Chicken-gizzard actin. Interaction with skeletal-muscle myosin

Interaction of actin from chicken gizzard and from rabbit skeletal muscle with rabbit skeletal muscle myosin was compared by measuring the rate of superprecipitation, the activation of the Mg-ATPase and inhibition of K-ATPase activity of myosin and heavy meromyosin, and determination of binding of heavy meromyosin in the absence of ATP. Both the rate of superprecipitation of the hybrid actomyosin and the activation of myosin ATPase by gizzard actin are lower than those obtained with skeletal muscle actin. The activation of myosin Mg-ATPase by the two actin species also shows different dependence on substrate concentration: with gizzard actin the substrate inhibition starts at lower ATP concentration. The double-reciprocal plots of the Mg-ATPase activity of heavy meromyosin versus actin concentration yield the same value of the extrapolated ATPase activity at infinite actin concentration (V) for the two actins and nearly double the actin concentration needed to produce half-maximal activation (Kapp) in the case of gizzard actin. A corresponding difference in the abilities of the two actin species to inhibit the K-ATPase activity of heavy meromyosin in the absence of divalent cations was also observed. The results are discussed in terms of the effect of substitutions in the amino acid sequence of gizzard and skeletal muscle actins on their interaction with myosin.     

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     

Ca2+ dependence of the ATPase activity of phosphorylated smooth muscle myosin: effects of tropomyosin and actin

Calcium ions produce a 3-4-fold stimulation of the actin-activated ATPase activities of phosphorylated myosin from bovine pulmonary artery or chicken gizzard at 37 degrees C and at physiological ionic strengths, 0.12-0.16 M. Actins from either chicken gizzard or rabbit skeletal muscle stimulate the activity of phosphorylated myosin in a Ca2+-dependent manner, indicating that the Ca2+ sensitivity involves myosin or a protein associated with it. Partial loss of Ca2+ sensitivity upon treatment of phosphorylated gizzard myosin with low concentrations of chymotrypsin and the lack of any change on similar treatment of actin supports the above conclusion. Although both actins enhance ATPase activity, activation by gizzard actin exhibits Ca2+ dependence at higher temperatures or lower ionic strengths than does activation by skeletal muscle actin. The Ca2+ dependence of the activity of phosphorylated heavy meromyosin is about half that of myosin and is affected differently by temperature, ionic strength and Mg2+, being independent of temperature and optimal at lower concentrations of NaCl. Raising the concentration of Mg2+ above 2-3 mM inhibits the activity of heavy meromyosin but stimulates that of myosin, indicating that Mg2+ and Ca2+ activate myosin at different binding sites.     

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.     

Proteolysis of smooth muscle myosin by Staphylococcus aureus protease: preparation of heavy meromyosin and subfragment 1 with intact 20 000-dalton light chains

The proteolysis of gizzard myosin by Staphylococcus aureus protease produces both heavy meromyosin and subfragment 1 in which the 20 000-dalton light chains are intact, and conditions are suggested for the preparation of each. Cleavage of the myosin heavy chain to produce subfragment 1 is dependent on the myosin conformation. Proteolysis of myosin in the 10S conformation yields predominantly heavy meromyosin, and myosin in the 6S conformation yields mostly subfragment 1 and some heavy meromyosin. Two sites are influenced by myosin conformation, and these are located at approximately 68 000 and 94 000 daltons from the N-terminus of the myosin heavy chain. The latter site is thought to be located at the subfragment 1-subfragment 2 junction, and cleavage at this site results in the production of subfragment 1. The time courses of phosphorylation of both heavy meromyosin and subfragment 1 can be fit by a single exponential. The actin-activated Mg2+-ATPase activity of heavy meromyosin is markedly activated by phosphorylation of the 20 000-dalton light chains. From the actin dependence of Mg2+-ATPase activity the following values are obtained: for phosphorylated heavy meromyosin, Vmax approximately 5.6 s-1 and Ka (the apparent dissociation constant for actin) approximately 2 mg/mL; for dephosphorylated heavy meromyosin, Vmax approximately 0.2 s-1 and Ka approximately 7 mg/mL. The actin-activated ATPase activity of subfragment 1 is not influenced by phosphorylation, and Vmax and Ka for both the phosphorylated and dephosphorylated forms are 0.4 s-1 and 5 mg/mL, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of cytoplasmic actin isolated from Acanthamoeba castellanii by a new method.

