrin that is a potent cross-linker of actin filaments. In the presence of this cross-linker we observed a concentration- dependent enhancement of skeletal muscle actomyosin ATPase activity (150-560% of control; maximum enhancement at a 1:70-80 TW 260/240:actin molar ratio). TW 260/240 did not cause a similar enhancement of either acto-heavy meromyosin (HMM) ATPase or acto-myosin subfragment-one (S1) ATPase. Villin, a Ca2+-dependent filament capping and severing protein of the intestinal microvillus, was used to generate populations of actin filaments of various lengths from less than 20 nm to 2.0 microns; (villin:actin ratios of 1:2 to 1:4,000). The effect of filament length on actomyosin ATPase was biphasic. At villin:actin molar ratios of 1:2- 25 actin-activated myosin ATPase activity was inhibited to 20-80% of control values, with maximum inhibition observed at the highest villin:actin ratio. The ATPase activities of acto-HMM and acto-S1 were also inhibited at these short filament lengths. At intermediate filament lengths generated at villin:actin ratios of 1:40-400 (average lengths 0.26-1.1 micron) an enhancement of actomyosin ATPase was observed (130-260% of controls), with a maximum enhancement at average filament lengths of 0.5 micron. The levels of actomyosin ATPase fell off to control values at low concentrations of villin where filament length distributions were almost those of controls. Unlike intact myosin, the actin-activated ATPase of neither HMM nor S1 showed an enhancement at these intermediate actin filament lengths.     

A novel biosensor for mercuric ions based on motor proteins

We explored the potential of contractile proteins, actin and myosin, as biosensors of solutions containing mercuric ions. We demonstrate that the reaction of HgCl2 with myosin rapidly inhibits actin-activated myosin ATPase activity. Mercuric ions inhibit the in vitro analog of contraction, namely the ATP-initiated superprecipitation of the reconstituted actomyosin complex. Hg reduces both the rate and extent of this reaction. Direct observation of the propulsive movement of actin filaments (10 nm in diameter and 1 microm long) in a motility assay driven by a proteolytic fragment of myosin (heavy meromyosin or HMM) is also inhibited by mercuric ions. Thus, we have demonstrated the biochemical, biophysical and nanotechnological basis of what may prove to be a useful nano-device.