An immunological approach to myosin light-chain function in thick filament linked regulation. 1. Characterization, specificity, and cross-reactivity of anti-scallop myosin heavy- and light-chain antibodies by competitive, solid-phase radioimmunoassay.

Abstract

Antibodies specific for the regulatory light-chain (R-LC), "essential" light-chain (SH-LC), heavy-chain, and rod fragment of myosin from the striated adductor muscle of scallop (Aequipecten irradians) were prepared and characterized. A competitive, solid-phase radioimmunoassay on microtiter plates, a combination of two systems described earlier by Kuettner et al. [Kuettner, M. G., Wang, A. L., & Nisonoff, A. (1972) U. Exp. Med. 135, 579-595] and Klinman et al. [Klinman, N. R., Pickard, A. R., Sigal N. H., Gearhart, P. J., Metcalf, E. S., & Pierce, S. K. (1976) Ann. Immunol. (Paris) 127C, 489-502], was adapted and used for an immunological survey of different myosins and myosin light chains. Anti-myosin light-chain antibodies were specific for the homologous chain and did not cross-react with the heterologous one, i.e., regulatory and essential light chains of scallop myosin could be distinguished immunologically. These antibodies also had a high degree of species specificity. A partial cross-reactivity was obtained only for the light chains of two closely related molluscan species out of the over thirty invertebrate or vertebrates species tested. Two populations of anti-SH-LC antibodies were found which differed in their ability to abolish regulation of scallop myofibrils and also in their immunological reactivity with cyanogen bromide fragments of teh SH-LC. A comparison of the cross-reactivity of the intact SH-LC with its CNBr fragments showed that most antigenic sites of the SH-LC were available to the antibodies. Free light chains and light chains associated with myosin reacted with antibodies in a very similar manner, indicating that the association of the light chains with myosin may not be accompanied by major conformational changes. Antibodies against scallop myosin heavy chain and rod fragment cross-reacted to a variable extent with all invertebrate myosins but with none of the vertebrates species tested. The antibodies did not cross-react with platelet and Physarum myosins. The heavy and light chains of myosin from scallop striated adductor, mantle, and foot were found toi be immunologically identical, whereas myosin from smooth adductor showed some differences mainly in the heavy-chain portion which forms the subfragment-l region of the myosin molecule. Heavy and light chains of scallop heart muscle myosin differed significantly from those of striated adductor muscle. Cross-reactivity did not depend on the regulatory properties of myosin.