Microcalorimetric measurement of the enthalpy of binding of rabbit skeletal myosin subfragment 1 and heavy meromyosin to F-actin

The heat of binding of rabbit skeletal myosin subfragment 1 (myosin-S1) and heavy meromyosin (HMM) to F-actin has been measured by batch calorimetry. Proton release measurements in unbuffered solutions indicate that less than 0.1 mol of protons is absorbed or released per mol of myosin head bound to actin. Hence, the measured heats are approximately equal to the enthalpy of myosin-S1 and HMM binding to actin. The enthalpy of binding of myosin-S1 to actin was +22 +/- 3 and +27 +/- 5 kJ/mol of myosin-S1 in two series of experiments at 12 degrees C and +26 +/- 5 kJ/mol of myosin-S1 at 0 degrees C, indicating that delta Cp for this reaction in the range of 0-12 degrees C is small (-80 J/mol/K). The enthalpy of binding of HMM to actin at 12 degrees C was found to be +26 +/- 1 kJ/mol of myosin head. The enthalpies determined here and the equilibrium constants obtained from the literature for measurements at 20 degrees C under identical solvent conditions were used to estimate the entropy of the association of myosin S1 and HMM with F-actin: +235 J/mol/K for myosin-S1 and +190 J/mol of myosin head/K for HMM. Thermodynamic parameters of the interaction of myosin-S1 with actin and ADP or AMP-PNP can be evaluated using the enthalpy of association of myosin-S1 with actin determined here, together with literature values for the equilibrium constants and enthalpies of binding of these nucleotides to myosin-S1. The calculated enthalpies of binding of ADP or AMP-PNP to actomyosin-S1 are small and negative.     

Phosphorylation of light chains of myosin from rabbit skeletal muscles affects the type of conformation changes of F-actin induced by heavy meromyosin

The changes in F-actin conformation in myosin-free single ghost fiber induced by the binding of heavy meromyosin (HMM) with dephosphorylated or phosphorylated light chains-2 (LC2) have been studied by measuring intrinsic tryptophan polarized fluorescence of F-actin. It has been found that at low concentrations of Ca2+ (pCa greater than or equal to 8), the binding of HMM with dephosphorylated LC2 to F-actin in ghost fibres increases, whereas the binding of HMM with phosphorylated LC2 decreases the anisotropy of polarized tryptophan fluorescence. The effect is reversed at high concentrations of Ca2+ (pCa = 5). It has been assumed that this effect of myosin light chains phosphorylation may be due to its influence on the type of myosin head binding to F-actin.     

Binding of phosphorylated and dephosphorylated heavy meromyosin to F-actin

The effect of myosin light chain phosphorylation in skeletal muscle was investigated with respect to the binding affinity of phosphorylated and dephosphorylated heavy meromyosin (HMM) for F-actin in the absence of ATP. For phosphorylated HMM the affinity was 2.5-times weaker in the presence of Ca²⁺ as in its absence (HMM divalent binding sites saturated only with Mg). For dephosphorylated HMM the reverse was true, the binding being 2.4-times higher in the presence of Ca²⁺.     

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.     

Calcium modulates conformational changes in F-actin induced by smooth muscle heavy meromyosin

The effect of Ca²⁺ on conformational changes in rhodamine-phalloidin-labeled F-actin induced by binding of smooth muscle heavy meromyosin (HMM) with either phosphorylated or dephosphorylated regulatory light chains (LC₂₀) was studied by polarized fluorimetry. LC₂₀ phosphorylation caused alterations in the F-actin structure typical of the force-producing (strong-binding) state, while dephosphorylation of the chains led to alterations typical of the formation of non-force-producing (weak-binding) state of the actomyosin complex. The presence of Ca²⁺ enhanced the effect of LC₂₀ phosphorylation and weakened the effect of LC₂₀ dephosphorylation. These data suggest that Ca²⁺ modulates actin-myosin interaction in smooth muscle by promoting formation of the strong-binding state.     

Effect of crosslinking by glutaraldehyde on interaction of F-actin with heavy meromyosin.

