Waygood E B, Mattoo R L, Peri K G
J Cell Biochem. 1984;25(3):139-59. doi: 10.1002/jcb.240250304.
Phosphoproteins produced by the incubation of crude extracts of Salmonella typhimurium and Escherichia coli with either [32P]phosphoenolpyruvate or [gamma 32P]ATP have been resolved and detected using sodium dodecyl sulphate polyacrylamide gel electrophoresis and autoradiography. Simple techniques were found such that distinctions could be made between phosphoproteins containing acid-labile or stable phosphoamino acids and between N1-P-histidine and N3-P-histidine. Phosphoproteins were found to be primarily formed from phosphoenolpyruvate, but because of an efficient phosphoexchange, ATP also led to the formation of the major phosphoenolpyruvate-dependent phosphoproteins. These proteins had the following apparent subunit molecular weights: 65,000, 65,000, 62,000, 48,000, 40,000, 33,000, 25,000, 20,000, 14,000, 13,000, 9,000, 8,000. Major ATP-dependent phosphoproteins were detected with apparent subunit molecular weights of 75,000, 46,000, 30,000, and 15,000. Other minor phosphoproteins were detected. The phosphorylation of the 48,000- and 25,000-MW proteins by phosphoenolpyruvate was independent of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The PTS phosphoproteins were identified as enzyme I (soluble; MW = 65,000); enzyme IIN-acetylglucosamine (membrane bound; MW = 65,000); enzyme IImannitol (membrane bound; MW = 62,000); IIIfructose (soluble; MW = 40,000); IIImannose (partially membrane associated; MW = 33,000); IIIglucose (soluble; MW = 20,000); IIIglucitol (soluble; MW = 13-14,000); HPr (soluble; MW = 9,000); FPr (fructose induced HPr-like protein (soluble; MW = 8,000). HPr and FPr are phosphorylated on the N-1 position of a histidyl residue while all the others appear to be phosphorylated on an N-3 position of a histidyl residue. These studies identify some previously unknown proteins of the PTS and show the phosphorylation of others, which although previously known, had not been shown to be phosphoproteins.
将鼠伤寒沙门氏菌和大肠杆菌的粗提物与[32P]磷酸烯醇丙酮酸或[γ32P]ATP一起温育所产生的磷蛋白，已通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳和放射自显影进行了分离和检测。发现了一些简单的技术，可区分含有酸不稳定或稳定磷酸氨基酸的磷蛋白，以及N1-P-组氨酸和N3-P-组氨酸。发现磷蛋白主要由磷酸烯醇丙酮酸形成，但由于有效的磷酸交换，ATP也导致了主要的磷酸烯醇丙酮酸依赖性磷蛋白的形成。这些蛋白具有以下表观亚基分子量：65,000、65,000、62,000、48,000、40,000、33,000、25,000、20,000、14,000、13,000、9,000、8,000。检测到主要的ATP依赖性磷蛋白，其表观亚基分子量为75,000、46,000、30,000和15,000。还检测到了其他一些次要的磷蛋白。磷酸烯醇丙酮酸对48,000和25,000分子量蛋白的磷酸化不依赖于磷酸烯醇丙酮酸：糖磷酸转移酶系统（PTS）。PTS磷蛋白被鉴定为酶I（可溶性；分子量= 65,000）；N-乙酰葡糖胺酶II（膜结合；分子量= 65,000）；甘露醇酶II（膜结合；分子量= 62,000）；果糖酶III（可溶性；分子量= 40,000）；甘露糖酶III（部分与膜相关；分子量= 33,000）；葡萄糖酶III（可溶性；分子量= 20,000）；葡糖醇酶III（可溶性；分子量= 13 - 14,000）；HPr（可溶性；分子量= 9,000）；FPr（果糖诱导的HPr样蛋白（可溶性；分子量= 8,000）。HPr和FPr在组氨酸残基的N-1位被磷酸化，而其他所有蛋白似乎在组氨酸残基的N-3位被磷酸化。这些研究鉴定了一些以前未知的PTS蛋白，并显示了其他一些蛋白的磷酸化，这些蛋白虽然以前已知，但尚未被证明是磷蛋白。
