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

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Abstract
Antibodies specific for the regulatory L-chain (R-LC), essential L-chain (SH-LC), H-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 2 systems described earlier by Kuettner et al. and Klinman et al. (1976), was adapted and used for an immunological survey of different myosins and myosin L-chains. Anti-myosin L-chain antibodies were specific for the homologous L chain and did not cross-react with the heterologous one, i.e., R-LC and SH-LC of scallop myosin were distinguished immunologically. These antibodies also had a high degree of species specificity. A partial cross-reactivity was obtained only for the L-chains of 2 closely related molluscan species out of the > 30 invertebrate or vertebrate 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 the 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 L-chains and L-chains associated with myosin reacted with antibodies in a very similar manner. The association of the L-chains with myosin may not be accompanied by major conformational changes. Antibodies against scallop myosin H-chain and rod fragment cross-reacted to a variable extent with all invertebrate myosins, but with none of the vertebrate species tested. The antibodies did not cross-react with platelet and Physarum myosins. The H- and L-chains of myosin from scallop striated adductor, mantle and foot were immunologically identical; myosin from smooth adductor showed some differences, mainly in the H-chain portion which forms the subfragment-1 region of the myosin molecule. H- and L-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.