The effects of substrate concentration on the Mg-adenosine triphosphatase activity of myosin.

Using myosin, heavy meromyosin, and subfragment-1 the steady state rate of Mg-modified adenosine triphosphatase (Mg-ATPase) was determined over a range of substrate concentrations between 10(-8) M and 5 X 10(-3)M, at 0.5 M and 0.05 M KC1 (pH 7.4 at 20 degrees C). At the substrate concentrations below 10(-5) M, myosin Mg-ATPase was observed to show that two active sites interact, as suggested by the analysis of transient kinetic studies (Walz, F. G., Jr.: J. Theor. Biol. 41, 357-373 (1973)). The increase in the activity at Mg-ATP concentrations higher than 10(-4) M corresponds to the binding of Mg-ATP to myosin sites not responsible for the catalytic action. With heavy meromyosin and subfragment-1, the activity was best expressed by the Michaelis equation. With heavy meromyonsin, the activation at high ATP concentrations is detectable, though not as pronounced as with myosin, but not with subfragment-1.     

Modification of the interactions of myosin with actin and 5''-adenylyl imidodiphosphate by substitution of ethylene glycol for water.

We studied the effect of replacing water by ethylene glycol as solvent on the properties of skeletal muscle myosin, myosin subfragment-1 (S1) and heavy meromyosin. Ethylene glycol (50%, v/v) had no detectable effect on the affinity of myosin or actomyosin for the substrate analogue 5'-adenylyl imidodiphosphate (AMPPNP). However, the rate constants for formation and dissociation of the myosin X MgAMPPNP complex were reduced 200-fold; the logarithm of the dissociation rate was roughly proportional to the fractional concentration of ethylene glycol. Nucleotide dissociation was accelerated at least 300-fold by pure actin but remained slow with regulated actin in the absence of Ca2+. Ethylene glycol substitution reduced the affinity of S1 and the S1 X MgAMPPNP complex for actin equally (100-fold at 50% ethylene glycol). These results show that ethylene glycol has specific effects on myosin's enzymic mechanism, which can account for its effect on the tension and stiffness of glycerinated muscle fibres.     

The effect of genetically expressed cardiac titin fragments on in vitro actin motility.

Titin is a striated muscle-specific giant protein (M(r) approximately 3,000,000) that consists predominantly of two classes of approximately 100 amino acid motifs, class I and class II, that repeat along the molecule. Titin is found inside the sarcomere, in close proximity to both actin and myosin filaments. Several biochemical studies have found that titin interacts with myosin and actin. In the present work we investigated whether this biochemical interaction is functionally significant by studying the effect of titin on actomyosin interaction in an in vitro motility assay where fluorescently labeled actin filaments are sliding on top of a lawn of myosin molecules. We used genetically expressed titin fragments containing either a single class I motif (Ti I), a single class II motif (Ti II), or the two motifs linked together (Ti I-II). Neither Ti I nor Ti II alone affected actin-filament sliding on either myosin, heavy meromyosin, or myosin subfragment-1. In contrast, the linked fragment (Ti I-II) strongly inhibited actin sliding. Ti I-II-induced inhibition was observed with full-length myosin, heavy meromyosin, and myosin subfragment-1. The degree of inhibition was largest with myosin subfragment-1, intermediate with heavy meromyosin, and smallest with myosin. In vitro binding assays and electrophoretic analyses revealed that the inhibition is most likely caused by interaction between the actin filament and the titin I-II fragment. The physiological relevance of the novel finding of motility inhibition by titin fragments is discussed.     

The mechanism of skeletal muscle myosin ATPase. The mechanism of ADP interaction

The mechanism of interaction between ADP and the myosin active center has been studied using a transient kinetic technique. The results show that the interaction of ADP with the myosin active center is a homogeneous process independent of the association state of the active centers; namely, whether ADP interacts with the monomeric myosin subfragment-1, or with the dimeric forms heavy meromyosin and myosin. The kinetics of the interaction conforms to a simple two-step reaction mechanism for ADP binding. The kinetic and thermodynamic constants for this mechanism have been determined. In addition, analysis of the binding isotherm indicates that the two active sites in heavy meromyosin and myosin function as identical and independent sites.     

