THE EFFECTS OF SMOOTH-MUSCLE CALPONIN ON THE STRONG AND WEAK MYOSIN BINDING-SITES OF F-ACTIN

We have investigated the mechanism of inhibition of the actomyosin MgA TPase by the smooth muscle protein calponin. We have shown previously the specific interaction of calponin with Glu(334) of actin (EL-Mezgue ldi, M., Fattoum, A., Derancourt, J., and Hassab, R. (1992) J. Biol. C hem. 267, 15943-15951). This residue is within the sequence 332-334, w hich has been proposed to be an important part of the strong myosin bi nding site (Rayment, L., Holden, H. M., Whittaker, M., Yohn, C. B., Lo renz, M., Holmes, K. C., and Milligan, R. A. (1993) Science 261, 58-65 ), Therefore, we suggested that calponin will affect the strong bindin g actin-myosin interaction, To test this hypothesis we have investigat ed the effect of calponin on the strong binding of S-1 . MgAMP-PNP (5' -adenylyl imidodiphosphate) and on the weak binding of S-1 . MgADP . P -i to actin, We found that an inhibitory concentration of calponin dec reased the binding of S-1 . MgAMP-PNP to actin but had no effect on th e binding of S-1 . MgADP . P-i. Similar results were obtained with ske letal muscle and smooth muscle S-1. In competition experiments calponi n was found to displace S-1 . MgAMP-PNP and S-1 . MgADP but not S-1 . MgADP . P-i from the actin filament, S-1 displaced calponin from actin in the rigor state, in the presence of MgADP, and in the presence of MgAMP-PNP. We conclude that calponin inhibits the actin activated S-1 ATPase by blocking a strong S-1 binding site on actin and does not blo ck the weak binding site.