Regulatory and essential light-chain interactions in scallop myosin: II. Photochemical cross-linking of regulatory and essential light-chains by heterobifunctional reagents

Abstract

From four to six lysine residues of isolated scallop regulatory light-chains were conjugated with N-hydroxysuccinimidyl-4-azidobenzoate; the single thiol group of isolated Mercenaria R-LC ‡ ‡ Abbreviations used: R-LC, regulatory light-chain; SH-LC, essential light-chain; HSAB, N-hydroxysuccinimidyl-4-azidobenzoate; p-APA, p-azidophenacylbromide ; Ig, immunoglobulin; EGTA, thyleneglycol-bis-(β-aminoethylether)- N, N′-tetraacetic acid; FITC, fluorescein isothiocyanate. , and the two thiol groups of isolated rabbit R-LCs were substituted with p-azidophenacylbromide. R-LCs modified by these heterobifunctional photosensitive cross-linkers combined readily with scallop myofibrils from which R-LCs had been removed by treatment with EDTA at 35 °C. The modified scallop and Mercenaria R-LCs restored the calcium sensitivity of the actin-activated Mg-ATPase. The complexes formed upon illumination between modified R-LCs and essential light-chains were visualized and quantitated by indirect antibody staining of electrophoretic nitrocellulose blots of sodium dodecyl sulfate/polyacrylamide gels using specific anti-scallop R-LC and anti-scallop SH-LC antibodies followed by fluorescein isothiocyanate-labeled or 125I-labeled goat anti-rabbit immunoglobulin G, respectively. The presence of SH-LC in bands of higher molecular weight complexes ( M r > 17,000) formed upon photolysis was taken as evidence for cross-linking between the substituted R-LCs and scallop SH-LCs. About one-third of the scallop SH-LC cross-linked with p-APA-substituted Mercenaria R-LC, when illumination generated a single new band at M r 35,000 consisting of one R-LC and one SH-LC. Higher molecular weight complexes were not formed. With the multi-substituted scallop and rabbit R-LCs, more than half of the scallop SH-LC was cross-linked; about one-third formed a one to one complex with the modified R-LC, and a significant amount was cross-linked to the myosin heavy-chain via the R-LC. Complexes of molecular weights of approximately 53,000 and 66,000 were also formed in small amounts with highly modified scallop and rabbit R-LCs, probably consisting of three and four cross-linked light-chains. Some cross-linking of the two R-LCs of the scallop myosin molecule was demonstrated with myofibrils treated with EDTA at 12 °C and containing one mole of intact scallop R-LC and one mole of p-APA-modified Mercenaria or rabbit R-LC. R-LCs did not significantly cross-link with actin or tropomyosin. Photolysis did not inactivate the actin-activated Mg-ATPase activity, but reduced calcium sensitivity by activation in the absence of calcium. These results show that R-LCs and SH-LCs of scallop myosin are in close proximity (no more than 6 to 8 Å apart) and overlap for at least half of their lengths. Cross-linking of R-LCs and SH-LCs interferes with regulation, consistent with a regulatory role for the SH-LCs.