Immunofluorescent and histochemical localization of AMP deaminase in skeletal muscle

Fluorescent antibody staining experiments with both isolated myofibrils and muscle fibers grown in culture show that AMP deaminase is bound to the myofibril in the A band. The strongest staining occurs at each end of the A band. The approximate width of the fluorescent stripes and their relation to the A band remains constant as a function of sarcomere length. Removal of enzyme from the myofibrils leads to loss of staining, and readdition of purified enzyme restores the original staining pattern. A histoenzymatic method for the detection of AMP deaminase activity in cultured fibers gives comparable localization. The results are consistent with the previous observation (Ashby, B. and C. Frieden. 1977.J. Biol. Chem. 252:1869--1872) that AMP deaminase forms a tight complex in solution with subfragment-2 (S-2) of myosin or with heavy meromyosin (HMM).     

Acrylamide fluorescence quenching studies on the actin-induced change in protein dynamics in the subfragment-1/subfragment-2 link region of cardiac myosin

In order to obtain information about the actin-induced conformational change around the subfragment-1/subfragment-2 link region of myosin, measurements of the fluorescence quenching by acrylamide were made on cardiac myosin and its heavy meromyosin, in which the reactive lysyl residue located in the link region was labeled with an extrinsic fluorophore, the N-methyl-2-anilino-6-naphthalenesulfonyl group. The results with the model compound indicated the involvement of a collisional quenching mechanism for the fluorophore. The quenching rate constant calculated from measured quenching constants using available lifetime data was extremely low for the labeled myosin (0.59 X 10(8) M-1 . S-1), suggesting that the fluorophore bound to myosin is surrounded by segments of proteins. This value was independent of the solvent viscosity, indicating that the quenching reaction is limited by fluctuations in the protein matrix, which produce the inward movement of acrylamide. Chymotryptic digestion of the labeled myosin, which yielded the light chain-2-deficient heavy meromyosin, made the bound fluorophore slightly exposed. Addition of F-actin resulted in about 40% reduction in the quenching rate constants for the labeled myosin and heavy meromyosin. The actin effect was reversed by adding ATP. These results suggest that the binding of actin to myosin makes the protein matrix around the subfragment-1/subfragment-2 link region less mobile.     

The mechanism of the skeletal muscle myosin ATPase. I. Identity of the myosin active sites.

In the present study, the question of whether the two myosin active sites are identical with respect to ATP binding and hydrolysis was reinvestigated. The stoichiometry of ATP binding to myosin, heavy meromyosin, and subfragment-1 was determined by measuring the fluorescence enhancement caused by the binding of MgATP. The amount of irreversible ATP binding and the magnitude of the initial ATP hydrolysis (initial Pi burst) was determined by measuring [gamma-32P]ATP hydrolysis with and without a cold ATP chase in a three-syringe quenched flow apparatus. The results show that, under a wide variety of experimental conditions: 1) the stoichiometry of ATP binding ranges from 0.8 to 1 mol of ATP/myosin active site for myosin, heavy meromyosin, and subfragment-1, 2) 80 to 100% of this ATP binding is irreversible, 3) 70 to 90% of the irreversibly bound ATP is hydrolyzed in the initial Pi burst, 4) the first order rate constant for the rate-limiting step in ATP hydrolysis by heavy meromyosin is equal to the steady state heavy meromyosin ATPase rate only if the latter is calculated on the basis of two active sites per heavy meromyosin molecule. It is concluded that the two active sites of myosin are identical with respect to ATP binding and hydrolysis.     

