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 MgATPase by the smooth muscle protein calponin. We have shown previously the specific interaction of calponin with Glu334 of actin (EL-Mezgueldi, M., Fattoum, A., Derancourt, J., and Kassab, R. (1992) J. Biol. Chem. 267, 15943-15951). This residue is within the sequence 332-334, which has been proposed to be an important part of the strong myosin binding site (Rayment, I., Holden, H. M., Whittaker, M., Yohn, C. B., Lorenz, M., Holmes, K. C., and Milligan, R. A. (1993) Science 261, 58-65). Therefore, we suggested that calponin will affect the strong binding actin-myosin interaction. To test this hypothesis we have investigated 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.Pi to actin. We found that an inhibitory concentration of calponin decreased the binding of S-1. MgAMP-PNP to actin but had no effect on the binding of S-1.MgADP.Pi. Similar results were obtained with skeletal muscle and smooth muscle S-1. In competition experiments calponin was found to displace S-1. MgAMP-PNP and S-1.MgADP but not S-1.MgADP.Pi 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 block the weak binding site.
Department of Cardiac Medicine, Imperial College School of Medicine at the National Heart and Lung Institute, Dovehouse Street, London SW3 6LY, United Kingdom. m.elmezgueldi@ic.ac.uk
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D000199:Actins / Q000378:metabolism*; D000205:Actomyosin / Q000037:antagonists & inhibitors; D000244:Adenosine Diphosphate / Q000378:metabolism; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D017301:Ca(2+) Mg(2+)-ATPase / Q000037:antagonists & inhibitors; D002135:Calcium-Binding Proteins / Q000494:pharmacology*; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002645:Chickens; D008840:Microfilament Proteins / Q000378:metabolism*; D009124:Muscle Proteins / Q000378:metabolism*; D009130:Muscle, Smooth / Q000737:chemistry*; D009218:Myosins / Q000378:metabolism*; D009994:Osmolar Concentration; D011487:Protein Conformation; D000096985:Calponins
D000199:Actins; D000205:Actomyosin; D000244:Adenosine Diphosphate; D000266:Adenylyl Imidodiphosphate; D017301:Ca(2+) Mg(2+)-ATPase; D002135:Calcium-Binding Proteins; D002148:Calmodulin-Binding Proteins; D008840:Microfilament Proteins; D009124:Muscle Proteins; D009218:Myosins; D000096985:Calponins