Phosphorylation of a 15 K axonemal protein is the trigger initiating trout sperm motility
Phosphorylation of a 15 K axonemal protein is the trigger initiating trout sperm motility
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15 K 轴丝蛋白的磷酸化是引发鳟鱼精子活力的触发器
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发表时间：
1985
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影响因子：
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通讯作者：
H. Hayashi
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作者：
M. Morisawa;H. Hayashi
用TritonX-100处理鳟鱼精子，去除质膜后与[γ-32 P]ATP孵育，用SDS-聚丙烯酰胺凝胶电泳分析精子蛋白的磷酸化。当去膜精子的运动被cAMP激活时，15 K蛋白在1秒内被强烈磷酸化，这一时间段与精子运动的正常启动相一致。当精子首先被允许游泳，然后去膜，cAMP依赖的磷酸化的15 K蛋白没有观察到。结果表明，15 K蛋白，完全磷酸化在1秒内cAMP的存在下，触发精子运动的启动。我们以前证明，鲑鱼精子脱膜与Triton X-100保持静止的ATP单独存在下，但被激活的额外存在的cAMP。这表明cAMP是启动Sahnonidae精子运动的主要因素（9，10，13）。还有一个问题，在任何物种中都没有得到澄清，那就是cAMP磷酸化的蛋白质触发了精子运动的启动。为了阐明这一点，我们研究了HP与精子蛋白的结合。我们的研究结果表明，磷酸化的15 K蛋白是触发启动精子活力。在含有非离子去污剂Triton X-100的提取培养基中除去从虹鳟萨尔莫盖尔德内里收集的精子的质膜（9），将脱膜的精子重悬并在含有ATP和[y-32 P]ATP的再活化培养基中孵育1或5秒，其中存在或不存在cAMP。采用十二烷基硫酸钠（SDS）聚丙烯酰胺凝胶电泳（6）和放射自显影（7）检测无机磷酸盐（Pi）掺入精子蛋白。在再活化培养基中不存在cAMP的情况下，当通过相差显微镜检查时，未观察到轴丝运动（也参见9，10，13）。在此条件下，Pi轻微掺入分子量为15 K、20 K、24 K、31 K、38 K、42 K和45 K的7种蛋白质中（图1a）。当cAMP存在于再活化培养基中时，运动开始（另见9，10，13），并且在15 K蛋白中，在1秒内发生Pi掺入的伴随显著增加。cAMP依赖性磷酸化也见于42 K和45 K蛋白中，但不在20 K、24 K、31 K和38 K蛋白中（图1b）。在缺乏cAMP的情况下，与暴露1秒相比，轴丝暴露于[y-32 P]ATP 5秒并没有明显增加15 K、42 K或45 K蛋白中的Pi掺入（图1，a和c）。在15 K、42 K或45 K蛋白中暴露5秒后，在cAMP存在下的Pi掺入比在其不存在下大得多。然而，在存在或不存在cAMP的情况下，5秒的暴露没有增加20 K、24 K、31 K和38 K蛋白中的Pi掺入（图1，c和d）。当切下对应于15 K、42 K和45 K蛋白质的凝胶带时，在182 M液体中测定其放射性。Morisawa和H. Hayashi
The plasma membrane of trout sperm was removed by the treatment with Triton X-100 and phosphorylation of sperm protein was analyzed by SDS-polyacrylamide gel electrophoresis after incubating the demembranated spermwith [y-32P]ATP. When the motility ofdemembranated sperm was initiated by addition ofcAMP, a 15 K protein was strongly phosphorylated within 1 sec, a time period compatible with the normal initiation of sperm motility. When sperm were first allowed to swim and then demembranated, cAMP-dependent phosphorylation of the 15 K protein was not observed. The results suggest that the 15 K protein, completely phosphorylated in 1 sec in the presence of cAMP, triggers the initiation of sperm motility. We previously demonstrated that salmonid sperm demembranated with Triton X-100 remained quiescent in the presence of ATP alone but were activated by the additional presence of cAMP. This suggests that cAMP is the primary factor for the initiation ofsperm motility in Sahnonidae (9, 10, 13). A further question, which has not been clarified in any species, is which protein phosphorylated by cAMP triggers the initiation ofsperm motility. To clarify this, we have studied the incorporation ofHP into sperm proteins. Our results suggest that phosphorylation of a 15 K protein is the trigger to initiate sperm motility. The plasma membranes of spermatozoa collected from rainbow trout Salmo gairdneri were removed in an extracting medium containing the nonionic detergent Triton X-100 (9), and the demembranated sperm were resuspended and incubated for 1 or 5 sec in the reactivating medium containing ATP and [y-32P]ATP in the presence or absence of cAMP. Incorporation of inorganic phosphate (Pi) into sperm proteins was detected using sodium dodecyl sulfate (SDS)polyacrylamide gel electrophoresis (6) and autoradiography (7). In the absence of cAMP in the reactivating medium, axonemal movement was not observed when this was checked by phase contrast microscopy (see also 9, 10, 13). Under this condition, Pi was slightly incorporated into seven proteins with molecular weights of 15 K, 20 K, 24 K, 31 K, 38 K, 42 K and 45 K (Fig. la). When cAMP was present in the reactivating medium, motility was initiated (see also 9, 10, 13) and a concomitant marked increase in Pi incorporation occurred within 1 sec in a 15 K protein. The cAMP-dependent phosphorylation was also seen in 42 K and 45 K proteins, but not in 20 K, 24 K, 31 K and 38 K proteins (Fig. lb). In the absence of cAMP, exposure of the axoneme to [y-32P]ATP for 5 sec did not appreciably increase the Pi incorporation in 15 K, 42 K or 45 K proteins, compared with 1-sec exposure (Fig. 1, a and c). Pi incorporation following 5-sec exposure in the 15 K, 42 K or 45 K protein was much greater in the presence of cAMP than in its absence. However, 5-sec exposure did not increase the Pi incorporation in 20 K, 24 K, 31 K and 38 K proteins in either the presence or absence of cAMP (Fig. 1, c and d). When the gel bands corresponding to the 15 K, 42 K and 45 K proteins were cut out, and their radioactivity was determined in a liquid 182 M. MORISAWA and H. HAYASHI