Preparation and characterization of heavy meromyosin and subfragment 1 from vertebrate cytoplasmic myosins

The soluble fragments of myosin, heavy meromyosin (HMM), and subfragment 1 (S-1) have been instrumental in elucidating the kinetic mechanisms of the actin-activated MgATPase activity of both skeletal and smooth muscle myosin. To date, relatively little has been published on these fragments from vertebrate cytoplasmic myosins. We now describe the preparation and steady-state kinetic characterization of S-1 and HMM from human platelet and avian intestinal epithelial brush border myosin. The HMM prepared from each of these tissues was similar both in their SDS-polyacrylamide gel pattern and in their steady-state kinetic properties. The Vmax of the actin-activated MgATPase activity varied between 0.8 and 2.5 s-1, and the KATPase (the apparent dissociation constant derived from a double-reciprocal plot of the MgATPase activity) was about 1-2 microM. This low value for the apparent dissociation constant was similar to the dissociation constant of HMM for actin directly measured under similar conditions and is about 40 times lower than that determined with avian smooth muscle HMM. The KATPase of the cytoplasmic HMM was only slightly increased when the ionic strength was raised from 12 to 112 mM.     

Interaction of AMP-aminohydrolase with myosin and its subfragments.

We have shown that purified rabbit skeletal muscle AMP-aminohydrolase binds to rabbit muscle myosin, heavy meromyosin, and Subfragment 2 but does not bind to light meromyosin nor to Subfragment 1. The dissociation constant for binding to myosin was determined to be 0.14 muM. A new sedimentation boundary, presumably reflecting formation of a complex between AMP-aminohydrolase and heavy meromyosin or Subfragment 2, can be observed using the analytical ultracentrifuge. Binding of AMP-aminohydrolase to myosin, heavy meromyosin, or Subfragment 2 is abolished by phosphate (less than 10 mM), an inhibitor of AMP-aminohydrolase. No other rabbit muscle enzyme tested showed any interaction with myosin under the same conditions and there was no indication of complex formation between AMP-aminohydrolase and phosphofructokinase or phosphocreatine kinase in the analytical ultracentrifuge.     

An enzyme-probe study of motile domains in the subfragment-2 region of myosin

The temperature-dependence of local melting within the subfragment-2 region of rabbit skeletal muscle myosin has been investigated using an enzyme-probe technique. Rate constants of fragmentation of two long subfragment-2 particles (61,000 Mr and 53,000 Mr per polypeptide chain) and a short subfragment-2 particle (34,000 Mr per polypeptide chain) by three different enzymes (alpha-chymotrypsin, trypsin and papain) have been determined over the temperature range 5 to 40 degrees C. We followed the time-course of digestion at specific sites at high (I = 0.50, pH 7.3) and low (physiological, I = 0.15, pH 7.3) ionic strengths by electrophoresis of the digestion products on sodium dodecyl sulfate-containing gels. All rate constants were corrected for the intrinsic temperature-dependence of the enzymes by comparison with model substrates. Normalized rate constant versus temperature profiles for the three enzyme-probes are similar in showing that local melting in long subfragment-2 (61,000 Mr) occurs in two distinct stages as was observed earlier for the intact myosin rod. Over the temperature range 5 to 25 degrees C a restricted region at Mr = 53,000 to 50,000 from the N terminus of the rod (the light meromyosin/heavy meromyosin junction) shows the highest susceptibility to proteolytic cleavage. At temperatures above 25 degrees C local melting was detected by all three enzymes at several specific sites within the hinge domain (Mr = 53,000 to 34,000). Activation energies for cleavage at the susceptible sites were similar for the three enzyme probes. They suggest that this region of the myosin rod has significantly lower thermal stability than the flanking light meromyosin and short subfragment-2 segments. These results, together with other physico-chemical studies, point to the hinge domain of the myosin cross-bridge as an important functional element in the mechanism of force generation in muscle.     

Temperature-dependence of local melting in the myosin subfragment-2 region of the rigor cross-bridge

