Chemical cross-linking of myosin. Disposition of the globular heads.
The interaction of a series of bifunctional reagents with skeletal muscle myosin has been studied. In the di-imido ester series dimethylmalonimidate failed to generate any cross-linked species, whereas the adipic and higher analogues gave dimers of myosin heavy chains. Analysis of free amino groups after reaction with these reagents and with the reducible species dimethyldithiobis(propionimidate) showed that no more than two to three cross-links per molecule were introduced. By contrast, the bifunctional reducible acylating agent, dithiobis(succinimidylpropionate), reacted with annihilation of about 10% of the amino groups under mild conditions that precluded the formation of intermolecularly linked species. Digestion of the intramolecularly cross-linked myosin with papain, followed by analysis of the fragments by gel electrophoresis, revealed extensive cross-linking between the globular heads of the myosin molecules. The subfragment 1 dimers regenerated subfragment 1 on reduction, as shown by the electrophoretic mobility and amino acid analysis. The extent of cross-linking, and therefore presumably the average relative orientation or freedom of the two heads, was unaffected by the addition of ADP and calcium ions. The internally cross-linked myosin retains practically its full calcium-activated adenosine triphosphatase activity, but in contrast to native myosin is soluble even at very low ionic strength. Circular dichroism measurements show that the alpha helical conformation is undisturbed in cross-linked myosin, but the sedimentation coefficient is considerably higher than that of the native protein, possibly due to freezing of the heads in a "closed" configuration. The light chaiins are not cross-linked to the heavy chains, except under extreme conditions that leads to intermolecular cross-linking and inactivation. The presence of calcium ions protects dithiobisnitrobenzoate light chains against degradation by papain.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001665:Binding Sites; D055598:Chemical Phenomena; D002621:Chemistry; D004120:Dimethyl Suberimidate; D004220:Disulfides; D004591:Electrophoresis, Polyacrylamide Gel; D046911:Macromolecular Substances; D009132:Muscles; D009218:Myosins; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D013388:Succinimides
D000596:Amino Acids; D004220:Disulfides; D046911:Macromolecular Substances; D013388:Succinimides; D004120:Dimethyl Suberimidate; D009218:Myosins