Evans J A, Eisenberg E
Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892.
Biochemistry. 1989 Sep 19;28(19):7741-7. doi: 10.1021/bi00445a033.
Considerable effort has been devoted to understanding the mechanism of 18O exchange in skinned skeletal and insect muscle fibers. However, a full understanding of the mechanism of 18O exchange in muscle fibers requires an understanding of the mechanism of 18O exchange in the simpler in vitro systems employing myosin subfragment 1 (S-1) and heavy meromyosin (HMM). In the present study, using both S-1 and S-1 covalently cross-linked to actin, we show first that over a wide range of temperature, ionic strength, and actin concentration there is only one pathway of 18O exchange with S-1. This is also the case with HMM except at very low ionic strength and low actin concentration, and even here, the data can be explained if 20% of the HMM is denatured in such a way that it shows no 18O exchange. Our results also show that actin markedly decreases the rate of 18O exchange. If it is assumed that Pi release is rate limiting, the four-state kinetic model of the actomyosin ATPase cannot fit these 18O exchange data. However, if it is assumed that the ATP hydrolysis step is rate limiting and Pi release is very fast, the four-state kinetic model can qualitatively fit these data although the fit is not perfect. A better fit to the 18O exchange data can be obtained with the six-state kinetic model of the actomyosin ATPase, but this fit requires the assumption that, at saturating actin concentration, the rate of Pi rotation is about 9-fold slower than the rate of reversal of the ATP hydrolysis step.
人们已付出巨大努力来理解皮肤化骨骼肌和昆虫肌纤维中18O交换的机制。然而，要全面理解肌纤维中18O交换的机制，需要先理解在使用肌球蛋白亚片段1（S-1）和重酶解肌球蛋白（HMM）的更简单体外系统中18O交换的机制。在本研究中，我们使用与肌动蛋白共价交联的S-1和S-1，首先表明在很宽的温度、离子强度和肌动蛋白浓度范围内，与S-1进行18O交换只有一条途径。HMM也是如此，除了在非常低的离子强度和低肌动蛋白浓度下，即便在此情况下，如果20%的HMM以不显示18O交换的方式变性，数据也能得到解释。我们的结果还表明，肌动蛋白显著降低18O交换速率。如果假设Pi释放是限速步骤，肌动球蛋白ATP酶的四态动力学模型无法拟合这些18O交换数据。然而，如果假设ATP水解步骤是限速步骤且Pi释放非常快，四态动力学模型虽不能完美拟合但能定性拟合这些数据。肌动球蛋白ATP酶的六态动力学模型能更好地拟合18O交换数据，但这种拟合需要假设在肌动蛋白浓度饱和时，Pi旋转速率比ATP水解步骤的逆转速率慢约9倍。
