Wolenski J S, Hayden S M, Forscher P, Mooseker M S
Department of Biology, Yale University, New Haven, Connecticut 06511.
J Cell Biol. 1993 Aug;122(3):613-21. doi: 10.1083/jcb.122.3.613.
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.
我们通过探究钙调蛋白（CM）轻链的可能作用，研究了刷状缘（BB）肌球蛋白-I的钙离子依赖性调节。使用1-10微摩尔/升的钙离子，并在添加外源CM的情况下，评估了BB肌球蛋白-I的MgATP酶活性、对胰凝乳蛋白酶消化的敏感性以及机械化学性质，因为有人提出这种肌球蛋白受钙离子诱导的CM从119-kD重链上解离的调节。这些BB肌球蛋白-I的特性每一个都在2-3微摩尔/升的相同阈值下发生了显著改变。缺乏CM结合结构域的BB肌球蛋白-I的酶活性NH2末端蛋白水解片段（78-kD肽）与含CM的肽不同，前者对钙离子完全不敏感。此外，78-kD肽表现出高水平的MgATP酶活性，这与在钙离子存在下观察到的BB肌球蛋白-I的活性相当。这表明钙离子通过直接结合到CM轻链上来调节BB肌球蛋白-I的MgATP酶活性，并且CM起到抑制内源性MgATP酶活性的作用。添加外源CM可以防止钙离子诱导的CM从BB肌球蛋白-I上解离。在这些条件下，钙离子会导致运动性可逆性减慢。相比之下，在没有外源CM的情况下，钙离子会使运动停止。我们证明这种可逆性减慢不是由于存在无活性的BB肌球蛋白-I分子对运动细丝产生“制动”作用。然而，我们确实观察到酸性磷脂会导致与钙离子无关的运动性减慢，这表明除了钙离子和CM含量之外的其他因素也会影响BB肌球蛋白-I的机械化学性质。
