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酶或酶 使肌球蛋白去磷酸化。 将致力于实现的具体目标， 研究血管平滑肌肌球蛋白去磷酸化是：1） 血管平滑肌磷酸酶活性的纯化和鉴定 对分离的肌球蛋白轻链表现出活性的肌肉，或 完整肌球蛋白; 2）根据肌球蛋白磷酸酶活性分类， 其他已知的基质，并检查重要的关系， 使用生物化学和免疫化学技术的磷酸酶; 3） 确定肌球蛋白轻链的化学计量之间的关系 磷酸化和力学参数，等长力和 轻载缩短速度，在化学皮肤血管光滑 肌肉; 4）检查特定磷酸酶抑制剂对 肌球蛋白磷酸化和机械性质（即，等距力和 轻负荷缩短速度）在化学皮肤血管平滑 肌肉;和5）检查纯化的肌球蛋白磷酸酶对 化学皮肤血管平滑肌的机械性能。 肌球 或肌球蛋白轻链磷酸酶将通过标准蛋白纯化 分离方法和广泛表征。 一个主要的尝试将是 以确定多亚基磷酸酶是否具有共同的亚基 而其它酶显示不同的底物特异性。 在 此外，纯化的磷酸酶的肌动蛋白结合特性将是 研究了 最终，将纯化的磷酸酶直接加入到 含有甘油的小肌肉浴（即，化学皮肤） 血管平滑肌 对肌球蛋白轻链磷酸化的影响 通过甘油-尿素凝胶放射免疫印迹测定水平。 在 此外，将监测力和轻载缩短速度 使用和电磁测力计。 这样，我们将确定 该机械参数对肌球蛋白轻链的变化敏感 磷酸化和这种敏感性的定量关系。 它 预计研究将在五年内进行， 物理科学家 总的来说，这些研究被认为是重要的。 由于血管疾病在人类发病率中起着重要作用 and mortality. 我们的建议将调查的基本机制， 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.