INTERACTION OF MALEIMIDOBENZOYL ACTIN WITH MYOSIN SUBFRAGMENT-1 AND TROPOMYOSIN TROPONIN

A chemical modification of G-actin with (m-maleimidobenzoyl)-N-hydroxysuccinimide ester (MBS) impairs actin polymerization [Bettache, N., Bertrand, R., & Kassab, R. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 6028-6032]. MBS-actin recovers the ability to polymerize when a 2-fold molar excess of phalloidin is added in 30 mM KCl/2 mM MgCl2/20 mM Tris-HCl (pH 7.6). The resulting polymer (MBS-P-actin) is highly potentiated so that it activates the Mg2+-ATPase of S1 more strongly than native F-actin. The affinity of MBS-P-actin for S1 in the presence of ATP (K(ATPase)) is about four times higher than that of native F-actin, although the maximum velocity at infinite actin concentration (V(max)) is almost the same. This high activation is not due to a cross-linking between MBS-P-actin and the S1 heavy chain, since no substantial amount of cross-linking was observed in SDS gel electrophoresis. Direct binding studies and ATPase measurements showed that the modification of actin with MBS impairs the binding of tropomyosin. Tropomyosin binding can be improved considerably by the addition of troponin. However, the regulation mechanism of the acto-S1 ATPase activity by troponin-tropomyosin is damaged. The addition of troponin-tropomyosin reduces the S1 ATPase activation by MBS-P-actin to the same level as that of native F-actin in 30 mM KCl/2.5 mM ATP/2 mM MgCl2, but there is no difference in the ATPase activation in the presence and absence of Ca2+. When the concentration of Mg2+ is increased to 5 mM, the regulation is partially restored. There is a significant difference in the ATPase activation in the presence and absence of Ca2+. The properties of MBS-actin, except for its extremely high activation of S1 ATPase activity, are very similar to those of FITC-labeled actin in which Lys-61 is selectively labeled as previously reported [Miki, M. (1989) J. Biochem. 106, 651-655].