Cytoplasmic actin has been isolated from Acanthamoeba castellanii by a new method, employing chromatography on DEAE-cellulose, that improves the yield by more than 20-fold over the previously reported method. This procedure should be particularly useful for isolating actin from cells in which it is present in relatively low concentration because the method does not depend initially on the polymerization of actin or its interaction with myosin. Systematic comparison of the properties of purified Acanthamoeba actin and rabbit skeletal muscle actin shows them to be similar in many ways: viscosity of F-actin, stoichiometry of bound nucleotide, stoichiometry of binding of muscle heavy meromyosin and myosin subfragment 1 in the absence of ATP, and ability to inhibit the KATPase activity of heavy meromyosin. The amino acid compositions of Acanthamoeba and muscle actin are also quite similar, but significant differences, especially the presence of epsilon-N-methyllysines in Acanthamoeba actin, have been confirmed. In addition to this structural difference, we find that Acanthamoeba actin is only one-third as effective as muscle actin as an activator of the MgATPase of muscle heavy meromyosin and subfragment 1. For Acanthamoeba actin, as for muscle actin, this activation exhibits hyperbolic dependence on actin concentration; i.e. the double reciprocal plot of ATPase activation versus actin concentration is linear. From these plots we find that the two actins give the same extrapolated ATPase activity at infinite actin concentration (Vmax) but differ by a factor of three in the concentration of actin needed to produce half-maximal activation (Kapp).     

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.     

3-Methylhistidine in actin and other muscle proteins

1. By the use of the extended elution system for basic amino acid analysis, 3-methylhistidine has been detected in hydrolysates of actin isolated from mammalian, fish and bird skeletal muscle. 2. Evidence is presented to indicate that 3-methylhistidine forms part of the primary structure and that in rabbit actin this residue is restricted to one peptide fraction obtained from the tryptic digest. 3. Rabbit skeletal-muscle actin has a 3-methylhistidine:histidine ratio 1:7.6, indicating a minimum molecular weight of 47600. 4. Adult rabbit myosin contains approximately 2 3-methylhistidine residues/mol. These residues are localized in the heavy meromyosin part of the molecule, and are restricted to the major component obtained after succinylation.     

Inhibition of the relative movement of actin and myosin by caldesmon and calponin.

Contractile activity of myosin II in smooth muscle and non-muscle cells requires phosphorylation of myosin by myosin light chain kinase. In addition, these cells have the potential for regulation at the thin filament level by caldesmon and calponin, both of which bind calmodulin. We have investigated this regulation using in vitro motility assays. Caldesmon completely inhibited the movement of actin filaments by either phosphorylated smooth muscle myosin or rabbit skeletal muscle heavy meromyosin. The amount of caldesmon required for inhibition was decreased when tropomyosin is present. Similarly, calponin binding to actin resulted in inhibition of actin filament movement by both smooth muscle myosin and skeletal muscle heavy meromyosin. Tropomyosin had no effect on the amount of calponin needed for inhibition. High concentrations of calmodulin (10 microM) in the presence of calcium completely reversed the inhibition. The nature of the inhibition by the two proteins was markedly different. Increasing caldesmon concentrations resulted in graded inhibition of the movement of actin filaments until complete inhibition of movement was obtained. Calponin inhibited actin sliding in a more "all or none" fashion. As the calponin concentration was increased the number of actin filaments moving was markedly decreased, but the velocity of movement remained near control values.     

