Open AccessDetermination of the Absolute Number of Escherichia coli Membrane Vesicles That Catalyze Active Transport
Author(s) -
Steven A. Short,
H. Ronald Kaback,
Gregory J. Kaczorowski,
Joan M. Fisher,
Christopher T. Walsh,
Samuel C. Silverstein
Publication year - 1974
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 5.011
H-Index - 771
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.71.12.5032
Subject(s) - vesicle , chemistry , membrane , dinitrophenol , biochemistry , membrane transport , electron transport chain , chromatography , valinomycin , escherichia coli , biophysics , biology , gene
Transport of vinylglycolate (2-hydroxy-3-butenoic acid) via the lactate transport system is the limiting step for covalent labeling of membrane vesicles prepared fromE. 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. [3 H]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.