Dey C S, Majumder G C
Indian Institute of Chemical Biology, Calcutta.
Biochem Cell Biol. 1990 Feb;68(2):459-70. doi: 10.1139/o90-065.
Several lines of evidence have demonstrated conclusively the presence of cAMP-dependent protein kinase (ecto-RC) activity on the external surface of goat cauda-epididymal intact spermatozoa. The intact-cell ecto-kinase that caused transfer of the terminal phosphate of exogenous ATP to the serine and threonine residues of exogenous histone was specifically activated by cAMP. As well, the ecto-kinase caused phosphorylation of the synthetic peptide Kemptide. The isolated spermatozoa, before or after incubation with reaction mixture for ecto-kinase assays, were approximately 99.5% viable, as shown by the analyses of ethidium bromide fluorescence and the cytosolic marker enzymes lactic dehydrogenase and 3-phosphoglycerate kinase. The ecto-kinase activity was not due to contamination of epididymal plasma and damaged cells or to protein kinase that may have leaked from the cells. There was little uptake of ATP and histone by the cells. The intact-cell kinase activity was strongly (80-90%) inhibited by treatment with membrane nonpenetrating surface probes: p-chloromercuriphenylsulfonic acid (2 microM), diazonium salt of sulfanilic acid (DSS, 0.5 mM), and proteases such as trypsin, chymotrypsin, and pronase (each 125 micrograms/mL). Disruption of sperm plasma membrane by sonication or Triton X-100 (0.2%) caused about a fivefold increase of the intact sperm kinase activity. Highly purified sperm plasma membrane (PM) possessed ecto-kinase activity that was resolved into type I and II kinases by DEAE-cellulose chromatography, the type I isoenzyme being the major (approximately 70%) enzymic species. Treatment of the intact spermatozoa with DSS prior to isolation of PM caused a marked loss of the activities of both the isoenzymes, indicating thereby the "ecto" nature of the PM-bound type I and II kinases. Preparations of vigorously forward-motile spermatozoa with 100% intactness had approximately fourfold higher specific activity of the ecto-kinase than the "composite" cells from which the former cells were isolated. However, the profiles of the type I and II ecto-kinases of the composite, as well as forward-motile spermatozoa, were nearly identical. The data are consistent with the view that ecto-kinases may have role in the regulation of flagellar motility.
多条证据已确凿证明，在山羊附睾尾完整精子的外表面存在环磷酸腺苷（cAMP）依赖性蛋白激酶（ecto - RC）活性。这种完整细胞外激酶能将外源性ATP的末端磷酸基团转移至外源性组蛋白的丝氨酸和苏氨酸残基上，且能被cAMP特异性激活。此外，该外激酶还能使合成肽肯普肽（Kemptide）发生磷酸化。通过溴化乙锭荧光分析以及胞质标记酶乳酸脱氢酶和3 - 磷酸甘油酸激酶分析表明，在用于外激酶活性检测的反应混合物中孵育之前或之后的分离精子，其活力约为99.5%。外激酶活性并非由于附睾血浆和受损细胞的污染，也不是由于可能从细胞中泄漏的蛋白激酶所致。细胞对ATP和组蛋白的摄取很少。用膜非穿透性表面探针处理可强烈抑制（80 - 90%）完整细胞激酶活性，这些探针包括对氯汞苯磺酸（2 microM）、磺胺酸重氮盐（DSS，0.5 mM）以及蛋白酶如胰蛋白酶、糜蛋白酶和链霉蛋白酶（各125微克/毫升）。通过超声处理或用0.2%的 Triton X - 100破坏精子质膜，可使完整精子激酶活性增加约五倍。高度纯化的精子质膜（PM）具有外激酶活性，通过DEAE - 纤维素色谱法可将其解析为I型和II型激酶，I型同工酶是主要的（约70%）酶种类。在分离PM之前用DSS处理完整精子，会导致两种同工酶的活性显著丧失，从而表明与PM结合的I型和II型激酶具有“外”的性质。活力旺盛、完整性为100%的向前游动精子制剂的外激酶比分离出前者的“混合”细胞具有约四倍高的比活性。然而，混合细胞以及向前游动精子的I型和II型外激酶谱几乎相同。这些数据与外激酶可能在鞭毛运动调节中起作用的观点一致。
