Interaction of the N-terminal part of the A1 essential light chain with the myosin heavy chain.

The kinetics of actin-dependent MgATPase activity of skeletal muscle myosin subfragment 1 (S1) isoform containing the A1 essential light chain differ from those of the S1 isoform containing the A2 essential light chain. The differences are due to the presence of the extra N-terminal peptide comprising 42 amino acid residues in the A1 light chain. This peptide can interact with actin; heretofore, there have no been reports of the direct interaction between this peptide and the heavy chain of S1. Here, using the zero-length cross-linker 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and S. aureus V8 protease, we show for the first time that the N-terminal part of the A1-light chain can interact with the 22-kDa fragment of the S1 heavy chain. No such interaction has been observed for the S1(A2) isoenzyme. Localization of residues which can possibly react with the cross-linker suggests that the interaction might involve the N-terminal residues of the A1 light chain and the converter region of the heavy chain.

Nucleotide-induced movements in the myosin head near the converter region.

Structural changes in subfragment 1 of skeletal muscle myosin were investigated by cross-linking trypsin-cleaved S1 with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. In the absence of nucleotide the alkali light chains are cross-linked to the 27 kDa heavy chain fragment; the presence of MgATP reduces the efficiency of this reaction. On the other hand, MgATP promotes the cross-link formation between the N-terminal 27 kDa and C-terminal 20 kDa fragments of the heavy chain. The chemical cleavage of the cross-linked heavy chains fragments with N-chlorosuccinimide and hydroxylamine indicates that the cross-links are formed between the regions spanning residues 131-204 and 699-809. These results indicate that the two regions of the heavy chain that are relatively distant in nucleotide-free skeletal S1 [Rayment et al. (1993) Science 261, 50-58] can potentially interact upon addition of nucleotide.

Influence of actin binding on the conformation of the central segment of the heavy chain of skeletal myosin subfragment 1.

Chymotryptic subfragment 1 (S1) of fast skeletal muscle myosin was digested with trypsin in a low ionic strength buffer in the presence of actin. Under these conditions, leading to S1-induced polymerization of actin (Cooke, R. and Morales, M.F. (1971) J. Mol. Biol. 60, 249-261), the S1 heavy chain was cleaved between Lys-561 and Ser-562, generating the C-terminal fragment with apparent mass of 31 kDa. In the absence of actin, this peptide bond was inaccessible to trypsin. The yield of the 31 kDa fragment decreased with the increase in the ionic strength of the medium. The cleavage was also partially inhibited by magnesium or calcium chloride at millimolar concentrations. The data suggest that in low salt conditions and at low concentrations of divalent cations, actin induces a conformational change in the C-terminal portion of the 50 kDa central segment of the S1 heavy chain.

Mapping of the region of the heavy chain of myosin subfragment 1 that can be crosslinked to the alkali light chains.

Earlier studies have shown that the presence of MgATP considerably diminishes the efficiency of crosslinking of the alkali light chains to the heavy chain of myosin subfragment 1 (S1) with dithiobis (succinimidylpropionate) (DSP). To localize the region of the nucleotide-induced change, a non-cleavable analog of DSP, disuccinimidyl suberate (DSS) was used. The products of crosslinking between the alkali light chains and the 27 kDa tryptic fragment of the heavy chain were isolated and further cleaved with N-chlorosuccinimide (NCS) or with formic acid. Mapping of the resulting peptides on SDS gels locates the crosslinking site(s) in the heavy chain region spanning residues 42-112.

Influence of nucleotide on chemical crosslinking between alkali light chains and the heavy chain of myosin subfragment 1.

When chymotryptic myosin subfragment 1 (S1) of fast skeletal muscle myosin is treated with dithiobis(succinimidylpropionate) (DSP), the alkali light chains A1 and A2 become intramolecularly crosslinked to the N-terminal 27 kDa fragment of the S1 heavy chain (Labbé et al. (1981) Biochem. Biophys. Res. Commun. 102, 466-475). The results presented here show that in the presence of MgATP the efficiency of the crosslinking is markedly reduced. The results may indicate a nucleotide-induced structural rearrangement within the myosin head. It was also observed that crosslinking depressed the nucleotide-promoted tryptic conversion of the 27 kDa fragment to its 22 kDa derivative, suggesting that the crosslinks are in the vicinity of the additional tryptic cleavage site in the 27 kDa fragment or that the crosslinking prevents nucleotide-induced conformational changes in this region of the S1 heavy chain.

Dependence of the length of the heavy chain of chymotryptic subfragment 1 on the temperature of myosin digestion.

Limited digestion of filamentous myosin with chymotrypsin at 0 degrees C in the absence of divalent cations generates two forms of subfragment 1 (S1), with heavy chains of 95 kDa and 98 kDa. The difference is at the C-terminal end of the chain. The 98 kDa form prevails, in contrast to the preparations obtained by digestion at room temperature which consist of the shorter species and only traces of the longer one. The results support the idea of a temperature-dependent conformational transition at the head-rod junctional region of the myosin heavy chain.

Crosslinking of trypsin digested acto-heavy meromyosin as a probe of the affinity of the two myosin heads to actin.

The interaction of the two heads of the myosin molecule with actin was studied by tryptic digestion of HMM in the presence of actin, followed by crosslinking the two nicked heavy chains with Nbs2 at the S2 region. In view of the protection by actin of the 50/60 kDa junction against proteolysis, the percentage of the heads interacting with actin was estimated from the proportion of the 110 kDa to the 60 kDa digestion product. Under conditions such that about 50% of HMM heads were protected by actin (at an actin to HMM head molar ratio of 1:1 in the absence of nucleotide, or 3:1 in the presence of 5 mM ADP), the crosslinking of the digestion products yielded a 230 kDa (110 + 110 kDa), 125 kDa (60 + 60 kDa) and 175 kDa (60 + 110 kDa) species. Since the latter should be the only crosslinking product when only one head of HMM molecule is protected by actin, it is concluded that there is no preferential binding of one of the two HMM heads to actin in the presence of ADP or at equimolar actin to myosin heads ratio.