Crosslinking of F-actin by a bifunctional reagent glutaraldehyde resulted in a marked decrease of viscosity and length of F-actin filaments. The extent and rate of superprecipitation of actomyosin reconstituted from the modified actin were lower than those of unmodified actin-myosin complex, but activation of heavy meromyosin ATPase by the crosslinked actin was higher than by unmodified one. Heavy meromyosin ATPase activated by the crosslinked actin was distinctly less dependent on KCl concentration than that activated by unmodified actin. Turbidity of the modified acto-heavy meromyosin in the presence of ATP exceeded the sum of turbidities of actin and heavy meromyosin, whereas in the case of unmodified acto-heavy meromyosin the turbidity was comparable to that for noninteracting system. The difference in activation of heavy meromyosin. ATPase by the cross-linked and unmodified actin, clearly seen at room temperature, significantly diminished when temperature was lowered to 0 degrees C.     

Difference between subfragment-1 and heavy meromyosin in their interaction with F-actin

To elucidate the difference between subfragment-1 and heavy meromyosin in their interaction with F-actin, we used limited tryptic digestion and cross-linking with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. The binding of actin to subfragment-1 lowers the susceptibility of the 50K-20K junction of its heavy chain to tryptic digestion. At a molar ratio of one actin to one subfragment-1, all the sites were gradually cleaved by trypsin whereas the sites were completely protected in the presence of a 2-fold molar excess of actin over subfragment-1. In the case of heavy meromyosin, nearly half of the sites were protected completely by the presence of an equimolar amount of actin to its heads suggesting that the two heads of heavy meromyosin bound actin in a different manner. The rate of the cross-linking reaction between subfragment-1 heavy chain and actin with 1-ethyl-3-[3-(dimethylamino) propyl]carbodiimide also depended on the molar ratio of actin to subfragment-1. The rate was maximum at a molar ratio of about 5 actin to 1 subfragment-1. When heavy meromyosin was cross-linked to actin, the maximum rate was observed at a molar ratio of about 3 actin to 1 heavy meromyosin head, the level being about 60% that for subfragment-1 and actin. It was suggested that the presence of the subfragment-2 portion of heavy meromyosin caused these differences by restricting the motion of the two heads.     

Cooperativity between the two heads of rabbit skeletal muscle heavy meromyosin in binding to actin.

An extensive series of experiments in this laboratory has shown that the binding of actin to rabbit skeletal muscle myosin subfragment-1 (a single-headed subfragment) can be described by a two-step model, with formation of a weakly bound complex, the A-state, followed by an isomerization to a more tightly bound complex, the R-state. In this paper, we report on additional experiments comparing the subfragment-1 with heavy meromyosin (a two-headed subfragment). Using a modeling approach, we have quantitated the two-step binding for each of the two heads. This indicates that the binding is cooperative and leads to a more complex view of the acto-myosin interaction than has previously been acknowledged. Implications for the dynamic behavior of the two heads during muscle contraction are discussed.     

Two pathways for oxygen exchange by heavy meromyosin and their dependence on actin

In the hydrolysis of MgATP by acto heavy meromyosin (HMM) there are two enzymatic pathways that differ in the properties of their intermediate oxygen exchange; one of these is designated the low exchange pathway (P1); the other is designated the high exchange pathway (P2). A plot of the P1 flux versus the actin concentration gives a sigmoid curve, whereas the corresponding curve for the P2 flux rises in an approximately hyperbolic manner. At low concentrations of actin, where the sigmoid curve of the P1 flux is in a lag phase, the major flux is along P2; but at higher concentrations of actin, as the P1 curve rises sharply, the flux along P1 comes to predominate. Even at the highest levels of actin, at saturating levels for both pathways, the kinetics of exchange along P1 and P2 are significantly different. In addition to these differences in the actin dependence, the flux of P1 relative to P2 is markedly inhibited by KCl. Therefore, which of the two pathways dominates during the hydrolysis of MgATP by HMM is strongly dependent on experimental conditions. The findings suggest that P1 involves the interaction of HMM with two actin units whereas P2 involves the interaction of HMM with one actin unit. The results are discussed in relation to a kinetic scheme based on this proposal.     