We have used alpha-chymotrypsin as an enzyme-probe to detect local melting in the subfragment-2 region of the cross-bridges of rigor myofibrils and glycerinated psoas fibers. The kinetics of proteolysis and the sites of cleavage were determined at various temperatures over the range 5 to 40 degrees C by following the decay of the myosin heavy chain and the rates of appearance of light meromyosin fragments, using electrophoresis on sodium dodecyl sulfate-containing polyacrylamide gels. Cleavage occurs primarily at the 72,000 Mr and 64,000 Mr (per polypeptide chain from the C terminus of myosin) sites within the light meromyosin-heavy meromyosin hinge domain of the subfragment-2 region, under all experimental conditions. At pH 8.2 to 8.3 and at low divalent metal ion (0.1 mM), where the actin-bound cross-bridges are thought to be released from the thick filament surface, the intrinsic cleavage rate constant (k) increases markedly as the temperature is raised. This suggests substantial thermal destabilization of the released cross-bridge in the intact contractile apparatus. Addition of divalent metal ion (10 mM) lowers the cleavage rate and shifts the k versus temperature profile to higher temperatures. Normalized rate constants for chymotryptic cleavage within the subfragment-2 hinge region of released cross-bridges (pH 8.2, low divalent metal) of rigor fibers were markedly lower than activated fibers at all temperatures investigated (5 to 40 degrees C). Results show that conformational melting within the subfragment-2 hinge region is amplified on activation and is well above that observed when the actin-attached rigor bridge is passively released from the thick filament surface.     

What limits the velocity of fast-skeletal muscle contraction in mammals?

In rat skeletal muscle the unloaded shortening velocity (Vo) is defined by the myosin isoform expressed in the muscle fibre. In 2001 we suggested that ADP release from actomyosin in solution (controlled by k(-AD)) was of the right size to limit Vo. However, to compare mechanical and solution kinetic data required a series of corrections to compensate for the differences in experimental conditions (0.5 M KCl, 22 degrees C for kinetic assays of myosin, 200 mM ionic strength, 12 degrees C to measure Vo). Here, a method was developed to prepare heavy meromyosin (HMM) from pure myosin isoforms isolated from single muscle fibres and to study k(-AD) (determined from the affinity of the acto-myosin complex for ADP, KAD) and the rate of ATP-induced acto-HMM dissociation (controlled by K1k+2) under the same experimental condition used to measure Vo). In fast-muscle myosin isolated from a wide range of mammalian muscles, k(-AD) was found to be too fast to limit Vo, whereas K1k+2 was of the right magnitude for ATP-induced dissociation of the cross-bridge to limit shortening velocity. The result was unexpected and prompted further experiments using the stopped-flow approach on myosin subfragment-1 (S1) and HMM obtained from bulk preparations of rabbit and rat muscle. These confirmed that the rate of cross-bridge dissociation by ATP limits the velocity of contraction for fast myosin II isoforms at 12 degrees C, while k(-AD) limits the velocity of slow myosin II isoforms. Extrapolating our data to 37 degrees C suggests that at physiological temperature the rate of ADP dissociation may limit Vo for both isoforms.     

Interaction of rat muscle AMP deaminase with myosin. I. Biochemical study of the interaction of AMP deaminase and myosin in rat muscle.

AMP deaminase was completely solubilized from rat skeletal muscle with 50 mM Tris-HCl buffer (pH 7.0) containing KCl at a concentration of 0.3 M or more. The purified enzyme was found to be bound to rat muscle myosin or actomyosin, but not to F-actin at KCl concentrations of less than 0.3 M. Kinetic analysis indicated that 1 mol of AMP deaminase was bound to 3 mol of myosin and that the dissociation constant (Kd) of this binding was 0.06 micrometer. It was also shown that AMP deaminase from muscle interacted mainly with the light meromyosin portion of the myosin molecule. This finding differs from that of Ashby and coworkers on rabbit muscle AMP deaminase, probably due to a difference in the properties of rat and rabbit muscle AMP deaminase. AMP deaminase isozymes from rat liver, kidney and cardiac muscle did not interact with rat muscle myosin. The physiological significance of this binding of AMP deaminase to myosin is discussed.     

Myosin and actomyosin from human skeletal muscle.

