Calcium-calmodulin and regulation of brush border myosin-I MgATPase and mechanochemistry.
We examined the Ca(2+)-dependent regulation of brush border (BB) myosin-I by probing the possible roles of the calmodulin (CM) light chains. BB myosin-I MgATPase activity, sensitivity to chymotryptic digestion, and mechanochemical properties were assessed using 1-10 microM Ca2+ and in the presence of exogenously added CM since it has been proposed that this myosin is regulated by calcium-induced CM dissociation from the 119-kD heavy chain. Each of these BB myosin-I properties were dramatically altered by the same threshold of 2-3 microM Ca2+. Enzymatically active NH2-terminal proteolytic fragments of BB myosin-I which lack the CM binding domains (the 78-kD peptide) differ from CM-containing peptides in that the former is completely insensitive to Ca2+. Furthermore, the 78-kD peptide exhibits high levels of MgATPase activity which are comparable to that observed for BB myosin-I in the presence of Ca2+. This suggests that Ca2+ regulates BB myosin-I MgATPase by binding directly to the CM light chains, and that CM acts to repress endogenous MgATPase activity. Ca(2+)-induced CM dissociation from BB myosin-I can be prevented by the addition of exogenous CM. Under these conditions Ca2+ causes a reversible slowing of motility. In contrast, in the absence of exogenous CM, motility is stopped by Ca2+. We demonstrate this reversible slowing is not due to the presence of inactive BB myosin-I molecules exerting a "braking" effect on motile filaments. However, we did observe Ca(2+)-independent slowing of motility by acidic phospholipids, suggesting that factors other than Ca2+ and CM content can affect the mechanochemical properties of BB myosin-I.
Wolenski
J S
JS
Department of Biology, Yale University, New Haven, Connecticut 06511.
Hayden
S M
SM
Forscher
P
P
Mooseker
M S
MS
D008841:Actin Cytoskeleton / Q000502:physiology; D000199:Actins / Q000494:pharmacology; D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology*; D002147:Calmodulin / Q000378:metabolism* / Q000494:pharmacology; D002148:Calmodulin-Binding Proteins; D002645:Chickens; D004789:Enzyme Activation; D007700:Kinetics; D008871:Microvilli / Q000201:enzymology*; D010743:Phospholipids / Q000378:metabolism; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*
D000199:Actins; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D010743:Phospholipids; D011506:Proteins; D017301:Ca(2+) Mg(2+)-ATPase; D002118:Calcium