Sugar Transport

作者：Werner Kundig, Saul Roseman · 发表于：Journal of Biological Chemistry · 年份：1971 · DOI：10.1016/s0021-9258(19)76987-7 · 被引用次数：288 · 研究领域：Amino Acid Enzymes and Metabolism、Metabolism and Genetic Disorders、Enzyme Structure and Function

Abstract 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...