Human skeletal natural actomyosin contained actin, tropomyosin, troponin and myosin components as judged by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Purified human myosin contained at least three light chains having molecular weights (+/-2000) of 25 000, 18 000 and 15 000. Inhibitory and calcium binding components of troponin were identified in an actin-tropomyosin-troponin complex extracted from acetone-dried muscle powder at 37 degrees C. Activation of the Mg-ATPase activity of Ca2+-sensitive human natural or reconstituted actomyosin was half maximal at approximately 3.4 muM Ca2+ concentration (CaEGTA binding constant equals 4.4 - 10(5) at pH 6.8). Subfragment 1, isolated from the human heavy meromyosin by digestion with papain, appeared as a single peak after DEAE-cellulose chromatography. In the pH 6-9 range, the Ca2+-ATPase activity of the subfragment 1 was 1.8- and 4-fold higher that the original heavy meromyosin and myosin, respectively. The ATPase activities of human myosin and its fragments were 6-10 fold lower than those of corresponding proteins from rabbit fast skeletal muscle. Human myosin lost approximately 60% of the Ca2+-ATPase activity at pH 9 without a concomitant change in the number of distribution of its light chains. These findings indicate that human skeletal muscle myosin resembles other slow and fast mammalian muscles. Regulation of human skeletal actomyosin by Ca2+ is similar to that of rabbit fast or slow muscle.     

Two-headed binding of the unphosphorylated nonmuscle heavy meromyosin.ADP complex to actin

The enzymatic and motor function of smooth muscle and nonmuscle myosin II is activated by phosphorylation of the regulatory light chains located in the head portion of myosin. Dimerization of the heads, which is brought about by the coiled-coil tail region, is essential for regulation since single-headed fragments are active regardless of the state of phosphorylation. Utilizing the fluorescence signal on binding of myosin to pyrene-labeled actin filaments, we investigated the interplay of actin and nucleotide binding to thiophosphorylated and unphosphorylated recombinant nonmuscle IIA heavy meromyosin constructs. We show that both heads of either thiophosphorylated or unphosphorylated heavy meromyosin bind very strongly to actin (K(d) < 10 nM) in the presence or absence of ADP. The heads have high and indistinguishable affinities for ADP (K(d) around 1 microM) when bound to actin. These findings are in line with the previously observed unusually loose coupling between nucleotide and actin binding to nonmuscle myosin IIA subfragment-1 (Kovács et al. (2003) J. Biol. Chem. 278, 38132.). Furthermore, they imply that the structure of the two heads in the ternary actomyosin-ADP complex is symmetrical and that the asymmetrical structure observed in the presence of ATP and the absence of actin in previous investigations (Wendt et al. (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 4361) is likely to represent an ATPase intermediate that precedes the actomyosin-ADP state.     

Determination of quantitative parameters of the binding of F-protein (phosphofructokinase) with myosin and localization of binding sites on the myosin molecule

To determine the localization of F-protein binding sites on myosin, the interaction of F-protein with myosin and its proteolytic fragments in 0.1 M KCl, 10 mM K-phosphate pH 6.5 was studied, using sedimentation, electron microscopic and optical diffraction methods. Sedimentation experiments showed that F-protein binds to myosin and myosin rod rather than to light meromyosin or S-1. The F-protein binding to myosin and rod is of a similar character. The calculated values of the constants of F-protein binding to myosin and rod are 2.6 X 10(5) M-1 and 2.1 X 10(5) M-1, respectively. The binding sites are probably located on the subfragment-2 portion of the myosin molecule. The number of F-protein binding sites on myosin calculated per chain weight of 80 000 is 5 +/- 1. The sedimentation results were confirmed by electron microscopic data. F-protein does not bind to light meromyosin paracrystals, but decorates myosin and rod filaments with the interval of 14.3 nm regardless of whether F-protein is added before or after filamentogenesis. A comparison of optical diffraction patterns obtained from myosin and rod filaments with those from decorated ones revealed a marked enhancement of meridional reflection at (14.3 nm)-1 in the latter case.     

