PROTEIN PHOSPHORYLATION IN SPERM FLAGELLAR MOTILITY

蛋白磷酸化在精子鞭毛调控中的作用 将研究cAMP和钙的运动性。 一种热稳定的NP-40可溶性 56，000 Mr蛋白，其cAMP依赖性磷酸化是 鞭毛能动性已被鉴定。 此外，钙-钙调蛋白 似乎在精子膜水平上调节鞭毛运动 它控制细胞内的钙水平。 的具体目标， 下一个预算期的项目是：（1）纯化和提高抗体， 56，000 Mr磷蛋白和膜钙调素结合蛋白; (2)确定56，000 Mr的作用部位和机制 磷蛋白在鞭毛运动调节中的作用及其变化 蛋白质的磷酸化状态影响其功能;（3） 研究这种蛋白质与时间的关系， 精子发生过程中的合成和磷酸化， 在精子成熟过程中获得活力;（4）鉴定和确定 精子膜钙调素结合蛋白在精子凋亡中的作用 鞭毛运动的调节以及这些蛋白的磷酸化是否 蛋白质影响其钙调素依赖性和功能;（5）探索 其他cAMP依赖性和非依赖性磷蛋白的可能作用， 钙调素结合蛋白在cAMP和钙依赖性调节 鞭毛运动，并将这些蛋白质与建议的 钙对运动的钙调素非依赖性抑制。 主要 实现这些具体目标的实验方法包括使用 用（α-32 P）ATP再活化洗涤剂渗透的精子。 具体 鞭毛运动的参数将通过添加 并通过视频图像定量 分析. 蛋白质磷酸化将在相同条件下测量。 高分辨二维聚丙烯酰胺凝胶电泳实验条件 电泳、放射自显影和数字图像处理。 结果 获得的信息应有助于更好地了解基本监管 参与轴丝运动控制的机制和鉴定 以轴丝改变为代表的潜在疾病状态的可能异常 能动性

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.