Reactivities of thiols in myosin rod: effect of magnesium and ionic strength.

There are six cysteines in each chain of myosin rod of rabbit skeletal muscle: three are in the S-2 portion, at residues 66, 108 and 410 (Lu, R.C. and Lehrer, S. (1984) Biochemistry 23, 5975-5981). The other three are in the light meromyosin portion, assigned at residues 572, 600 and 770 on the basis of homology between the amino acid sequence in the vicinity of these thiols and that of the rod of nematode myosin (McLachlan, A.D. and Karn, J. (1982) Nature 299, 226-231). Since the thiols are distributed in different regions of the rod, measuring their reactivities under various conditions may provide information on the conformations of these regions. Myosin rod was carboxymethylated with radioactive iodoacetic acid under various conditions. The cysteine-containing peptides were isolated using HPLC from the tryptic digests, and the radioactivity incorporated into each thiol was measured. In the denatured state all six thiols were equally reactive. In the native state, all thiols have low reactivity, the reactivity of Cys-108 or -410 is only 0.1% of that in the denatured state, Cys-600 exhibited the highest reactivity, about 20-times that of Cys-410; Cys-66, -572 and -770 had 2-4-times that of Cys-410. When the rods formed filaments, the reactivities of all cysteines further decreased: Cys-66, -108 and -770 were reduced to 50%, while Cys-410, -572 and -600, located in the middle of the rod, were reduced to 20-30% of their reactivities in the monomeric form. In the presence of Mg2+ the reactivity of Cys-108 increased by 20%, whereas Cys-572 decreased by 50%. The results are consistent with the view that metal ions affect the conformation of the rod. This may play a role in the mechanism of filament formation and the movement of crossbridges.

Unusual features of the Ca2+-ATPase activity of myosin from fast skeletal muscle of the frog: effect of actin and SH1 thiol group modification.

The K+-ATPase and actin-activated Mg2+-ATPase activity of myosin from fast skeletal muscle of the frog, Rana esculenta or Rana temporaria, are comparable to the respective activities of rabbit fast skeletal muscle. On the other hand, the Ca2+-ATPase activity of the same preparations of frog myosin is 6-7-fold lower than that of myosin from rabbit muscle. Various control experiments indicate that the small extent of Ca2+ stimulation is an intrinsic property of frog muscle myosin. Unlike myosin from rabbit muscle, the Ca2+-ATPase activity of frog myosin is strongly activated by actin; at high actin concentrations it approaches the level of the Ca2+-ATPase activity of rabbit myosin. The levels of Ca2+-ATPase activity of frog and rabbit myosins also become comparable upon modification of myosin SH1 thiol groups; this means that the modification of the SH1 groups results in a much higher activation of the Ca2+-ATPase of frog myosin than that of rabbit myosin. The results suggest a difference in the active site conformation in frog and rabbit muscle myosins. The effects of actin and SH1 group modification are discussed in terms of allosteric changes which diminish the difference in the active site conformation of the two myosins. We have also observed a difference in the reactivity of thiol groups which are not essential for the enzymatic activity in frog and rabbit myosin, indicating structural differences in regions other than the active site.

Comparison of myosin isoenzymes from slow-tonic and fast-twitch fibers of frog muscle.

Myosin from distal cruralis bundle of triceps femoris composed of slow-tonic and fast-twitch fibers in an about 1 to 1 ratio, from rectus abdominis containing a lower proportion of slow-tonic fibers, and from a fast-twitch sartorius muscle of the frog, was characterized by means of polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate (SDS), electrophoresis in non-dissociating conditions, and determination of the ATPase activity. SDS-polyacrylamide gel electrophoresis failed to reveal the presence of any specific light chain in myosin from slow-tonic fibers. Light chains having the same mobilities as fast-twitch LC1 and LC2 were observed; as previously described by Focant and Reznik [15], myosin from tonic fibers appears, on the other hand, devoid of LC3. By electrophoresis in non-dissociating conditions myosin from sartorius muscle was resolved into three components which comigrated with the three myosin isoenzymes from the fast-twitch posterior latissimus dorsi muscle of the chicken. Preparations from rectus abdominis and from cruralis bundle were shown to contain an additional component of lower electrophoretic mobility. Its relative proportion in these two muscles suggests that it represents the slow-tonic fiber myosin isoenzyme. Analysis of proteolytic digestion patterns revealed differences in the heavy chain structure of the isoenzymes from slow and fast fibers, respectively. As indicated by ATPase measurements, fast-twitch myosin exhibits a higher catalytic activity than myosin from slow-tonic fibers, the difference being of the same order as that reported for myosins from slow and fast muscles of higher vertebrates.

Changes in the ultrastructure of actomyosin gel during hydrolysis of ATP under various ionic conditions.

The interaction of myosin and actin in the presence of MgATP under various ionic conditions was investigated by a simultaneous determination of changes in turbidity, liberation of inorganic phosphate, distribution of the two proteins between the supernatant and precipitate obtained after a short-time centrifugation, and by electron microscopy. The results confirm the view that the extent of association between myosin and actin filaments is low not only in the clear phase but also under the conditions when the addition of MgATP results in superprecipitation without a detectable clear phase. Extensive formation of aggregates of parallelly aligned myosin and actin filaments coincides with the depletion of ATP, indicating that these aggregates represent rigor complexes. Relation between ATP-induced turbidity changes and structural changes in the actomyosin system under various ionic conditions is discussed.