The two main proteins involved in muscular contraction and cell motility, myosin and actin, possess the intrinsic property of being able to form filamentous structures. This property poses a serious impediment to the study of their structures and interactions, and a considerable effort has thus been made to isolate their functional domains. The globular part of myosin, subfragment-1 (S1), which possesses ATPase and actin-binding sites as well as supporting the movement of actin filaments during in vitro assays, has been isolated. But because S1 is efficient in inducing actin polymerization, as is myosin, it has not been possible to prepare and characterize a complex of S1 with monomeric actin (G-actin). We have now used chromatographically purified proteins to show that only the S1 isoenzyme carrying the A1 light-chain subunit promotes actin polymerization. The other isoenzyme, S1 (A2), carrying the A2 light-chain subunit, binds to actin, forming a tight complex of G-actin and S1 in a 1:1 ratio. This new functional difference between myosin isoforms directly implicates the A1 light-chain in myosin-induced actin polymerization. Additionally, this finding should lead to the purification of the stable G-actin-S1 complex needed to resolve the structure and to understand the molecular dynamics of the actin-myosin system.

In previous work, we studied the early steps of the Mg(2+)-ATPase activity of Ca(2+)-activated myofibrils [Houadjeto, M., Travers, F., & Barman, T. (1992) Biochemistry 31, 1564-1569]. The myofibrils were free to contract, and the results obtained refer to the ATPase cycle of myofibrils contracting with no external load. Here we studied the ATPase of myofibrils contracting isometrically. To prevent shortening, we cross-linked them with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC). SDS-PAGE and Western blot analyses showed that the myosin rods were extensively cross-linked and that 8% of the myosin heads were cross-linked to the thin filament. The transient kinetics of the cross-linked myofibrils were studied in 0.1 M potassium acetate, pH 7.4 and 4 degrees C, by the rapid-flow quench method. The ATP binding steps were studied by the cold ATP chase and the cleavage and release of products steps by the Pi burst method. In Pi burst experiments, the sizes of the bursts were equal within experimental error to the ATPase site concentrations (as determined by the cold ATP chase methods) for both cross-linked (isometric) and un-cross-linked (isotonic) myofibrils. This shows that in both cases the rate-limiting step is after the cleavage of ATP. When cross-linked, the kcat of Ca(2+)-activated myofibrils was reduced from 1.7 to 0.8 s-1. This is consistent with the observation that fibers shortening at moderate velocity have a higher ATPase activity than isometric fibers.(ABSTRACT TRUNCATED AT 250 WORDS)