Bookbinder L H, Moy G W, Vacquier V D
Marine Biology Research Division, Scripps Institution of Oceanography, University of California, San Diego, La Jolla 92093.
Mol Reprod Dev. 1991 Feb;28(2):150-7. doi: 10.1002/mrd.1080280208.
Addition of [gamma -32P]ATP to a 2% Brij-78 40,000g supernatant of sea urchin sperm results in the cAMP-dependent phosphorylation of eight to ten proteins. One phosphoprotein of Mr 190 kD is sperm adenylate cyclase (AC). An antiserum to the AC immunoprecipitates the Mr 190 kD protein. Peptide maps of immunoprecipitates show that the AC is the only phosphoprotein present in the Mr 200 kD range. With respect to the in vitro phosphorylation of AC, the endogenous kinase has a Km for ATP of 5.2 microM and is maximally stimulated by 4-8 microM cAMP. The protein kinase inhibitors H8 (9 microM) and PKI (30 U/ml) inhibit the phosphorylation of the AC. The catalytic subunit of bovine cAMP-dependent protein kinase phosphorylates the AC on the same peptides as the endogenous protein kinase. Cyanogen bromide generated peptide maps of the phosphorylated AC show a minimum of five sites of phosphorylation. No change in the Km or Vmax of the sperm AC resulted from the additional phosphorylation by bovine kinase. Calcium ions at submicromolar concentrations completely block the in vitro phosphorylation of the AC, suggesting the presence in the preparation of a Ca2(+) -activated protein phosphatase. To our knowledge, this is the first report of the phosphorylation of an AC by cAMP-dependent protein kinase.
向海胆精子的2% Brij - 78 40,000g上清液中添加[γ - 32P]ATP会导致8至10种蛋白质发生cAMP依赖性磷酸化。一种分子量为190 kD的磷蛋白是精子腺苷酸环化酶（AC）。针对AC的抗血清可免疫沉淀分子量为190 kD的蛋白质。免疫沉淀物的肽图谱表明，AC是分子量在200 kD范围内唯一存在的磷蛋白。就AC的体外磷酸化而言，内源性激酶对ATP的Km为5.2 microM，在4 - 8 microM cAMP的刺激下达到最大活性。蛋白激酶抑制剂H8（9 microM）和PKI（30 U/ml）可抑制AC的磷酸化。牛cAMP依赖性蛋白激酶的催化亚基与内源性蛋白激酶在相同的肽段上使AC发生磷酸化。溴化氰生成的磷酸化AC的肽图谱显示至少有五个磷酸化位点。牛激酶的额外磷酸化并未导致精子AC的Km或Vmax发生变化。亚微摩尔浓度的钙离子完全阻断了AC的体外磷酸化，这表明制剂中存在一种Ca2(+)激活的蛋白磷酸酶。据我们所知，这是关于cAMP依赖性蛋白激酶使AC发生磷酸化的首次报道。
