Determination of the Absolute Number of Escherichia coli Membrane Vesicles That Catalyze Active Transport

Abstract

Transport of vinylglycolate (2-hydroxy-3-butenoic acid) via the lactate transport system is the limiting step for covalent labeling of membrane vesicles prepared from E. coli ML 308-225. Thus, the rate and extent of vinylglycolate labeling is stimulated about 10-fold by ascorbate-phenazine methosulfate, and stimulation is abolished by 2,4-dinitrophenol and by phospholipase treatment, neither of which affect the rate of vinylglycolate oxidation. [³H]Vinylglycolate of high specific activity has been prepared, and vesicles have been labeled with this compound in the presence of ascorbate-phenazine methosulfate. Examination of these preparations by high resolution radioautography in the electron microscope demonstrates that virtually all of the vesicles are labeled. The experiments provide a strong indication that most, if not all, of the membrane vesicles in these preparations catalyze active transport.