MECHANISM FOR THE INHIBITION OF ACTO-HEAVY MEROMYOSIN ATPASE BY THE ACTIN CALMODULIN BINDING DOMAIN OF CALDESMON

Caldesmon, an actin/calmodulin binding protein, inhibits acto-heavy meromyosin (HMM) ATPase, while it increases the binding of HMM to actin, presumably mediated through an interaction between the myosin subfragment 2 region of HMM and caldesmon, which is bound to actin. In order to study the mechanism for the inhibition of acto-HMM ATPase, we utilized the chymotryptic fragment of caldesmon (38-kDa fragment), which possesses the actin/calmodulin binding region but lack the myosin binding portion. The 38-kDa fragment inhibits the actin-activated HMM ATPase to the same extent as does the intact caldesmon molecule. In the absence of tropomyosin, the 38-kDa fragment decreased the K(ATPase) and K(binding) without any effect on the V(max). However, when the actin filament contained bound tropomyosin, the caldesmon fragment caused a 2-3 fold decrease in the V(max), in addition to lowering the K(ATPase) and the K(binding). The 38-kDa fragment-induced inhibition is partially reversed by calmodulin at a 10:1 molar ratio to caldesmon fragment; the reversal was more remarkable in 100 mM ionic strength at 37-degrees-C than in 20 or 50 mM at 25-degrees-C. Results from these experiments demonstrate that the 38-kDa domain of caldesmon inhibits the binding of myosin head to actin; however, when the actin filaments contain bound tropomyosin, caldesmon fragment affects not only the binding of HMM to actin but also the catalytic step in the ATPase cycle. The interaction between the 38-kDa domain of caldesmon and tropomyosin-actin is likely to play a role in the regulation of actomyosin ATPase and contraction in smooth muscle.