Analysis of specific protein phosphorylation after infection with bacteria

当体外培养小鼠腹腔巨噬细胞并感染沙门氏菌、沙门氏菌等细菌时，细胞内出现一组磷酸化蛋白。一些蛋白质，如65 kDa（pp65），对细菌多糖（LPS）是特异性的，而其他几种蛋白质，如85 kDa（pp85）和72 kDa（pp72）对活沙门氏菌感染是特异性的。细胞松弛素D处理能部分抑制吞噬作用，但不能抑制pp85和pp72的磷酸化。当氯霉素或萘啶酸抑制感染沙门氏菌的细胞内生长时，磷酸化不出现。这些结果表明，磷酸化是由于活生物体的细胞内生长，而不是吞噬过程。感染卡介苗、嗜肺军团菌、胞内分枝杆菌、金黄色葡萄球菌、单核细胞增生李斯特菌、铜绿假单胞菌后，pp85和pp72均未发生磷酸化，而感染LPS或热灭活革兰氏阴性菌后，pp85和pp72均发生磷酸化。pp65在巨噬细胞胞浆中占优势。测定了pp65的氨基酸序列。它是丝氨酸激酶的底物，是纤维蛋白酶家族的成员。

When murine peritoneal macrophages were cultured in vitro and infected with bacteria such as Salmonella typhimurium, S.enteritidis and others, a set of phosphorylated proteins appeared inthe cells. Some of the proteins, such as 65 kDa(pp65), are specific to bacterial lipipolysaccharide (LPS), while several others, such as 85 kDa (pp85) and 72 kDa (pp72) are specific to the live Salmonella infection. Cytochalacin D-treatment inhibited partially the phagocytosis oforganisms, but it did not inhibit the phosporylations of pp85 and pp72. When intracellular growth of infected Salmonella was inhibited by chloram-phenicol or nalidixic acid, the phosphorylation did not appear. These findings suggest that the phosphorylation is due to the intracellular growth of live organisms rather than phagocytic process. The phosphorylation of pp85 and pp72 was not observed by the infections with Mycobacterium bovis BCG,Legionella pneumophila, Mycobacterium intracellulare, Stapylococcus aureus, Listeria monocytogenes, Pseudomonas aeruginosa.Many phosphorylated proteins were observed by stimulation with LPS or heat-killed gram negative organisms. pp65 was the most dominant one in cytoplasm of macrophages. Amino acid sequences of pp65 was determined. It was a substrate of serine-kinase and a member of plastin family.