Duong A M, Reisler E
J Biol Chem. 1987 Mar 25;262(9):4124-8.
The initial rates of tryptic digestion at the 50/20-kDa junction in myosin subfragment 1 (S-1) were determined for free S-1, acto-S-1, and acto-S-1 in the presence of magnesium adenyl-5'-yl imidodiphosphate (Mg AMP-PNP) and MgATP under ionic strength conditions ranging from 30 to 124 mM. The percentage of S-1 bound to actin in the presence of Mg AMP-PNP and MgATP was calculated from these rates for each set of digestion experiments. Parallel experiments carried out in an Airfuge centrifuge on identical acto-S-1 solutions yielded independent information on the binding of S-1 to actin. The results of binding measurements by these two methods were in excellent agreement in all cases tested, covering the range from 15 to 95% binding of S-1 to actin. Tryptic digestions of synthetic mixtures of S-1 and p-phenylenedimaleimide S-1 in the presence of actin demonstrated that a two-component system of myosin heads with different affinities for actin can be resolved into its constituents by the proteolytic rates method. The results of this work justify applications of the proteolytic rates method to actomyosin binding studies in more complex systems.
在离子强度范围为30至124 mM的条件下，测定了肌球蛋白亚片段1（S-1）中50/20-kDa连接处胰蛋白酶消化的初始速率，分别针对游离S-1、肌动蛋白-S-1以及存在镁腺苷-5'-亚氨基二磷酸（Mg AMP-PNP）和MgATP时的肌动蛋白-S-1。根据每组消化实验的这些速率，计算了在Mg AMP-PNP和MgATP存在下与肌动蛋白结合的S-1的百分比。在Airfuge离心机中对相同的肌动蛋白-S-1溶液进行的平行实验，得出了关于S-1与肌动蛋白结合的独立信息。在所有测试的情况下，这两种方法的结合测量结果都非常吻合，涵盖了S-1与肌动蛋白结合从15%到95%的范围。在肌动蛋白存在的情况下，对S-1和对苯二马来酰亚胺S-1的合成混合物进行胰蛋白酶消化，结果表明，通过蛋白水解速率法可以将对肌动蛋白具有不同亲和力的肌球蛋白头部的双组分系统解析为其组成成分。这项工作的结果证明了蛋白水解速率法在更复杂系统中应用于肌动球蛋白结合研究的合理性。