Temperature-dependent change in rate-limiting step of the magnesium-stimulated ITPase of myosin.

The effects of temperature on Mg-ITPase activity of heavy meromyosin and myosin subfragment 1 were measured in 0.1 M KC1. The initial burst of Pi liberation was one mol per mol of heavy meromyosin or two mol of myosin subfragment 1, i.e. one mol per two mol of myosin active sites, at 20 degrees C. However, it was almost zero mol below 8degrees C. Effects of KC1 concentration and pH on ITPase activity of heavy meromyosin at 20 degrees C were different from those below 8 degrees C, suggesting that the rate-limiting step in the Mg-ITP hydrolysis of myosin depends on temperature. The effect of temperature on the actin activation of heavy meromyosin Mg-ITPase was analyzed by measuring the temperature dependence of double-reciprocal plots of ITPase activity against actin concentration. The extent of actin activation was larger at low temperture. The results presented in this paper might be explained by assuming the existence of two kinds of active sites on a myosin molecule.     

Temperature-dependent transitions of the myosin-product intermediate at 10 degrees during Mn(II)-ATP hydrolysis by myosin from rabbit psoas muscle.

The initial burst of Pi liberation during the hydrolysis of Mn(II)-ATP by heavy meromyosin from rabbit psoas muscle was investigated. Below 10 degrees, the initial burst of Pi liberation was inhibited by the pre-addition of ADP without any change in the steady-state activity, but it was not inhibited above 10 degrees. The burst size was about one mole per mole of heavy meromyosin. The initial burst of Pi liberation in Mg-ATP hydrolysis at 8 degrees, however, was not inhibited by the pre-addition of ADP. These results, obtained with psoas muscle heavy meromyosin, were almost the same as those obtained with heavy meromyosin from rabbit leg and back muscles (Hozumi and Tawada (1975) Biochim. Biophys. Acta 376, 1-12) and, therefore, indicate that in Mn-ATP above 10 degrees there is at the burst site a predominant myosin -product complex generated by ATP hydrolysis. Similarly, below 10 degrees there is a myosin-product complex identical with the one generated by adding ADP (and Pi) to myosin.     

Cross-linking of contractile proteins from skeletal muscle by treatment with microbial transglutaminase.

The action on muscle proteins of microbial transglutaminase (MTGase), which catalyzes the formation of a "zero-length" covalent cross-link between glutamine and lysine residues in peptides, was studied in order to define a basis for future application of MTGase cross-linking to the study of muscle protein interaction. We examined the cross-linking of skeletal muscle myosin, myosin subfragments, actin, and myofibrils by treatment with MTGase and the possible side-effects of the cross-linking on the enzymic activity of myosin, and found that the rod portions of myosin in myosin filaments were quickly cross-linked to each other by the action of MTGase, but myosin subfragment 1 was not cross-linked to actin. The MgATPase activities at 0.5 M KCl of myosin, heavy meromyosin, subfragment 1, and subfragment 1-actin were not significantly affected by the MTGase reaction. A very small fraction of the head portion of heavy meromyosin was cross-linked to actin in their rigor complexes by MTGase, and the ATPase activity at 0.5 M KCl of the cross-linked heavy meromyosin-actin complexes was slightly enhanced.     

Complex formation with heavy meromyosin of the isolated actin-like component of thrombosthenin,the contractile protein from blood platelets

Heavy meromyosin from rabbit skeletal muscle myosin was added on grids to the filamentous polymer of highly purified thrombosthenin A, tho actin-like protein in blood platelets. This resulted in an arrowhead complex formation between heavy meromyosin and the polymer, providing evidence that the polymer has a helical tertiary structure similar to muscle actin. The complex formation was inhibited by ATP.     

Affinity chromatography of myosin, heavy meromyosin, and heavy meromyosin subfragment one on F-actin columns stabilized by phalloidin.

