Barbara Pliszka

Subunit composition and some other properties of myosin from skeletal muscles of the frog rana esculenta

Abstract 1. 1. Two out of three light chains of myosin from mixed hind leg muscles of the frog (LC 1 and LC 2 ) undergo a proteolytic cleavage during preparation with procedures routinely used to obtain myosin from skeletal muscles of other vertebrates. 2. 2. The intact LC 1 and LC 3 comigrate on SDS-polyacrylamide gels with the respective light chains of rabbit fast skeletal muscle; LC 2 has lower mobility than the rabbit LC 2 . All the three light chains differ from their counterparts in the rabbit myosin in mobilities on urea-polyacrylamide gels. 3. 3. The LC 2 of frog myosin is released upon DTNB-EDTA † treatment more easily than LC 2 of rabbit fast skeletal muscle. 4. 4. Differences in the fragmentation of the heavy chains of myosin from hind leg and from rabbit fast skeletal muscles by trypsin indicate some differences in primary and/or secondary structure. 5. 5. Fast inactivation accompanied by aggregation of frog myosin was confirmed. Pyrophosphate was shown to have a protective effect. 6. 6. Myosin from mixed hind leg muscles of the frog is similar to myosin from rabbit slow skeletal muscle in its instability under mild alkaline conditions.

Influence of locomotor training on the structure and myosin heavy chains of the denervated rat soleus muscle

Abstract Objective: Mechanism of denervation atrophy remains poorly understood. In particular, the question about irreversibility of the late atrophy is still open. Therefore, in the present study, we investigated whether and how a passive movement can affect a progress of atrophy in rat soleus muscle. To address this issue, a locomotor training on a treadmill was applied to rats with their right hindlimb muscles denervated. Methods: The hindlimb muscles were denervated by cutting the sciatic nerve. Starting either 7 days or 1 month after the surgery, the animals were trained on a treadmill. Two months after denervation, the soleus muscle was investigated using light and electron microscopy and biochemical methods. Control soleus muscles were obtained from non-trained animals: the untreated and the 2-month denervated. Results: Locomotor training caused slight increase in denervated rat soleus muscle weight and significant increase in its fiber diameter. The training positively affected some of the factors that were believed to be the reasons of atrophy irreversibility, because of significant increase in the number of capillary blood vessels and muscle fiber nuclei with the concomitant decrease in the number of severely damaged muscle fibers and amount of collagen. Morphology of the contractile apparatus was also improved as more regular organization of sarcomeres and the hexagonal arrangement of myosin filaments was evident. Moreover, the amount of myosin heavy chains (MHC) significantly increased after training. The effects were more evident in the animals with longer training. Conclusion: Passive movement seems to attenuate some of the pathologic processes within the denervated muscle.

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 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°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.

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.

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 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.

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

SummaryThe K+-ATPase and actin-activated Mg2+-ATPase activity of myosin from fast skeletal muscle of the frog,Rana esculenta orRana 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.

Changes at the interface of the N- and C-terminal parts of the heavy chain of myosin subfragment 1.

It has been previously shown that in the M-MgADP-P(i) state, where the myosin head adopts a pre-power stroke conformation, treatment of trypsin-split subfragment 1 of skeletal muscle myosin with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) results in cross-linking of the C-terminal fragment of the heavy chain of S1 -- most probably its converter region -- to the N-terminal S1 heavy-chain fragment, generating a product of 44 kDa [Biochim. Biophys. Acta 1481 (2000) 55]. The results described here show that this product is neither generated in the absence of nucleotide nor in the presence of MgADP or MgPP(i). The 44 kDa cross-linking product can be formed when S1 treated with EDC is complexed with MgADP-AlF(4) or MgADP-V(i) (MgADP-P(i) analogs) and with MgADP-BeF(x), MgATP gamma S or MgAMPPNP (MgATP analogs). The results suggest structural differences between MgATP- or MgADP-P(i)-bound S1, and MgADP-bound or nucleotide-free S1, in spatially close regions of their N- and C-terminal heavy-chain fragments.