IN VITRO FUNCTIONAL STUDIES OF HUMAN CARDIAC MYOSIN

人心肌肌球蛋白调节轻链(RLC) 突变与一种罕见的心室肥厚有关， 家族性肥厚型心肌病的骨骼肌异常 (HCM)。为了了解这些产品的功能效果 突变，我们已经表达了RLC突变，Ala13Thr， Glu22Lys，Pro94Arg，以及 细菌中的可磷酸化丝氨酸(Ser15Ala，Ser15Asp) 表达系统。然后将表达的RLCs交换到 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 然后在体外测定RLC的运动能力。纯净人 心肌肌球蛋白也已用木瓜酶消化，以获得 用于ATPase测定的亚片段1。初步数据表明， 轻链交换过程没有损害肌球蛋白 通过体外运动率来评估其功能。Glu22Lys和 Pro94Arg突变导致运动速度减慢，而 没有改变Ala13Thr的。谷氨酸22Lys的ATPase活性 似乎显著升高，而其他突变体则没有 更改ATPase活动。然而，我们无法做好准备 木瓜酶消化足够量的亚片段-1(S-1) 人心肌肌球蛋白进行所需的动力学 实验和制备的S一号是异质的 经十二烷基硫酸钠凝胶电泳法分析。因此，我们求助于 以高度同源的猪心肌肌球蛋白为起点 程序。大量的肌球蛋白可以从 屠宰场获得的原料和均匀的制剂 S一号研制成功。这种材料将用于制造轻链。 交换以产生用于瞬变动力学分析的材料 HCM引起的基本和非必需基因突变的后果 监管轻链。

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.