Removal Of Regulatory Light Chains Research Articles

A folded (10 S) conformer of myosin from a striated muscle and its implications for regulation of ATPase activity

Myosin from the striated adductor muscle of the scallop Pecten maximus is shown to fold into a compact 10 S conformer under relaxing conditions, as has been characterized for smooth and non-muscle myosins. The folding transition is accompanied by the trapping of nucleotide at the active site to give a species with a half-life of about an hour at 20 °C. Ca 2+ binding to the specific, regulatory sites on a myosin head promotes unfolding to the extended 6 S conformer and activates product release by 60-fold. The unfolding transition, however, remains much slower than the contraction-relaxation cycle of scallop striated muscle and could not play a role in the regulation of these events. The dissociation of products from myosin heads in native thick filaments is Ca 2+-regulated, but under relaxing conditions the nucleotide is released at least an order of magnitude faster than from the 10 S monomeric myosin, at a rate similar to that observed with heavy meromyosin. Thus, there is no evidence for any intermolecular interaction between neighbouring molecules in the filament analogous to the head-neck intramolecular interaction in the 10 S conformer. It is possible that the 10 S myosin state represents an inert form involved in the control of filament assembly during muscle growth and development. Removal of regulatory light chains or labelling the reactive heavy chain thiol of myosin prevents, or at least disfavours, formation of the folded 10 S conformer and allows separation of the modified protein from the native molecules.

Electron microscopy of negatively stained scallop myosin molecules: Effect of regulatory light chain removal on head structure

The heads of myosin molecules from the striated adductor muscle of scallop have been studied by electron microscopy after negative staining. In common with vertebrate skeletal muscle myosin visualized by this method, the scallop myosin heads were pear-shaped and often showed pronounced curvature. Staining suggestive of two or, more frequently, three domains could often be observed. Removal of regulatory light chains (R-LCs) resulted in a reduction in the length of the heads of about 2.6 nm, with no significant change in maximum width. In desensitized preparations a majority of heads displayed anticlockwise curvature, whereas intact heads were usually seen curved clockwise. Analysis of the head curvature in both intact and desensitized molecules was consistent with an ability of each head to rotate about its long axis. Desensitization resulted in an increased incidence of heads showing two domains. It seems likely that the reduction in length upon removal of the R-LC is due to the two small domains located in the neck region of the head collapsing into one.

Biochemical properties of akazara scallop myosin hybridized with the foreign regulatory light chains.

The roles of regulatory light chains in the biochemical function of molluscan myosin were studied by hybridizing “desensitized” akazara scallop myosin with the regulatory light chains of rabbit skeletal, chicken gizzard, rainbow trout dorsal, and crayfish abdominal myosins. By measuring the Mg-ATPase activities of myosin and actomyosin, and the superprecipitation activity of actomyosin, the following results were obtained:1) All the foreign regulatory light chains we tested, led to the inhibition of Mg-ATPase activities of both desensitized myosin and acto-desensitized myosin in the absence of Ca2+. Whereas, whether the inhibition of Mg-ATPase activities was neutralized by Ca2+ or not, i.e. Ca2+-sensitivity of the Mg-ATPase activities recovered or not, was dependent on the species of the regulatory light chain. In this respect, these regulatory light chains could be classified into two groups; One includes the regulatory light chains of akazara scallop, chicken gizzard, and crayfish abdominal myosins, which afford the Ca2+-sensitivity to desensitized myosin, and the other includes those of rabbit skeletal and rainbow trout dorsal myosins, which afford no Ca2+-sensitivity.2) The superprecipitation activity of skeletal acto-akazara scallop myosin was remarkably decreased by the removal of regulatory light chains. However, the decreased superprecipitation activity recovered on the binding of either akazara scallop regulatory light chains or foreign regulatory light chains. These results may indicate that foreign regulatory light chains as well as molluscan regulatory light chains can maintain the myosin conformation that is indispensable for actin-myosin interaction.

