Boylan M H, Edmondson D E
Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322.
Biochem J. 1990 Jun 15;268(3):745-9. doi: 10.1042/bj2680745.
Previous studies have shown the flavodoxin from Azotobacter vinelandii (strain OP, Berkeley) to contain a covalently bound disubstituted phosphate residue [Edmondson & James (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 3786-3789]. Phosphorylation of the protein in vivo was investigated by the addition of [32P]phosphate to cells grown under N2-fixing conditions, under conditions of nif-gene repression and under conditions of nif-gene de-repression. Rocket immunoelectrophoresis of cell extracts showed an approx. 5-fold decrease in the concentration of flavodoxin expressed in cells grown in the presence of NH4+ as compared with those grown under N2-fixing conditions. A similar increase in flavodoxin concentration was observed on nif-gene de-repression. Incorporation of [32P]phosphate occurs only into newly synthesized flavodoxin, as observed on SDS/PAGE of immunoprecipitates of cell extracts. Western blots demonstrated no observable precursor forms of flavodoxin. These data provide conclusive evidence for the phosphorylation of Azotobacter strain OP flavodoxin in vivo and suggest that the covalently bound phosphate residue does not exchange with cellular phosphate pools. Thus the role of this phosphodiester cross-link is proposed to be structural rather than regulatory.
先前的研究表明，来自棕色固氮菌（菌株OP，伯克利）的黄素氧还蛋白含有一个共价结合的二取代磷酸残基[埃德蒙森和詹姆斯（1979年）《美国国家科学院院刊》76, 3786 - 3789]。通过向在固氮条件下生长的细胞、在固氮基因阻遏条件下生长的细胞以及在固氮基因去阻遏条件下生长的细胞中添加[³²P]磷酸盐，对该蛋白在体内的磷酸化进行了研究。细胞提取物的火箭免疫电泳显示，与在固氮条件下生长的细胞相比，在NH₄⁺存在下生长的细胞中表达的黄素氧还蛋白浓度大约降低了5倍。在固氮基因去阻遏时观察到黄素氧还蛋白浓度有类似的增加。如在细胞提取物免疫沉淀物的SDS/聚丙烯酰胺凝胶电泳上所观察到的，[³²P]磷酸盐仅掺入新合成的黄素氧还蛋白中。蛋白质免疫印迹法显示未观察到黄素氧还蛋白的前体形式。这些数据为棕色固氮菌菌株OP黄素氧还蛋白在体内的磷酸化提供了确凿证据，并表明共价结合的磷酸残基不会与细胞内的磷酸盐池进行交换。因此，这种磷酸二酯交联的作用被认为是结构性的而非调节性的。
