Stoichiometry of actin X S-1 cross-linked complex.

Abstract

Mornet et al. ( Mornet , D., Bertrand , R., Pantel , P., Audemard , E., Kassab , R. (1981) Nature (Lond.) 292, 301-306) have shown that myosin subfragment 1 (S-1) can be covalently linked to F-actin by the zero-length cross-linker 1-ethyl-3-[3-(dimethylamino)-propyl]carbodiimide. Their results indicated that the stoichiometry of the cross-linked complex is one S-1 to two actin monomers. However, Sutoh ( Sutoh , K. (1983) Biochemistry 22, 1579-1585) reported that S-1 is cross-linked to only one actin monomer. In both of these measurements, the stoichiometry was determined by separating the cross-linked complex from free actin on sodium dodecyl sulfate-polyacrylamide gels and then determining the concentration of actin and S-1 in the complex. In this study, a new approach was used to determine the stoichiometry of actin to S-1 in the cross-linked complex. The cross-linked actin X S-1 preparation, which was composed of [14C]iodoacetamide-modified S-1 and [3H]N-ethylmaleimide-modified actin, was passed through several cycles of actin depolymerization and centrifugation. This had no effect on the ATPase activity of the cross-linked S-1, but it preferentially removed noncross-linked actin which in turn increased the ratio of S-1 to total actin from 1:5 to 1:2 in the recycled cross-linked preparation. The stoichiometry of the cross-linked complex could then be determined by measuring the amount of free actin in the 42-kDa band on sodium dodecyl sulfate-polyacrylamide gels. The amount of free actin in the 42-kDa band was equal to the amount of cross-linked S-1. This establishes that the stoichiometry of the cross-linked complex is one S-1/one F-actin monomer, in agreement with the results of Sutoh .