Harwalkar V A, White M P, Annis D T, Zervou F, Stein L A
Department of Medicine, State University of New York, Stony Brook 11790.
Biophys J. 1990 May;57(5):1065-74. doi: 10.1016/S0006-3495(90)82624-2.
We have investigated the effect of limited trypsin digestion of chymotryptic myosin Subfragment-1 (S-1) on its kinetic properties. We find that Vmax (i.e., the extrapolated maximal ATPase activity at infinite actin) remains approximately constant, independent of the period of digestion. We also find that the apparent actin activation constant, KATPase, and the apparent dissociation constant, Kbinding, are both significantly weakened by trypsin digestion of S-1, and that these kinetic parameters change in concert. In addition, we investigated the effect of limited trypsin digestion on the initial phosphate burst. We find that trypsin digestion has no effect on the rate of the tryptophan fluorescence enhancement that occurs after ATP binds to digested S-1, but that the magnitude of the fluorescence enhancement falls approximately 40% with digestion. Digested S-1 also showed anomalous behavior in that the fluorescence magnitude increased and the fluorescence rate dropped in the presence of actin. Trypsin digestion also decreased the magnitude of the chemically measured Pi burst approximately 35%, but this magnitude was essentially unaffected by actin. A possible explanation for this behavior is discussed.
我们研究了用胰蛋白酶对胰凝乳蛋白酶肌球蛋白亚片段-1（S-1）进行有限消化对其动力学性质的影响。我们发现，Vmax（即无限肌动蛋白浓度下外推得到的最大ATP酶活性）保持大致恒定，与消化时间无关。我们还发现，S-1经胰蛋白酶消化后，表观肌动蛋白激活常数KATPase和表观解离常数Kbinding均显著降低，且这些动力学参数协同变化。此外，我们研究了有限胰蛋白酶消化对初始磷酸盐爆发的影响。我们发现，胰蛋白酶消化对ATP与消化后的S-1结合后发生的色氨酸荧光增强速率没有影响，但荧光增强幅度随消化而下降约40%。消化后的S-1还表现出异常行为，即在肌动蛋白存在下荧光幅度增加而荧光速率下降。胰蛋白酶消化还使化学测定的Pi爆发幅度降低约35%，但该幅度基本不受肌动蛋白影响。文中讨论了对此行为的一种可能解释。
