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-HM ATPase, we utilized the chymotryptic fragment of caldesmon (38-kDa fragment), which possesses the actin/calmodulin binding region but lacks 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 KATPase and Kbinding without any effect on the Vmax. However, when the actin filament contained bound tropomyosin, the caldesmon fragment caused a 2-3-fold decrease in the Vmax, in addition to lowering the KATPase and the Kbinding. 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 fragment of myosin head to actin; however, when the actin filament contains 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.
Horiuchi
K Y
KY
Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, Philadelphia 19104.
Samuel
M
M
Chacko
S
S
D000199:Actins / Q000378:metabolism*; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D002147:Calmodulin / Q000494:pharmacology*; D002148:Calmodulin-Binding Proteins / Q000494:pharmacology*; D002645:Chickens; D002918:Chymotrypsin; D004789:Enzyme Activation / Q000187:drug effects; D005895:Gizzard, Avian / Q000187:drug effects / Q000201:enzymology*; D006868:Hydrolysis; D007700:Kinetics; D009218:Myosins / Q000378:metabolism*
D000199:Actins; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D002918:Chymotrypsin; D017301:Ca(2+) Mg(2+)-ATPase; D009218:Myosins