Phosphoproteins and the phosphoenolpyruvate: Sugar phosphotransferase system in salmonella typhimurium and escherichia coli: Evidence for IIIMannose, IIIFructose, IIIGlucitol, and the phosphorylation of enzyme IIMannitol and enzyme IIN‐acetylglucosamine

Abstract

Phosphoproteins produced by the incubation of crude extracts of Salmonella typhimurium and Escherichia coli with either [³²P]phosphoenolpyruvate or [γ³²P]ATP have been resolved and detected using sodium dodecyl sulphate poly‐acrylamide 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 phos‐phoenolpyruvate was independent of the phosphoenolpyruvate:sugar phospho‐transferase system (PTS). The PTS phosphoproteins were identified as enzyme I (soluble; MW = 65,000); enzyme II^{N‐acetylglucosamine} (membrane bound; MW = 65,000); enzyme II^mannitol (membrane bound; MW = 62,000); III^fructose (soluble; MW = 40,000); III^mannose (partially membrane associated; MW = 33,000); III^glucose (soluble; MW = 20,000); III^glucitol (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.