アカザラガイ横紋筋および平滑筋筋原線維の脱感作と再感作

By using the myofibrils prepared from akazara scallop striated and smooth adductor muscles, dissociation of myosin regulatory light chains and the relation of the regulatory light chain con-tent to Mg-ATPase activity were investigated. The regulatory light chain dissociation was found to occur more readily from the striated myofibrils than from the smooth ones. Moreover, it was confirmed that the complete removal of regulatory light chains was required for the complete loss of Ca-sensitivity of Mg-ATPase; and that the Mg-ATPase activity both in the presence and absence of Ca2+ changed in proportion to the regulatory light chain content. These results are very similar to those obtained with akazara myosin.

Rapid removal of regulatory light chains from scallop heavy meromyosin

Rapid removal of regulatory light chains from scallop heavy meromyosin

Movement of scallop myosin on Nitella actin filaments: regulation by calcium.

In order to determine if Ca2+ regulates scallop myosin movement on actin, we have measured motility of scallop myosin along actin filaments using a direct visual assay. This procedure consists of covalently linking myosin to 1-micron beads and pipetting them onto a parallel array of actin filaments located on the cytoplasmic face of a Nitella internodal cell. In the absence of Ca2+, scallop myosin-coated beads exhibit no directed motion; however, in the presence of pCa2+ of greater than 5.84, these beads undergo linear translocations with average velocities of 2.0 micron/s. This Ca2+ -sensitive motility requires the presence of regulatory light chains on the scallop myosin. Removal of regulatory light chains with 10 mM EDTA produces a "desensitized" myosin, no longer sensitive to Ca2+, which moves at rates of 0.09-0.3 micron in the presence or absence of Ca2+. Readdition of regulatory light chains to preparations of desensitized myosin once again confers Ca2+-sensitive motility. The Ca2+ dependence of scallop-myosin motility shows a sharp transition, consistent with the Ca2+ activation sensitivity of the actin-activated ATPase. Furthermore, relative rates of movement of calcium-regulated myosins from various molluscan species are consistent with their respective rates of ATP hydrolysis. Thus, myosin motility along actin filaments provides a sensitive and direct assay of myosin activity and is suitable for studying myosin regulation.

Heat (30 degrees C)-desensitization of Akazara striated adductor myosin, and its resensitization.

In a previous report (J. Biochem. 89, 1333-1335, 1981) we showed that 30 degrees C-treatment (pCa less than 6 and 2 mM MgCl2), like EDTA-treatment, caused a reversible removal of regulatory light-chains from Akazara adductor myosin. Utilizing the heat-treatment, we now show (a) that not half but the total removal of regulatory light-chains from Akazara myosin is required for a complete loss of calcium sensitivity of myosin-ATPase, and (b) that recombination of not 1 but 2 mol of regulatory light-chains is required for a full recovery of calcium sensitivity of both myosin-ATPase and actomyosin-superprecipitation. These (a, b) are what we showed previously with EDTA-treatment (J. Biochem. 85, 1543-1546, 1979 and 86, 663-673, 1979), thus establishing that in all respects we tested, the heat-treatment is as good as EDTA-treatment for reversible removal of regulatory light-chains. We also show that the presence of actin during heat-treatment of myosin prevented regulatory light-chains from being released (at pCa 7) and that how well the release was prevented depended on the MgCl2 concentration during the heat-treatment.

Reversible loss of calcium control of tension in scallop striated muscle associated with the removal of regulatory light chains.

THE control by calcium of the ATPase activities of myosin, actomyosin and myofibrils from molluscan muscles requires the presence of a particular ‘regulatory’ light chain of myosin1,2. In these muscles troponin is not found in stoichiometric amounts and does not function as a major regulatory component2,3. In scallop muscles one of the two regulatory light chains per myosin is readily and reversibly removed by reducing the divalent cation concentration with EDTA2,4, with the result that the preparations become ‘desensitised’ to calcium, that is, the ATPase activity is maximal whether or not calcium is present. ‘Resensitising’ can be achieved by the recombination of regulatory light chains with myosin or myofibrils. We have extended these studies to a scallop muscle preparation (chemically ‘skinned’ fibre bundles) in which the contractile machinery is intact, and we show here that the regulatory light chains can be removed and recombined, with a concomitant loss and recovery of calcium control over tension production.