Stock J B, Waygood E B, Meadow N D, Postma P W, Roseman S
J Biol Chem. 1982 Dec 10;257(23):14543-52.
We have previously reported that glucose can be phosphorylated by phospho-HPr and two sugar-specific pairs of proteins of the Escherichia coli and Salmonella typhimurium phosphoenolpyruvate:glycose phosphotransferase system. Each of the sugar-specific complexes comprises two proteins, lipid, and divalent cation, and each is present in membranes isolated from wild type cells. For reasons described in this report, one of the complexes is designated IIGlc and the other IIMan. The IIMan complex has previously been separated into its protein components, II-A and II-B (Kundig, W., and Roseman, S. (1971) J. Biol. Chem. 246, 1407-1418), while the accompanying reports describe dissociation of the IIGlc complex into its components, IIIGlc and II-BGlc. Curtis and Epstein (Curtis, S. J., and Epstein, W. (1975) J. Bacteriol. 122, 1189-1199) first showed that there are two phosphotransferase systems in whole cells responsible for glucose uptake and obtained the respective mutants, now designated ptsG and ptsM. The present studies provide kinetic conditions for assaying each activity separately (in vivo and in vitro), when both are present in the same membrane preparation. The IIGlc system is responsible for the uptake and phosphorylation of glucose and methyl alpha-glucoside, whereas the IIMan system is less specific and utilizes glucose, mannose, and 2-deoxyglucose. With high sugar concentrations in vitro, IIMan is also capable of phosphorylating methyl alpha-glucoside, fructose, and N-acetylmannosamine, while IIGlc phosphorylates fructose and mannose. The in vivo transport results were qualitatively consistent with the in vitro phosphorylation results, and several of the kinetic parameters also showed good quantitative agreement. The levels of the two activities depended on the growth conditions. In addition, transport studies showed that initial uptake rates of methyl alpha-glucoside and steady state levels of this analogue depended on the energy state of the cells and that these two parameters did not necessarily change in the same direction when metabolic inhibitors were used. A series of E. coli and S. typhimurium mutants were characterized both with respect to their ability to transport the glucose analogues and to phosphorylate them in vitro. The original mutants of Curtis and Epstein, ptsG and ptsM, were found to be defective in II-BGlc and the IIMan complex, respectively.
我们之前报道过，葡萄糖可被磷酸化的HPr以及大肠杆菌和鼠伤寒沙门氏菌磷酸烯醇丙酮酸：葡萄糖磷酸转移酶系统中的两对糖特异性蛋白磷酸化。每对糖特异性复合物都由两种蛋白质、脂质和二价阳离子组成，且每种复合物都存在于从野生型细胞中分离出的膜中。基于本报告中所述的原因，其中一种复合物被命名为IIGlc，另一种为IIMan。IIMan复合物先前已被分离为其蛋白质组分II - A和II - B（昆迪格，W.，和罗斯曼，S.（1971年）《生物化学杂志》246卷，1407 - 1418页），而随附的报告描述了IIGlc复合物解离为其组分IIIGlc和II - BGlc。柯蒂斯和爱泼斯坦（柯蒂斯，S. J.，和爱泼斯坦，W.（1975年）《细菌学杂志》122卷，1189 - 1199页）首先表明，全细胞中有两种负责葡萄糖摄取的磷酸转移酶系统，并获得了各自的突变体，现在分别命名为ptsG和ptsM。本研究提供了在同一膜制剂中同时存在这两种活性时分别测定每种活性（体内和体外）的动力学条件。IIGlc系统负责葡萄糖和α - 甲基葡萄糖苷的摄取和磷酸化，而IIMan系统的特异性较低，可利用葡萄糖、甘露糖和2 - 脱氧葡萄糖。在体外高糖浓度下，IIMan也能够磷酸化α - 甲基葡萄糖苷、果糖和N - 乙酰甘露糖胺，而IIGlc可磷酸化果糖和甘露糖。体内转运结果在定性上与体外磷酸化结果一致，并且一些动力学参数在定量上也显示出良好的一致性。这两种活性的水平取决于生长条件。此外，转运研究表明，α - 甲基葡萄糖苷的初始摄取速率和该类似物的稳态水平取决于细胞的能量状态，并且当使用代谢抑制剂时，这两个参数不一定沿相同方向变化。一系列大肠杆菌和鼠伤寒沙门氏菌突变体在转运葡萄糖类似物的能力以及在体外对其进行磷酸化的能力方面进行了表征。柯蒂斯和爱泼斯坦最初的突变体ptsG和ptsM分别被发现存在II - BGlc和IIMan复合物缺陷。
