PROTEIN PHOSPHORYLATION IN SPERM FLAGELLAR MOTILITY

精子鞭毛运动中的蛋白质磷酸化

• 批准号: 3277134
• 负责人: JOSEPH S TASH
• 金额: $ 16.2万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1982
• 资助国家: 美国
• 起止时间: 1982-07-01 至 1989-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3277134
• 关键词: affinity chromatography; autoradiography; calcium; cilium /flagellum motility; computer data analysis; cyclic AMP; enzyme substrate; gel electrophoresis; immunoelectron microscopy; immunofluorescence technique; monoclonal antibody; phosphorylation; radiotracer; sperm motility; spermatogenesis

The role of protein phosphorylation in the regulation of sperm flagellar motility by cAMP and calcium will be studied. A heat-stable NP-40-soluble 56,000 Mr protein whose cAMP-dependent phosphorylation is required for flagellar motility has been identified. In addition, calcium-calmodulin appears to regulate flagellar motility at the level of the sperm membranes where it controls intracellular calcium levels. The specific aims for the next budget period of the project are: (1) Purify and raise antibodies to the 56,000 Mr phosphoprotein and the membrane calmodulin binding proteins; (2) Determine the site and mechanism of action of the 56,000 Mr phosphoprotein in the regulation of flagellar motility and how alterations in the phosphorylation state of the protein affects its function; (3) Investigate the significance of this protein with respect to time of synthesis and phosphorylation during spermatogenesis in relation to the acquisition of motility during sperm maturation; (4) Identify and determine the role of the sperm membrane calmodulin binding proteins in the regulation of flagellar motility and whether the phosphorylation of these proteins affects their calmodulin dependency and function; (5) Explore the possible role of other cAMP-dependent and -independent phosphoproteins and calmodulin binding proteins in the cAMP and calcium-dependent regulation of flagellar motility and relate these proteins to the suggested calmodulin-independent inhibition of motility by calcium. The major experimental approach to pursue these specific aims comprises the use of detergent-permeabilized sperm reactivated with (alpha-32P)ATP. Specific parameters of flagellar motility will be modified by the addition of specific protein and antibody probes and be quantitated by video image analysis. Protein phosphorylation will be measured under identical experimental conditions by high resolution 2-dimensional polyacrylamide gel electrophoresis, autoradiography and digital image processing. The results obtained should yield a greater understanding of the basic regulatory mechanisms involved in the control of axonemal motility and identify possible abberations underlying disease states typified by altered axonemal motility.

蛋白质磷酸化在精子鞭毛调控中的作用 将研究cAMP和钙的运动性。一种耐热的NP-40可溶物 56,000 mR蛋白需要cAMP依赖的磷酸化 鞭毛的运动性已被鉴定。此外，钙-钙调蛋白 似乎在精子膜水平上调节鞭毛的运动 它控制着细胞内的钙水平。该计划的具体目标 该项目的下一个预算期是：(1)提纯和提高抗体 56,000个MR磷酸蛋白和膜钙调素结合蛋白； (2)确定56,000名MR的作用地点和机制 磷蛋白在鞭毛运动调节中的作用及其变化 蛋白质处于磷酸化状态会影响其功能； 研究该蛋白在时间上的意义。 精子发生过程中的合成和磷酸化与 精子成熟过程中运动能力的获得；(4)鉴定和测定 精子膜钙调素结合蛋白在精子死亡中的作用 鞭毛运动的调节以及它们的磷酸化是否 蛋白质影响其钙调蛋白依赖性和功能；(5)探索 其他cAMP依赖性和非依赖性磷酸蛋白的可能作用 CAMP中的钙调素结合蛋白及其钙依赖调节 鞭毛的运动性，并将这些蛋白质与建议的 钙对运动的抑制不依赖于钙调蛋白。少校 实现这些特定目标的实验方法包括使用 (α-~(32)P)-三磷酸腺苷激活了洗涤剂渗透的精子。特定的 鞭毛运动参数将通过添加 特异性蛋白质和抗体探针，并通过视频图像进行定量 分析。蛋白质的磷酸化将在相同的条件下测量 高分辨率二维聚丙烯酰胺凝胶的实验条件 电泳学、放射自显影和数字图像处理。结果是 所获得的信息应能更好地理解基本的监管 参与轴丝运动控制和识别的机制 以轴线改变为典型的疾病状态下可能存在的幻觉 能动性。