Effect of limited trypsin digestion on the biochemical kinetics of skeletal myosin subfragment-1.
Effect of limited trypsin digestion on the biochemical kinetics of skeletal myosin subfragment-1.
复制标题
有限胰蛋白酶消化对骨骼肌球蛋白亚片段 1 生化动力学的影响。
DOI：
10.1016/s0006-3495(90)82624-2
复制
发表时间：
1990
影响因子：
3.4
通讯作者：
Stein,LA
中科院分区：
文献类型：
--
作者：
Harwalkar,VA;White,MP;Annis,DT;Zervou,F;Stein,LA
我们研究了有限胰酶消化乳糜胰蛋白酶肌球蛋白亚段-1(S-1)对其动力学性质的影响。我们发现，Vmax(即外推的无限大肌动蛋白的最大ATPase活性)大致保持不变，与消化时间无关。我们还发现，S-1的表观肌动蛋白激活常数KATPase和表观解离常数Kbinding值均被胰酶消化显著减弱，且这两个动力学参数的变化是一致的。此外，我们还研究了有限胰酶消化对初始磷酸盐破裂的影响。我们发现，胰酶消化对三磷酸腺苷与S-1结合后的色氨酸荧光增强速率没有影响，但消化后荧光增强的幅度下降了约40%。经消化的S-1也表现出异常行为，在肌动蛋白存在下，荧光强度增加，荧光速率下降。胰酶消化也使化学测量的PI猝发的幅度降低约35%，但这一幅度基本上不受肌动蛋白的影响。讨论了这种行为的一种可能的解释。
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.