IN VITRO FUNCTIONAL STUDIES OF HUMAN CARDIAC MYOSIN

人心肌肌球蛋白调节轻链 突变与罕见的心室肥大有关， 家族性肥厚型心肌病骨骼肌异常 （HCM）。为了了解这些功能的影响， 突变，我们已经表达了RLC突变，Ala 13Thr， Glu 22 Lys、Pro 94 Arg、沿着人工突变的 细菌中的可磷酸化丝氨酸（Ser 15 Ala，Ser 15 Asp） 表达系统然后将表达的RLC交换为 正常新鲜纯化的人心肌肌球蛋白与天然RLC 耗尽了携带不同突变的重组肌球蛋白 然后在体外运动性测定中测试RLC。纯化的人 还用木瓜蛋白酶消化心肌肌球蛋白， 亚片段1用于ATP酶测定。初步数据显示， 轻链交换程序不损害肌球蛋白 通过体外运动率评估功能。Glu 22 Lys和 Pro 94 Arg突变导致运动速率减慢， Ala 13Thr的表达没有改变。Glu 22 Lys的ATP酶活性 似乎显着升高，而其他突变体没有 改变ATP酶活性。然而，我们无法准备 通过木瓜蛋白酶消化足够量的亚片段1（S-1 进行所需的动力学 实验和制备的S-1是异质的 通过SDS凝胶电泳分析。因此，我们转向 高度同源的猪心肌肌球蛋白作为起始 procedure.大量的肌球蛋白可以从 屠宰场获得的材料和均质制剂 制备S-1。该材料将用于形成轻链 交换，以产生用于瞬态动力学分析的材料， HCM引起的突变的后果，在这两个基本和 调节轻链。

Human cardiac myosin regulatory light chain (RLC) mutations have been linked to a rare ventricular hypertrophy and skeletal muscle abnormality in familial hypertrophic cardiomyopathy (HCM). In order to understand the functional effects of these mutations, we have expressed the RLC mutations, Ala13Thr, Glu22Lys, Pro94Arg, along with artificial mutations on the phosphorylatable serine (Ser15Ala, Ser15Asp) in a bacteria expression system. The expressed RLCs were then exchanged into normal freshly purified human cardiac myosin with the native RLC depleted. The reconstituted myosins carrying different mutations on RLC were then tested in an in vitro motility assay. Purified human cardiac myosin has also been digested with papain to obtain subfragment 1 for ATPase assay. The preliminary data indicate that the light chain exchange procedures did not impair the myosin function as assessed by in vitro motility rate. Both Glu22Lys and Pro94Arg mutations resulted in slower motility rates whereas that of Ala13Thr was not altered. The ATPase activity for Glu22Lys seemed to be elevated significantly while other mutants did not change the ATPase activities. However, we were unable to prepare sufficient quantities of subfragment-one (S-1) by papain digestion of human cardiac myosin to carry out the desired kinetic experiments and the S-1 that was prepared was heterogeneous upon analysis by SDS gel electrophoresis. Therefore, we turned to the highly homologous porcine cardiac myosin as a starting procedure. Large quantities of myosin can be purified from slaughter house obtained material and a homogeneous preparation of S-1 was prepared. This material will be used to for light chain exchange to produce material for transient kinetic analysis of the consequences of HCM-causing mutations in both the essential and regulatory light chains.