Saha A K, Dowling J N, Mukhopadhyay N K, Glew R H
Department of Microbiology, Biochemistry and Molecular Biology, University of Pittsburgh School of Medicine, Pennsylvania 15261.
J Bacteriol. 1989 Sep;171(9):5103-10. doi: 10.1128/jb.171.9.5103-5110.1989.
Legionella micdadei, a pathogen which enters into host phagocyte phagolysosomal structures, contains at least two protein kinases. We have purified to homogeneity the predominant, nucleotide-independent protein kinase and examined its ability to catalyze the transfer of phosphate from ATP to acceptors in human neutrophils. The L. micdadei protein kinase catalyzed the phosphorylation of proteins of 11.5, 14, 19, 23, 28, 34, and 38 kilodaltons (kDa) present in a Triton X-100 extract of neutrophil membranes and of 11.5, 13.5, 25, and 38 kDa in the neutrophil cytosol. Tubulin was a good substrate for the L. micdadei protein kinase in vitro. The bacterial kinase also catalyzed the phosphorylation of phosphatidylinositol (PI) at about half the rate at which histones were phosphorylated; phosphatidylinositol-4-phosphate (PIP) was not phosphorylated by the kinase. The PI kinase activity of the L. micdadei enzyme was optimum at pH 7.0, and the divalent cation requirement was satisfied best by Mg2+ and Ca2+. The maximum rate of PI phosphorylation was obtained with 0.6 mM PI; in the presence of MgCl2 (10 mM), the Km for PI was 0.9 mM and the Km for ATP was 1.5 mM. The detergents octyl-beta-D-glucoside (10 to 20 mM) and Triton X-100 (0.5%) stimulated kinase activity twofold when PI was the phosphate acceptor; however, only octyl glucoside stimulated histone kinase activity. Various membrane phospholipids inhibited PI kinase activity. The most potent phospholipid inhibitor was the product of the PI kinase reaction, PIP, which at a 0.6 mM concentration inhibited both PI and tubulin phosphorylation by 80%. The inhibition of kinase activity by PIP when histone served as the acceptor was noncompetitive in character. The L. micdadei kinase also phosphorylated PI in intact. (3H)inositol-labeled neutrophils. The PI kinase and histone kinase activities of teh L. micdadei kinase copurified and cofucused (pI, 5.8) when subjected to isoelectric focusing, suggesting that the two enzymatic activities reside in a single protein.
米德戴军团菌是一种能进入宿主吞噬细胞吞噬溶酶体结构的病原体，它至少含有两种蛋白激酶。我们已将主要的、不依赖核苷酸的蛋白激酶纯化至同质，并检测了其催化磷酸基团从ATP转移至人中性粒细胞中受体的能力。米德戴军团菌蛋白激酶催化了中性粒细胞膜的Triton X-100提取物中11.5、14、19、23、28、34和38千道尔顿（kDa）蛋白质以及中性粒细胞胞质溶胶中11.5、13.5、25和38 kDa蛋白质的磷酸化。微管蛋白在体外是米德戴军团菌蛋白激酶的良好底物。该细菌激酶还能催化磷脂酰肌醇（PI）的磷酸化，其速率约为组蛋白磷酸化速率的一半；磷脂酰肌醇-4-磷酸（PIP）不能被该激酶磷酸化。米德戴军团菌酶的PI激酶活性在pH 7.0时最佳，二价阳离子需求以Mg2+和Ca2+满足得最好。PI磷酸化的最大速率在PI浓度为0.6 mM时获得；在存在MgCl2（10 mM）的情况下，PI的Km为0.9 mM，ATP的Km为1.5 mM。当PI作为磷酸受体时，去污剂辛基-β-D-葡萄糖苷（10至20 mM）和Triton X-100（0.5%）可使激酶活性提高两倍；然而，只有辛基葡萄糖苷能刺激组蛋白激酶活性。各种膜磷脂抑制PI激酶活性。最有效的磷脂抑制剂是PI激酶反应的产物PIP，其浓度为0.6 mM时可抑制PI和微管蛋白磷酸化达80%。当组蛋白作为受体时，PIP对激酶活性的抑制具有非竞争性。米德戴军团菌激酶还能使完整的、用（3H）肌醇标记的中性粒细胞中的PI磷酸化。当进行等电聚焦时，米德戴军团菌激酶的PI激酶和组蛋白激酶活性共纯化且共聚焦（pI，5.8），这表明这两种酶活性存在于单一蛋白质中。
