Cytoplasmic Membrane Vesicles of Escherichia coli

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Abstract
Orientation of cytoplasmic membrane vesicles (IM vesicles) of Escherichia coli K12 prepared by the method of Yamato, Anraku and Hirosawa ( (1975) J. Biochem. 77, 705–718) was studied by determining uptake activities for calcium ion and proline and by estimating localization of certain membrane marker enzymes. The IM vesicles showed respiration-driven uptake activities of Ca2+ and proline. Calcium ion was taken up by the vesicles with NADH but the NADH-dependent Ca2+ uptake was not inhibited by D-lactate. Effects of NADH oxidation on the active, D-Iactate-dependent proline uptake was found to be additive. These findings suggested strongly that orientation of IM vesicles was heterogeneous and that the IM preparation contained right-side out and inverted vesicles but no mosaic vesicles. About 60% of the D-lactate and g]ycerol-3-phosphate oxidase activities were inhibited by the antibody against D-lactate dehydrogenase [EC 1.1.1.28] and ferricyanide, respectively. About 70% of the glycerol-3-phosphate-ferricyanide reductase was detectable without tolueniza-tion. D-Lactate- and glycerol-3-phosphate-dependent proline uptakes were inhibited by 40%, by the antibody and ferricyanide, respectively. From these data and the available criteria for orientation, the populations of IM vesicles with different orientations were estimated: 70% of the total population were right-side out closed vesicles and 30% were inverted vesicles or unsealed membrane fragments. In addition, about 40% of the D-lactate dehydrogenase of the right-side out closed vesicles was suggested to be dislocated from the original location and found on the outside.