COOPERATIVE EFFECTS IN SMOOTH MUSCLE REGULATION

尽管平滑肌在健康和疾病中的重要性， 其运作和监管的细节基本上仍不清楚。这 续期申请是一项持续的努力，旨在获得更多 这方面的信息。总体目标是使用分子和 细胞方法，以获得信息的功能作用， 各种平滑肌蛋白，并加强我们对 平滑肌收缩的调节机制。 程序 包括六个项目，两个设施核心和一个行政核心。 项目I-A涉及肌球蛋白磷酸化的作用， 结构以及肌动蛋白与肌球蛋白之间的相互作用。 项目 I-B与肌球蛋白轻链 激酶被Ca/钙调蛋白激活，肌球蛋白通过其发光 链被酶识别。 项目II-A和II-B研究 钙调素和钙调蛋白的结构和功能，分别在 我希望这些研究将导致阐明他们的作用， 基于薄顺应性的平滑肌收缩调节。 项目II-C寻找涉及 平滑肌中的丝裂原活化蛋白激酶。 在项目III中 粗细丝和细丝的集成系统将 研究，以及原肌球蛋白在合作中的可能作用 将探索平滑肌细丝的性质。 所有 除II-C和II外，其他项目均采用化学交联 和共振能量转移作为表征蛋白质-蛋白质的工具 相互作用，实际上每个项目都使用定向诱变 来生产蛋白质变体。 特别是项目II-A和II-C， 探索遗传学方法，以阐明 一些调节蛋白。 其他方法包括荧光 和圆二色性测量，化学和酶 蛋白水解，合成肽的应用，分析超离心 和电子显微镜。 生物物理/生物化学核心包括 蛋白质化学服务，分析超浓缩服务 以及电子显微镜和免疫细胞化学服务。 在 蛋白表达矿石，重组蛋白将产生的 昆虫细胞杆状病毒表达系统。 两个核心都将使用 所有项目。 这项计划的资金将使我们能够继续 我们对平滑肌调节机制的研究 系统

Despite the importance of smooth muscle in health and disease, the details of its operation and regulation remain largely unclear. This renewal application is a continued effort intended to gain more information in this area. The overall goal is to use molecular and cellular methodologies to gain information on the functional roles of various smooth muscle proteins and to enhance our understanding of the regulatory mechanism of smooth muscle contraction. The program contains six projects, two facility cores and an administrative core. Projects I- A deals with the effect of phosphorylation of the myosin structure and on the interaction between acting and myosin. Project I-B is concerned with the mechanism by which myosin light chain kinase is activated by Ca/calmodulin and that by which myosin light chain is recognized by the enzyme. Projects II-A and II-B study the structure and function of caldesmon and calponin, respectively, in the hope that such studies will lead to the elucidation of their roles in the thin filament-based regulation of smooth muscle contraction. Project II-C searches for the regulatory pathway involving the mitogen activated protein kinases in smooth muscle. In Project III the integrated system of the thick and the thin filaments will be examined, and the possible role of tropomyosin in the cooperative properties of smooth muscle thin filament will be explored. All projects, with the exception of II-C and II, use chemical crosslinking and resonance energy transfer as tools to characterize protein-protein interactions, and practically every project uses-directed mutagenesis to produce protein variants. Projects II-A II-C, in particular, will explore genetic approaches to elucidate the functional significance of some of the regulatory proteins. Other methods include fluorescence and circular dichroism measurements, chemical and enzymatic proteolysis, use of synthetic peptides, analytical ultracentrifugation and electron microscopy. The Biophysical/Biochemical Core includes Protein Chemistry service, the Analytical Ultracentrifugation service and the Electron Microscopy and Immunocytochemistry service. In the Protein Expression ore, recombinant proteins will be generated by baculovirus expression system in insect cells. Both cores will be used by all projects. Funding of this program will allow us to continue our investigation on the regulatory mechanisms in the smooth muscle system.