Phosphoprotein phosphatase activity of sea urchin spermatozoa.
Phosphoprotein phosphatase activity of sea urchin spermatozoa.
复制标题
海胆精子的磷蛋白磷酸酶活性。
DOI：
10.1095/biolreprod26.5.953
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发表时间：
1982
影响因子：
3.6
通讯作者：
Garbers,DL
中科院分区：
文献类型：
--
作者：
Swarup,G;Garbers,DL
海胆的精子中含有能够使磷酸组蛋白和磷酸化酶脱磷酸化的磷酸蛋白磷酸酶。磷酸组蛋白磷酸酶通过DEAE Sephacel、组蛋白- Sepharose和高压液体凝胶渗透色谱法纯化约400倍，达到每分钟每mg蛋白质形成0.3µmol磷酸盐的比活性。这种形式的表观分子量超过300，000，但它可以通过用有机溶剂处理转化为35，000 Mr形式。该酶不类似于来自肝脏或肠的碱性磷酸酶，因为磷酸-Ser-组蛋白比磷酸-Tyr-组蛋白是更有效的底物。当精子匀浆的上清液首先用丙酮处理以提取磷酸酶活性时，随后的层析导致几乎均一的35，000 Mr蛋白磷酸酶，并且没有较大分子量的形式。高度纯化的低分子量磷酸酶的比活性为每分钟每mg蛋白质形成4.0µmol磷酸盐。它被氟化物（50 mM）、锌（0.2 mM）和焦磷酸盐（2 mM）强烈抑制（>85%）。一种富含岩藻糖硫酸盐的因子，从S. purpuratuseggs（F-SP）引起完整精子中蛋白磷酸酶活性增加约3倍;释放的活性量约占总细胞磷酸酶活性的4%。细胞外Ca 2+所需的F-SP效应的一半最大响应在约4 mM。paclicin，一种离子载体，已知诱导精子顶体反应，也引起明显的释放，或暴露，磷蛋白磷酸酶活性在完整的精子，而speract，一种肽，已知刺激精子的呼吸，未能增加蛋白磷酸酶活性。这些结果表明，海胆精子含有磷蛋白磷酸酶活性类似于在许多脊椎动物细胞中发现的，这种活性的至少一部分可能驻留在精子顶体区域。
Spermatozoa of the sea urchinStrongylocentrotus purpuratuswere shown to contain phosphoprotein phosphatases capable of dephosphorylating phosphohistones and phosphorylasea. The phosphohistone phosphatase was purified approximately 400-fold by DEAE Sephacel, histone- Sepharose and high-pressure liquid-gel permeation chromatography to a specific activity of 0.3µmol phosphate formed per min per mg of protein. The apparent molecular weight of this form was in excess of 300,000, but it could be converted to a 35,000Mrform by treatment with organic solvents. The enzyme did not resemble alkaline phosphatases from liver or intestine since phospho-Ser-histones were more effective substrates than phospho-Tyr-histones. When the supernatant fluid from the sperm homogenate was first treated with acetone to extract phosphatase activity, subsequent chromatography resulted in a nearly homogeneous, 35,000Mrprotein phosphatase and none of the larger molecular weight form. The specific activity of the highly purified, low molecular weight phosphatase was 4.0µmol phosphate formed per min per mg of protein. It was strongly inhibited (>85%) by fluoride (50 mM), zinc (0.2 mM) and pyrophosphate (2 mM). A fucose sulfate rich factor obtained from the jelly coat ofS. purpuratuseggs (F-SP) caused approximately 3-fold increases in protein phosphatase activity in intact spermatozoa; the amount of released activity represented about 4% of the total cell phosphatase activity. Extracellular Ca2+was required for the F-SP effect with half maximal responses at approximately 4 mM. Nigericin, an ionophore known to induce the sperm acrosome reaction, also caused apparent release of, or exposure of, phosphoprotein phosphatase activity in intact spermatozoa, while speract, a peptide known to stimulate the respiration of spermatozoa, failed to increase protein phosphatase activity. These results suggest that sea urchin spermatozoa contain phosphoprotein phosphatase activity similar to that found in many vertebrate cells and at least a part of this activity may reside in the sperm acrosomal region.