A method of affinity chromatography based on the trapping of actin filaments within agarose gel beads is described. This method can be used for the purification of myosin and its active proteolytic subfragments, as well as for studies on the interaction between actin and these proteins. Actin columns stabilized by phalloidin bind myosin, heavy meromyosin (HMM), and heavy meromyosin subfragment 1 (HMM-S1) specifically and reversibly. The effect of pyrophosphate and KCl on the dissociation of actomyosin, acto-HMM, or acto-HMM-S1 complex is reported. We also describe the single-step purification of myosin from a crude rabbit psoas muscle extract.     

Degradation of smooth-muscle myosin by trypsin-like serine proteinases.

1. Hydrolysis of the myosins from smooth and from skeletal muscle by a rat trypsin-like serine proteinase and by bovine trypsin at pH 7 is compared. 2. Proteolysis of the heavy chains of both myosins by the rat enzyme proceeds at rates approx. 20 times faster than those obtained with bovine trypsin. Whereas cleavage of skeletal-muscle myosin heavy chain by both enzymes results in the generation of conventional products i.e. heavy meromyosin and light meromyosin, the heavy chain of smooth-muscle myosin is degraded into a fragment of mol. wt. 150000. This is dissimilar from heavy meromyosin and cannot be converted into heavy meromyosin. It is shown that proteolysis of the heavy chain takes place in the head region. 3. The 'regulatory' light chain (20kDa) of smooth-muscle myosin is degraded very rapidly by the rat proteinase. 4. The ability of smooth-muscle myosin to have its ATPase activity activated by actin in the presence of a crude tropomyosin fraction on introduction of Ca2+ is diminished progressively during exposure to the rat proteinase. The rate of loss of the Ca2+-activated actomyosin ATPase activity is very similar to the rate observed for proteolysis of the heavy chain and 3-4 times slower than the rate of removal of the so-called 'regulatory' light chain. 5. The significance of these findings in terms of the functional organization of the smooth muscle myosin molecule is discussed. 6. Since the degraded myosin obtained after exposure to very small amounts of the rat proteinase is no longer able to respond to Ca2+, i.e. the functional activity of the molecule has been removed, the implications of a similar type of proteolysis operating in vivo are considered for myofibrillar protein turnover in general, but particularly with regard to the initiation of myosin degradation, which is known to take place outside the lysosome (i.e. at neutral pH).     

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.     

Effect of F-actin upon the binding of ADP to myosin and its fragments.

The effect of F-actin upon the binding of ADP to rabbit skeletal muscle myosin, heavy meromyosin, and subfragment 1 was studied by equilibrium dialysis, ultracentrifuge transport, and light scattering techniques. Both myosin and H-meromyosin (HMM) bind a maximum of approximately 1.6 mol of ADP/mol of protein, while S-1 binds approximately 0.9 mol of ADP/mol of protein. The affinity for ADP of all three preparations was similar at a given ionic strength (approximately 10(6) M-1 at 0.05 M KCl) and decreased with increasing ionic strength. Under conditions similar to those used for the measurement of ADP binding, the binding sites of myosin, HMM, and subfragment 1 (S-1) are saturated with actin at molar ratios of 2, 2, and 1 mol of actin monomer/mol of protein, respectively, as determined by light scattering, ultracentrifuge transport, and in the case of myosin by ATPase measurements. F-actin was found to inhibit ADP binding, but even at an actin concentration at least twice that required for saturation of myosin, HMM, or S-1, significant ADP binding remained. This ADP binding was inhibited by 10(-4) M pyrophosphate. The observations are consistent with the formation of an actomyosin-ADP complex in which actin and ADP are bound to myosin at distinct but interacting sites.     