Myosin and actomyosin from human skeletal muscle

Human skeletal natural actomyosin contained actin, tropomyosin, troponin and myosin components as judged by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Purified human myosin contained at least three light chains having molecular weights (±2000) of 25 000, 18 000 and 15 000. Inhibitory and calcium binding components of troponin were identified in an actin-tropomyosin-troponin complex extracted from acetone-dried muscle powder at 37°C. Activation of the Mg-ATPase activity of Ca²⁺-sensitive human natural or reconstituted actomyosin was half maximal at approximately 3.4 μM Ca²⁺ concentration (CaEGTA binding constant = 4.4 · 10⁵ at pH 6.8). Subfragment 1, isolated from the human heavy meromyosin by digestion with papain, appeared as a single peak after DEAE-cellulose chromatography. In the pH 6–9 range, the Ca²⁺-ATPase activity of the subfragment 1 was 1.8-and 4-fold higher that the original heavy meromyosin and myosin, respectively. The ATPase activities of human myosin and its fragments were 6–10 fold lower than those of corresponding proteins from rabbit fast skeletal muscle. Human myosin lost approximately 60% of the Ca²⁺-ATPase activity at pH 9 without a concomitant change in the number of distribution of its light chains. These findings indicate that human skeletal muscle myosin resembles other slow and fast mammalian muscles. Regulation of human skeletal actomyosin by Ca²⁺ is similar to that of rabbit fast or slow muscle     

The interaction of C-protein with heavy meromyosin and subfragment-2.

C-protein has previously been shown to bind to the light-meromyosin region of the myosin tail. Examination of mixtures of C-protein with heavy meromyosin or subfragment-2 or subfragment-1 in the analytical ultracentrifuge shows that there is also a binding site for C-protein in the subfragment-2 region of the tail.     

The primary structure of skeletal muscle myosin heavy chain: IV. Sequence of the rod, and the complete 1,938-residue sequence of the heavy chain

In the preceding paper [Maita, T., Miyanishi, T., Matsuzono, K., Tanioka, Y., & Matsuda, G. (1991) J. Biochem. 110, 68-74], we reported the amino-terminal 837-residue sequence of the heavy chain of adult chicken pectoralis muscle myosin. This paper describes the carboxyl terminal 1,097-residue sequence and the linkage of the two sequences. Rod obtained by digesting myosin filaments with alpha-chymotrypsin was redigested with the protease at high KCl concentration, and two fragments, subfragment-2 and light meromyosin, were isolated and sequenced by conventional methods. The linkage of the two fragments was deduced from the sequence of an overlapping peptide obtained by cleaving the rod with cyanogen bromide. The rod contained 1,039 amino acid residues, but lacked the carboxyl-terminal 58 residues of the heavy chain. A carboxyl-terminal 63-residue peptide obtained by cleaving the whole heavy chain with cyanogen bromide was sequenced. Thus, the carboxyl terminal 1,097-residue sequence of the heavy chain was completed. The linkage of subfragment-1 and the rod was deduced from the sequence of an overlapping peptide between the two which was obtained by cleaving heavy meromyosin with cyanogen bromide. Comparing the sequence of the adult myosin thus determined with that of chicken embryonic myosin reported by Molina et al. [Molina, M.I., Kropp, K.E., Gulick, J., & Robbins, J. (1987) J. Biol. Chem. 262, 6478-6488], we found that the sequence homology is 94%.     

Three-dimensional reconstruction and averaging of 50 S ribosomal subunits of Escherichia coli from electron micrographs

The thermal stability and melting kinetics of the α-helical conformation within several regions of the rabbit myosin rod have been investigated. Cyanogen bromide cleavage of long myosin subfragment-2 produced one coiled-coil α-helical fragment corresponding to short subfragment-2 with molecular weight 90,000 (M_r = 45,000) and two fragments from the hinge region with molecular weights of 32,000 to 34,000 (M_r = 16,000 to 17,000) and 24,000 to 26,000 (M_r = 12,000 to 13,000). Optical rotation melting experiments and temperature-jump kinetic studies of long subfragment-2 and its cyanogen bromide fragments show that the hinge and the short subfragment-2 domains melt as quasi-independent co-operative units. The α-helical structure within the hinge has an appreciably lower thermal stability than the flanking short subfragment-2 and light meromyosin regions of the myosin rod. Two relaxation processes for helix-melting, one in the submillisecond range (τ_f) and the other in the millisecond range (τ_s), are observed in the light meromyosin and short subfragment-2 regions of the rod, but melting in the hinge domain is dominated by the fast (τ_f) process. Results suggest that the hinge domain of the subfragment-2 link may be the locus of force generation in a cycling cross-bridge.