Platt M W, Rottem S, Milner Y, Barile M F, Peterkofsky A, Reizer J
Department of Membrane and Ultrastructure Research, Hebrew University-Hadassah Medical School, Jerusalem, Israel.
Eur J Biochem. 1988 Sep 1;176(1):61-7. doi: 10.1111/j.1432-1033.1988.tb14251.x.
Incubation of the soluble fraction derived from Mycoplasma gallisepticum cells with [gamma-32P]ATP results in the phosphorylation of several endogenous proteins. One protein with an apparent molecular mass of 55 kDa was the acceptor of more than 95% of the radioactive phosphate. This protein was also found to be radiolabeled in intact cells grown in the presence of [32P]orthophosphate. Acid hydrolysis of the phosphorylated 55-kDa protein followed by two-dimensional electrophoresis revealed that the 32P-labeled material co-migrated with phosphoserine. The in vitro phosphorylation of the 55-kDa protein has an optimum pH of 5.5-6.0 and is not affected by various metabolites of glycolysis, by cAMP or by calmodulin with or without Ca2+. The phosphorylation is dependent upon divalent cations, a dependency that is best fulfilled by the simultaneous addition of Ca2+ and Zn2+ that act in a specific and cooperative manner. Of a variety of possible exogenous protein acceptors tested, the endogenous protein kinase was capable to phosphorylate only phosvitin. The phosphorylation of the 55-kDa protein is reversible through the activity of a phosphoprotein phosphatase present in the soluble fraction of M. gallisepticum. The phosphoprotein phosphatase has an optimum pH of 7.5-8.0, is inhibited by NaF and stimulated to a large extent by inorganic phosphate and arsenate and to a lesser extent by pyrophosphate ATP and ADP. The possible association of the reversible protein phosphorylation to cell shape and gliding motility of M. gallisepticum are discussed.
将源自鸡毒支原体细胞的可溶性组分与[γ-32P]ATP一起温育，会导致几种内源性蛋白质发生磷酸化。一种表观分子量为55 kDa的蛋白质接受了超过95%的放射性磷酸盐。在存在[32P]正磷酸盐的情况下生长的完整细胞中也发现该蛋白质被放射性标记。对磷酸化的55 kDa蛋白质进行酸水解，然后进行二维电泳，结果显示32P标记的物质与磷酸丝氨酸共迁移。55 kDa蛋白质的体外磷酸化的最适pH为5.5 - 6.0，不受糖酵解的各种代谢产物、cAMP或有无Ca2+时的钙调蛋白的影响。磷酸化依赖于二价阳离子，同时添加以特定协同方式起作用的Ca2+和Zn2+能最好地满足这种依赖性。在测试的多种可能的外源蛋白质受体中，内源性蛋白激酶仅能使卵黄高磷蛋白磷酸化。55 kDa蛋白质的磷酸化可通过鸡毒支原体可溶性组分中存在的一种磷蛋白磷酸酶的活性逆转。该磷蛋白磷酸酶的最适pH为7.5 - 8.0，受NaF抑制，在很大程度上受无机磷酸盐和砷酸盐刺激，在较小程度上受焦磷酸盐、ATP和ADP刺激。讨论了可逆蛋白质磷酸化与鸡毒支原体细胞形状和滑行运动的可能关联。
