Paupard M C, MacLeod J, Wasco W, Orr G A
Department of Molecular Pharmacology, Atran Foundation Laboratories, Albert Einstein College of Medicine, Bronx, New York 10461.
J Cell Biochem. 1988 Jun;37(2):161-75. doi: 10.1002/jcb.240370204.
It has been shown that cAMP-dependent phosphorylation of a soluble sperm protein is important for the initiation of flagellar motion. The suggestion has been made that this motility initiation protein, named axokinin, is the major 56,000-dalton phosphoprotein present in both dog sperm and in other cells containing axokinin-like activity. Since the regulatory subunit of a type II cAMP-dependent protein kinase is a ubiquitous cAMP-dependent phosphoprotein of similar subunit molecular weight as reported for axokinin, we have addressed the question of how many soluble 56,000-dalton cAMP-dependent phosphoproteins are present in mammalian sperm. We report that in bovine sperm cytosol, the ratio of the type I to type II cAMP-dependent protein kinase is approximately 1:1. The type II regulatory subunit is related to the non-neural form of the enzyme and undergoes a phosphorylation-dependent electrophoretic mobility shift. The apparent subunit molecular weights of the phospho and dephospho forms are 56,000 and 54,000 daltons, respectively. When bovine sperm cytosol or detergent extracts are phosphorylated in the presence of catalytic subunits, two major proteins are phosphorylated and have subunit molecular weights of 56,000 and 40,000 daltons. If, however, the type II regulatory subunit (RII) is quantitatively removed from these extracts using either immobilized cAMP or an anti-RII monoclonal affinity column, the ability to phosphorylate the 56,000- but not 40,000-dalton polypeptide is lost. These data suggest that the major 56,000 dalton cAMP-dependent phosphoprotein present in bovine sperm is the regulatory subunit of a type II cAMP-dependent protein kinase and not the motility initiator protein, axokinin.
已经表明，可溶性精子蛋白的cAMP依赖性磷酸化对于鞭毛运动的启动很重要。有人提出，这种名为轴动素的运动启动蛋白是存在于犬精子和其他具有轴动素样活性的细胞中的主要56,000道尔顿磷蛋白。由于II型cAMP依赖性蛋白激酶的调节亚基是一种普遍存在的cAMP依赖性磷蛋白，其亚基分子量与轴动素报道的相似，我们研究了哺乳动物精子中存在多少种可溶性56,000道尔顿的cAMP依赖性磷蛋白。我们报道，在牛精子胞质溶胶中，I型与II型cAMP依赖性蛋白激酶的比例约为1:1。II型调节亚基与该酶的非神经形式相关，并经历磷酸化依赖性的电泳迁移率变化。磷酸化和去磷酸化形式的表观亚基分子量分别为56,000和54,000道尔顿。当牛精子胞质溶胶或去污剂提取物在催化亚基存在下进行磷酸化时，两种主要蛋白质被磷酸化，其亚基分子量分别为56,000和40,000道尔顿。然而，如果使用固定化cAMP或抗RII单克隆亲和柱从这些提取物中定量去除II型调节亚基（RII），则磷酸化56,000道尔顿多肽而不是40,000道尔顿多肽的能力丧失。这些数据表明，牛精子中存在的主要56,000道尔顿cAMP依赖性磷蛋白是II型cAMP依赖性蛋白激酶的调节亚基，而不是运动启动蛋白轴动素。