Inhibition of palmitoyl CoA of EDTA- and Mg2+-ATPase of heavy meromyosin from rabbit skeletal muscle

Palmitoyl CoA inhibited EDTA-ATPase of heavy meromyosin (HMM) prepared from rabbit skeletal muscle. The concentration for half maximum inhibition of EDTA-ATPase was about 18 microM. Myristoyl CoA, the other long chain fatty acyl CoA, also inhibited EDTA-HMM ATPase, but CoA and short chain CoA thioesters, such as butyryl CoA, acetoacetyl CoA and acetyl CoA, at 40 microM hardly inhibited EDTA-ATPase. Less than 20% inhibition of EDTA-HMM ATPase was obtained with Na-palmitate and Na-myristate at 40 microM, whereas about 90% inhibition of the enzyme occurred in the presence of 40 microM palmitoyl CoA and myristoyl CoA. Palmitoyl carnitine, as well as carnitine, failed to inhibit EDTA-HMM-ATPase. The inhibition of palmitoyl CoA of EDTA-ATPase was reversed by bovine serum albumin and spermine. Mg2+-HMM ATPase activity was enhanced by palmitoyl CoA at 2, 5, and 10 microM. About a 25% increase in Mg2+-HMM ATPase activity was obtained at 5 and 10 microM. At higher concentrations than 20 microM, the enzyme was inhibited by palmitoyl CoA and the degree of inhibition was related to the concentration of the CoA thioester. At 80 microM, the activity was about 15% of the maximum value. The efficacy of myristoyl CoA on Mg2+-ATPase was almost the same as that of palmitoyl CoA. Mg2+-ATPase activity was not enhanced by CoA, butyryl CoA, acetoacetyl CoA, Na-myristate, Na-palmitate, palmitoyl carnitine, or carnitine at 10 microM, and was hardly reduced by these substances at 40 microM. Serum albumin and spermine also canceled, to some extent, these effects of palmitoyl CoA on Mg2+-ATPase.     

Carbodiimide-catalyzed cross-linking sites in the heads of gizzard heavy meromyosin attached to F-actin

In the rigor complex between rabbit skeletal muscle F-actin and chicken gizzard heavy meromyosin (HMM), the direct contact between two HMM heads was demonstrated by using a zero-length cross-linker 1-ethyl-3-[3-(dimethylamino)propyl]maleimide (EDC) [Onishi, H., Maita, T., Matsuda, G., & Fujiwara, K. (1989) Biochemistry (preceding paper in this issue)]. Here, the 60K peptide which was a product of the EDC cross-linking between two 24K heavy chain (tryptic) fragments of HMM was further fragmented with cyanogen bromide, and the location of the cross-linking sites on the amino acid sequence of the HMM heavy chain was investigated. The result showed that one site resided within the 77-residue peptide region (residues 1-77) on one head of HMM, whereas the other site belonged to the 40-residue peptide region (residues 164-203) on the other head. This finding suggests that the two HMM heads are in contact with each other at different sites. Ultracentrifugal fractionation revealed that the head-to-head cross-linked gizzard HMM could be reversibly released from F-actin in the presence of Mg-ATP. The yield of the head-to-head cross-linking was not significantly changed with the acto-HMM complex between actin/HMM head molar ratios of 1 and 4, and it was very slightly decreased even at a molar ratio of 8, where HMM molecules were attached sparsely to actin filaments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of tropomyosin, troponin and Ca on the interaction between F-actin and heavy meromyosin

1. In the absence of ATP, H-meromyosin (heavy meromyosin) bound with the F-actin-tropomyosin-troponin complex up to the molar ratio of H-meromyosin to actin of 1:1, independently of the concentration of Ca²⁺.