Conformational stability of the myosin rod

Chymotryptic cleavage patterns of myosin rods from pig stomach, chicken gizzard, and rabbit skeletal muscle indicate that short (approximately 45 nm) heavy meromyosin subfragment 2 (SF2) is a consistent product of all three rods, whereas long (approximately 60 nm) SF2 is derived only from skeletal muscle myosin. Differential scanning calorimetry was used to follow the thermally induced melting transition of the rods and certain of their subfragments. In 0.12 M KCl, sodium phosphate buffer, pH 6.2-7.6, the light meromyosin (LMM) and SF2 domains of each rod had essentially identical conformational stabilities. Temperature midpoints for the melting transitions were 54-56 degrees C for the two smooth muscle myosin rods and 50-53 degrees C for the skeletal muscle myosin rod. In 0.6 M K Cl buffer, melting transitions for the smooth muscle myosin rods were essentially unchanged, but skeletal muscle myosin rods showed multiphase melting, with major transitions at 43 degrees C and 52 degrees C. The first of these was tentatively attributed to LMM, and the second to SF2. In 0.12 M K Cl buffer, the LMM transition was stabilised so that it superimposed on that of SF2. No melting was observed in any of the rods at physiological temperature. These results indicate that, excluding a possible but only narrow hinge region, the entire myosin rod has essentially uniform conformational stability at physiological pH and ionic strength, and thus that the contractile and elastic properties of the cross-bridge exist in the heavy meromyosin subfragment 1 (SF1) domains of the molecule.     

Immunochemical evidence for the species-specificity of mammalian cardiac myosin and heavy meromyosin.

Structural differences between various myosins were investigated by means of antibodies to heavy meromyosin, a tryptic subfragment of myosin. Heavy meromyosin was purified from rabbit white skeletal and from pig and human cardiac muscles by gel filtration, and antisera were produced in guinea pigs. Analyses, carried out with the quantitative micro-complement fixation technique, indicated that the antibodies were specific to heavy meromyosin and myosin and not to other contractile proteins. For each muscle type, the corresponding intact myosin reacted, and the degree of dixation was always lower than with heavy meromyosin (50 and 70% fixation respectively). This vertical shift was the same for the three muscle types, indicating that the heavy meromyosin represent corresponding fragments of the myosin molecule from one muscle to the other. Antisera to pig or human cardiac heavy meromyosin clearly distinguished antigens (heavy meromyosins, myosins, or crude extracts) from the ventricles of various heterologous species. Relative to pig, the immunological distances were 50 for the rabbit, 73 for the rat and greater than 100 for human and mice. Relative to human, these values were 20 for the rat, 60 for the rabbit, 72 for the pig. These data provide direct evidence that mammalian cardiac myosin is species-specific.     

Modulation of the actin-activated adenosinetriphosphatase activity of myosin by tropomyosin from vascular and gizzard smooth muscles

Tropomyosins from bovine aorta and pulmonary artery exhibit identical electrophoretic patterns in sodium dodecyl sulfate but differ from tropomyosins of either chicken gizzard or rabbit skeletal muscle. Each of the four tropomyosins binds readily to skeletal muscle F-actin as indicated by their sedimentation with actin and by their ability to maximally stimulate or inhibit actin-activated ATPase activity at a molar ratio of one tropomyosin per seven actin monomers. Smooth and skeletal muscle tropomyosins differ in their effects on activity of skeletal myosin or heavy meromyosin (HMM); the former can enhance activity under conditions in which the latter inhibits. Gizzard and arterial tropomyosins are usually equally effective in stimulating ATPase activity of skeletal acto-HMM, but at high concentrations of Mg2+ gizzard tropomyosin is more effective, a result that cannot be attributed to differences in the binding of the two tropomyosins to F-actin. The effects of tropomyosin also depend on the type of myosin; tropomyosin enhances activity of gizzard myosin under conditions in which it inhibits that of skeletal myosin. Increasing the pH or the Mg2+ concentration can reverse the effect of tropomyosin on actin-stimulated ATPase activity of skeletal HMM from activation to inhibition, but this reversal is not found with gizzard myosin. Activity in the absence of tropomyosin is independent of pH, and the loss of activation with increasing pH is not accompanied by loss of binding of tropomyosin to actin.