Sugar Transport

The Enzymes II of a bacterial phosphotransferase system are sugar-specific membrane components, most of which are inducible. The present studies are concerned with the constitutive Enzymes II of Escherichia coli. In the presence of purified Enzyme I and HPr of the phosphotransferase system, an E. coli membrane fraction catalyzed the transfer of phosphate from phosphoenolpyruvate to d-glucose, d-mannose, and d-fructose. Extraction of the membrane fraction with a mixture of urea and 1-butanol gave a protein fraction (II-A), which catalyzed the reaction only in the presence of the residual pellet. Fraction II-A was purified free of lipid, and subfractionated by isoelectric focusing to three proteins, each being required for phosphorylation of one of the sugars. The pellet was treated with deoxycholate and fractionated, giving another protein fraction (II-B) which was purified free of lipid. The combination of II-A and II-B was inactive in the phosphorylation reaction unless supplemented with divalent cation and with crude E. coli lipid. A minor component of crude E. coli lipid, phosphatidylglycerol, was found to be the active lipid factor. Thus, reconstitution of Enzyme II activity, specific for one of the three sugars, was effected by combining the corresponding specific II-A with II-B, phosphatidylglycerol, and either Ca++ or Mg++. However, activity was dependent upon the sequence of mixing of the four components. The activity of reconstituted Enzyme II was found in a sedimentable pellet. Further study of this phenomenon showed that II-B, phosphatidylglycerol, and divalent cation interacted to give a sedimentable pellet which catalyzed the phosphorylation of the sugars in the presence of II-A and the phospho-HPr generating system. Activity of the pellet was studied as a function of the concentration of phosphatidylglycerol added to the mixture, and the optimum was reached when all of the lipid sedimented with the protein; additional quantities of lipid gave less active pellets. At the optimum, the pellet contained II-B and lipid in a ratio of 1:1 (w/w). Each of the II-A proteins appeared homogeneous when examined by disc gel electrophoresis. Protein II-B, which showed a marked tendency to aggregate in the absence of detergent, was studied by electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulfate, and only one protein band was detected, its mobility corresponding to a molecular weight of about 36,000. If II-B is homogeneous it represents at least 10% of the protein in the membrane preparation.