2. In the presence of free Ca²⁺ above about 1 μM, with an increasing amount of H-meromyosin bound to a fixed amount of the F-actin-tropomyosin-troponin complex, the degree of flow birefringence decreased and the extinction angle increased. The minimum value of the birefringence and the maximum value of the extinction angle were found at the molar ratio of H-meromyosin to actin of 1:2. A further increase of bound H-meromyosin to actin restored both the degree of birefringence and the extinction angle to nearly the same level as the F-actin-tropomyosin-troponin complex only. In the absence of free Ca²⁺, the birefringence did not change with the binding of H-meromyosin.

3. This sensitivity of birefringence to the concentration of Ca²⁺ appeared only in the presence of tropomyosin and troponin. At a fixed ratio of H-meromyosin, actin and tropomyosin, the birefringence in the absence of Ca²⁺ increased with increasing amount of added troponin up to the weight ratio of troponin to actin of 1:6; whereas the birefringence in the presence of Ca²⁺ did not change.

4. At a fixed ratio of H-meromyosin to actin, the birefringence changed with increasing amount of tropomyosin added up to the weight ratio of tropomyosin to actin of 1:6; above this ratio, the birefringence was constant.

5. Subfragment S-1, prepared by the chymotryptic digestion of myosin, bound to F-actin, but the birefringence did not change even in the presence of tropomyosin and troponin.     

Hybrid with rabbit skeletal DTNB-light chains of heavy meromyosin, myosin, and glycerinated fibers from Akazara scallop adductor

It was shown in our previous report (Ojima et al. (1983) J. Biochem. 94, 307-310) that hybridization of Akazara scallop "desensitized" myosin with rabbit skeletal DTNB-light chains led to inhibition of the Mg-ATPase activity of acto-desensitized myosin but to enhancement of its superprecipitation activity. The following are now found: Development of tension in desensitized glycerinated fibers of Akazara adductor is significantly improved when DTNB-light chains are added to the fiber bath. The actin-affinity of desensitized heavy meromyosin in the presence of ATP but in the absence of Ca2+ is decreased by hybridization with chicken gizzard 20K dalton-light chains but significantly increased by that with DTNB-light chains. It is therefore suggested that the increase in actin-binding may account for the enhancing effect of DTNB-light chains on the superprecipitation and on the tension development.     

Polarized fluorescence from epsilon-ADP incorporated into F-actin in a myosin-free single fiber: conformation of F-actin and changes induced in it by heavy meromyosin.

Fluorescent ADP analog, ε-ADP (1:N⁶-ethenoadenosine 5′-diphosphate), was incorporated into F-actin in a myosin-free ghost single fiber and polarized fluorescence measurements were performed under a microspectrophotometer to investigate the conformation of F-actin and the changes induced in it by heavy meromyosin and subfragment-1. Four components of polarized fluorescence were obtained by exciting the fiber with light polarized parallel and perpendicular to the long axis of the fiber and measuring the intensity of emission polarized parallel and perpendicular. From these data it was shown that F-actin in the fiber was not rigid but flexible, with a value for the elastic modulus for bending of 5.3 × 10^?17 dyn cm². The angles of absorption dipole and emission dipole of bound ε-ADP with the long axis of F-actin were both about 75 °.The binding of heavy meromyosin decreased the elastic modulus of F-actin by 30% and the angles of absorption and emission dipoles by 2.5 ° and 1.5 °, respectively. The molar ratios of bound heavy meromyosin and subfragment-1 to actin in the ghost fiber at saturation were 0.3 and 0.6, respectively, being smaller than those in solution.     

Characterization of cDNA coding for the complete light meromyosin portion of a rabbit fast skeletal muscle myosin heavy chain

Myosin-heavy-chain-specific cDNA clones have been isolated from a cDNA library prepared from hind leg muscle of a 14-day-old rabbit. According to restriction enzyme analysis these can be grouped into at least two, probably three different classes. RNA dot-blot hybridization shows that all of these clones correspond to mRNAs expressed in fast skeletal muscle. The clones of the most abundant form, class I, can be aligned to cover the complete light meromyosin portion of myosin heavy chain. The sequence of the coding and the 3'-untranslated region, together comprising 2143 base pairs, has been determined. The class I clone detects a multigene family of 8-12 members on a Southern blot of rabbit genomic DNA.