Cooperative inhibition of actin filaments in the absence of tropomyosin

We measured the inhibition of actin activated myosin subfragment- 1 MgATPase activity in a solution containing no added KCl ( 5 mM PIPES. K-2 ( pH 7.1), 2.5 mM MgCl2, 1 mM DTT, 1 mM NaN3, 5 mM MgATP). Maximal inhibition was observed with substoichiometric concentrations of caldesmon, caldesmon domain 4, troponin and troponin I. In six experiments using different preparations of actin, S- 1 and caldesmon 50% inhibition required 0.09 +/- 0.01 ( sem) caldesmon added per actin. This compares with 0.66 +/- 0.32 ( sem, n = 5) caldesmon per actin for 50% inhibition in the presence of 60 mM KCl. With caldesmon domain 4, 50% inhibition was achieved with 0.17 +/- 0.08 ( n = 11) domain 4 added per actin. We measured the amount of caldesmon bound at the same time as inhibition. Complete inhibition of actin activated ATPase needed only one caldesmon bound per 5.0 +/- 0.5 ( sem, n = 5) actin monomers or one caldesmon domain 4 bound per 3.9 +/- 0.6 ( sem, n = 3) actin monomers at zero KCl. We conclude that under these conditions inhibition of actin is cooperative despite the absence of tropomyosin. We measured the effect of caldesmon inhibition upon S- 1 binding to actin. S- 1. ADP. Pi ( weak binding) was not affected by caldesmon concentrations giving 80% inhibition, however S- 1. ADP ( strong binding) was highly cooperative, being very weak at < 0.3 μM but indistinguishable from uninhibited actin at > 2 muM S- 1. ADP. We conclude that actin can exist in two activity states corresponding to the ' on' and ' o.' states of actin - tropomyosin and inhibitory proteins function as allosteric- cooperative inhibitors of actin. The implications of these. findings for the role of tropomyosin in thin. lament regulation are discussed.