BIOCHEMICAL MECHANISMS OF SMOOTH MUSCLE CONTRACTION

在平滑肌中，肌球蛋白轻链(P-轻链)的磷酸化 钙和钙调蛋白依赖的肌球蛋白轻链激酶是 肌动蛋白激活对肌球蛋白镁-ATPase活性的影响。因此，肌球蛋白 磷酸化可能是平滑肌收缩所必需的。这个 本申请中描述的研究的长期目标是 测定肌球蛋白磷酸化系统的调节特性 并评价P-轻链磷酸化与蛋白表达的关系。 牛的力学性能(力、最大缩短速度) 气管和冠状动脉。可逆超渗透将是 用于引入肌球蛋白轻链激酶的特定抑制剂或 钙调素非依赖性肌球蛋白轻链激酶进入平滑肌细胞 从而影响P-轻链的磷酸化。蛋白激酶C的参与 肌球蛋白P-轻链的磷酸化与机械调节 还将对物业进行评估。生化研究将扩展到 培养的血管平滑肌细胞。肌醇1，4，5-三磷酸可能是第二个 药理激动剂的信使，动员体内储存 Ca2+。因此，P-轻链磷酸化的动力学性质 将测量与肌醇1，4，5-三磷酸的形成有关的 以及肌浆内钙离子浓度。递减的相对作用 肌浆钙浓度与肌球蛋白轻链激酶的关系 磷酸化是环核苷酸抑制的生化机制 对P-轻链磷酸化的影响进行分析。 猪肌球蛋白轻链激酶同工酶的生化性质 肌腱和牛气管的平滑肌肉将与 骨骼肌同工酶的性质。功能域中的 将通过有限的蛋白分解和鉴定特异性的蛋白来探测激酶 结合钙调蛋白、三磷酸腺苷类似物和单抗的多肽。这个 纯化的哺乳动物肌球蛋白轻链的催化性质 将用合成肽底物分析激酶以定义氨基 初级序列中可能解释标记的酸性决定因素 底物专一性。这些调查将提供关于 血管平滑肌肌球蛋白轻链激酶的生化特性 将与其他钙调蛋白依赖的酶和其他蛋白质相关 激活剂。

In smooth muscle, phosphorylation of myosin light chain (P-light chain) by Ca+-and calmodulin-dependent myosin light chain kinase is obligatory for actin activation of myosin Mg2+-ATPase activity. Thus, myosin phosphorylation may be essential for smooth muscle contraction. The long-range goals of the research described in this application are to determine the regulatory properties of the myosin phosphorylation system and to evaluate the relationships between P-light chain phosphorylation and mechanical performance (force, maximum shortening velocity) in bovine trachealis and coronary arteries. Reversible hyperpermeabilization will be used to introduce specific inhibitors of myosin light chain kinase or calmodulin-independent myosin light chain kinase into smooth muscle cells to affect P-light chain phosphorylation. Involvement of protein kinase C in myosin P-light chain phosphorylation and regulation of mechanical properties will also be assessed. Biochemical studies will be extended to cultured smooth muscle cells. Inositol 1,4,5-triphosphate may be a second messenger for pharmacological agonists that mobilize internal stores of ca2+. Therefore, the kinetic properties of P-light chain phosphorylation will be measured in relation to formation of inositol 1,4,5-triphosphate and to sarcoplasmic Ca2+ concentrations. The relative roles of decreased sarcoplasmic Ca2+ concentrations vs. myosin light chain kinase phosphorylation as biochemical mechanisms for cyclic nucleotide inhibiton of P-light chain phosphorylation will be analyzed. The biochemical properties of myosin light chain kinase isozymes from gizzard and bovine tracheal smooth muscles will be compared to the properties of the skeletal muscle isozymes. Functional domains within a kinase will be probed by limited proteolysis and identification of specific peptides that bind calmodulin, ATP analogs, and monoclonal antibodies. The catalytic properties of purified mammalian smooth muscle myosin light chain kinase will be analyzed with synthetic peptide substrates to define amino acid determinants in the primary sequence that may account for marked substrate specificity. These investigations will provide information on the biochemical properties of smooth muscle myosin ligh chain kinases which will be related to other calmodulin-dependent enzymes and other protein kinases.