Sugar transport by the bacterial phosphotransferase system. Isolation and characterization of a glucose-specific phosphocarrier protein (IIIGlc) from Salmonella typhimurium.

Abstract

The phosphocarrier protein, IIIGlc, of the phosphoenolpyruvate:glycose phosphotransferase system (PTS) was purified to homogeneity by two methods. The first method utilized ion exchange and gel filtration chromatography, isoelectric focusing, and polyacrylamide gel electrophoresis, and required several weeks for completion. The second method utilized and antibody affinity column plus two additional steps and could be completed in a few days. By both procedures, two forms of IIIGlc were isolated, which were called IIIGlc Slow and IIIGlc Fast on the basis of their relative mobilities in polyacrylamide gels. IIIGlc Fast is derived from IIIGlc Slow by cleavage of the seven NH2-terminal amino acids from the latter protein. Both IIIGlc Slow and IIIGlc Fast have Mr approximately 20,000; neither protein contains cysteine, tyrosine, or tryptophan. IIIGlc Slow is very stable to heat; only 50% of its sugar phosphorylating activity is lost after 1 h at 100 degrees C. The phosphoryl group in IIIGlc Slow appears to be linked to a histidinyl residue. Direct transfer of the phosphoryl group from HPr (the histidine-containing phosphocarrier protein of the PTS) to IIIGlc slow was demonstrated as well as the reverse reaction. In addition, phospho-IIIGlc Slow served as a phosphoryl donor to methyl alpha-glucoside (or glucose) in the absence of all other PTS components except the partially purified integral membrane protein specific for this sugar, II-BGlc. The loss of the seven amino acids from IIIGlc Slow (giving IIIGlc Fast) leads to a marked alteration in the kinetic properties of the protein in the phosphotransferase system. IIIGlc Slow accepts 1 mol of phosphate from phosphoenolpyruvate via Enzyme I and HPr (the histidine-containing phosphocarrier protein) and participates in the phosphorylation of glucose or methyl alpha-D-glucoside. IIIGlc Fast also accepts 1 mol of phosphate, but phospho-IIIGlc Fast is only 2-3% as active as phospho-IIIGlc Slow in the phosphorylation of sugar. IIIGlc Fast is found only in trace quantities in living cells, and may play a role in the regulation of non-PTS sugar transport systems.