CALCIUM-CALMODULIN AND REGULATION OF BRUSH-BORDER MYOSIN-I MGATPASE AND MECHANOCHEMISTRY

We examined the Ca2+-dependent regulation of brush border (BB) myosin- I by probing the possible roles of the calmodulin (CM) light chains. B B myosin-I MgATPase activity, sensitivity to chymotryptic digestion, a nd mechanochemical properties were assessed using 1-10 muM 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 1 19-kD heavy chain. Each of these BB myosin-I properties were dramatica lly altered by the same threshold of 2-3 muM Ca2+ . Enzymatically acti ve NH2-terminal proteolytic fragments of BB myosin-I which lack the CM binding domains (the 78-kD peptide) differ from CM-containing peptide s in that the former is completely insensitive to Ca2+. Furthermore, t he 78-kD peptide exhibits high levels of MgATPase activity which are c omparable to that observed for BB myosin-I in the presence of Ca2+. Th is suggests that Ca2+ regulates BB myosin-I MgATPase by binding direct ly to the CM light chains, and that CM acts to repress endogenous MgAT Pase activity. Ca2+-induced CM dissociation from BB myosin-I can be pr evented by the addition of exogenous CM. Under these conditions Ca2+ c auses a reversible slowing of motility. In contrast, in the absence of exogenous CM, motility is stopped by Ca2+. We demonstrate this revers ible slowing is not due to the presence of inactive BB myosin-I molecu les exerting a ''braking'' effect on motile filaments. However, we did observe Ca2+-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.