Study on the structural and functional significance of the double-heads of myosin with the use of selective and reversible affinity labeling.

肌球蛋白分子由两个球形头部(S1)组成：一个保守的结构。每个头部都有ATPase活性的活性部位。我们研究项目的最终目标是阐明这种双头结构的意义。我们发明了一种动力学方法，通过使用钒酸加ADP作为可逆的亲和标记，来测试肌球蛋白的活性部位是否为酶均一。该方法显示，经SH试剂修饰的S1至少含有两种(1：1)不同的物种。后来，我们用ADP柱层析成功地将这两种S1分离成两个组分。随后的研究表明，这两个S1在SH修饰前表现出“两种状态”。最近由其他研究小组对肌球蛋白头部进行的电子显微镜观察显示，肌球蛋白头部有两个不同的形状(1：1不同)，这可能对应于我们的两个S1组分。每个肌球蛋白头部都有一个高活性的赖氨酸残基(RL)，可以被TNBS修饰。先前的研究表明，在MgPPI的存在下，TNBS修饰的RL的数量减少到0.5(M/M)，这表明存在两个结构不同的S1。为了了解这一观察结果与我们上述发现的关系，我们表示要重新研究S1的TNBS修饰。为此，我们构建了一个用分光光度计和计算机相结合的系统，对TNBS反应进行精确的数据收集和分析。我们发现，在mgADP的存在下，只有一半添加的sl的RL被TNBS修饰，就像在mgPPI存在的情况下一样。这一结果表明存在两个不同的S1，它们在ADP结合效应方面是不同的。我们现在正试图通过柱层析分离这两个S1，以阐明这两个不同的S1与肌球蛋白分子的双头结构的关系。

A myosin molecule consists of two globular heads (S1): a conserved structure. Each head has an active site for ATPase activity. The final goal of our research project is to elucidate the significance of this double-headed structure. We invented a kinetic method to test whether the active site of myosin is enzymatically homogeneous or not, by using vanadate plus ADP as a reversible affinity labelling. The method revealed that S1, modified by an SH-reagent, contains at least two (1:1) different species. Later, we succeeded in separating these two species of S1 into two fractions by an ADP-column chromatography. Subsequent studies showed that these two S1s manifest "two states" S1 takes before the SH-modification. Recent electron microscopic observation of myosin heads by other research group has shown that myosin heads take two (1:1 different) distinct shapes, which may correspond to our two fractions of S1. Each myosin head has one highly reactive lysin residue (RL), which can be modified by TNBS. It was shown previously that the number of RL modified by TNBS is reduced to 0.5 (M/M) in the presence of MgPPi, and this observation suggested that two "structurally different" S1s exist. To see the relation of this observation with our above-mentioned finding, we stated to re-investigate the TNBS-modification of S1. To do it, we constructed a system for the precise data-collection and analysis of the TNBS reaction with a combination of a spectrophotometer with a computer. We found that RL of only half of added sl is modified by TNBS in the presence of mgADP as in the presence of MgPPi. This result suggests the existence of two distinct S1s which are different from each other with regards to the ADP-binding effect. We are now attempting to separate these two S1s by column chromatography, to elucidate the relationship of these two different S1s with the double-headed structure of the myosin molecule.