MODULATION OF VASCULAR CONTRACTION BY PHOSPHATASES

这项提案的长期目标是确定肌球蛋白的作用 轻链磷酸化在血管平滑肌调节中的作用 机械性能和研究特定的一种或多种酶 使肌球蛋白去磷酸化。将承担的具体目标 研究肌球蛋白在血管平滑肌中的去磷酸化是：1) 血管平滑肌磷酸酶活性的纯化与鉴定 显示出对分离的肌球蛋白轻链或 完整的肌球蛋白；2)对肌球蛋白磷酸酶活性进行分类 其他已知的底物并检查它们之间的重要关系 使用生化和免疫化学技术的磷酸酶；3) 肌球蛋白轻链化学计量比关系的确定 磷酸化与力学参数、等长力和 轻负荷缩短速度，在化学皮肤血管光滑 4)检测特异性磷酸酶抑制剂对大鼠骨骼肌的影响。 肌球蛋白的磷酸化和机械性能(即等轴力和等轴向力 轻负荷缩短速度)在化学性皮肤血管平滑中的作用 以及5)检测纯化的肌球蛋白磷酸酶对 化学剥皮血管平滑肌的力学特性。肌球蛋白 或用标准蛋白纯化肌球蛋白轻链磷酸酶 分离方法和广泛鉴定。一项重大尝试将是 用来确定多亚单位磷酸酶是否共享公共亚基 而其他酶表现出不同的底物特异性。在……里面 此外，纯化的磷酸酶的肌动蛋白结合特性将是 调查过了。最终，纯化的磷酸酶将直接添加到 含有甘油的小型肌肉浴缸(即化学蒙皮) 血管平滑肌。肌球蛋白轻链磷酸化的影响 水平将用甘油-尿素凝胶放射免疫印迹法测定。在……里面 此外，还将监测作用力和轻载缩短速度 使用和电磁测力仪。通过这种方式，我们将确定 哪个力学参数对肌球蛋白轻链的变化敏感 磷酸化与这种敏感性的定量关系。它 预计研究将在五年内由 医生-科学家。总的来说，这些研究被认为是重要的。 因为血管疾病在人类发病率中扮演着重要的角色 和死亡率。我们的提案将调查以下基本机制： 血管平滑肌收缩。

The long-term goals of this proposal are to determine the role of myosin light chain phosphorylation in the regulation of vascular smooth muscle mechanical properties and to investigate the specific enzyme or enzymes which dephosphorylate myosin. The specific aims that will be undertaken to investigate myosin dephosphorylation in vascular smooth muscle are: 1) to purify and characterize the phosphatase activities in vascular smooth muscle which demonstrate activity toward isolated myosin light chains or intact myosin; 2) to classify myosin phosphatase activities with respect to other known substrates and to examine important relationships among phosphatases using biochemical and immunochemical techniques; 3) to determine the relationship between the stoichiometry of myosin light chain phosphorylation and the mechanical parameters, isometric force and lightly-loaded shortening velocity, in chemically-skinned vascular smooth muscle; 4) to examine the effects of specific phosphatase inhibitors on myosin phosphorylation and mechanical properties (ie. isometric force and lightly-loaded shortening velocity) in chemically-skinned vascular smooth muscle; and 5) to examine the effects of purified myosin phosphatases on mechanical properties of chemically-skinned vascular smooth muscle. Myosin or myosin light chain phosphatases will be purified by standard protein isolation methods and extensively characterized. A major attempt will be made to determine whether multisubunit phosphatases share common subunits with other enzymes displaying different substrate specificities. In addition, the actin binding properties of purified phosphatases will be investigated. Ultimately, purified phosphatases will be added directly to small muscle baths containing glycerinated (ie. chemically-skinned) vascular smooth muscle. The effects on myosin light chain phosphorylation levels will be determined by glycerol-urea gel radioimmunoblot. In addition, force and lightly-loaded shortening velocity will be monitored using and electromagnetic ergometer. In this manner, we will determine which mechanical parameter is sensitive to changes in myosin light chain phosphorylation and the quantitative relationship of this sensitivity. It is anticipated that studies will be conducted over five years by a Physician-Scientist. In general, these studies are deemed to be important because of the important role blood vessel disease plays in human morbidity and mortality. Our proposal will investigate the basic mechanism of contraction in vascular smooth muscle.