Production of membrane vesicles by extrusion: size distribution, enzyme activity, and orientation of plasma membrane and chloroplast inner-envelope membrane vesicles.
A comparison of plasma membrane vesicles prepared by a freeze/thaw method was made with vesicles prepared by extrusion through a 100-nm polycarbonate filter. Based on ATPase measurements in the presence or absence of detergent, plasma membrane vesicles were approximately 30% right-side-out in freeze/thaw vesicles, whereas vesicles produced by extrusion were approximately 80% right-side-out. Chloroplast inner-envelope membrane vesicles were loaded with a membrane-impermeant, pH-sensitive fluorophore, pyranine, by either freeze/thaw or extrusion techniques. ATP-linked proton transport activity was considerably lower in vesicles prepared by extrusion compared to vesicles prepared by freeze/thaw. However, total ATPase activity measured as ADP release from ATP was equivalent in both preparations of vesicles. These results suggest that the inner-envelope vesicles produced by extrusion were predominantly oriented right-side-out. Inner-envelope vesicles were loaded internally with phosphate to study proton-linked transport of 3-phosphoglycerate. Vesicle acidification by 3-phosphoglycerate addition was similar in both freeze/thaw and extruded vesicle preparations, indicating that metabolite transport by the phosphate translocator is both functional and bidirectional. These results indicate that extrusion can be used as a method to produce proteoliposomes which are competent for transport studies.
Shingles
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Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218-2685, USA.
McCarty
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D000251:Adenosine Triphosphatases / Q000378:metabolism; D001693:Biological Transport, Active; D002458:Cell Fractionation / Q000379:methods*; D002462:Cell Membrane / Q000201:enzymology* / Q000648:ultrastructure*; D002736:Chloroplasts / Q000201:enzymology* / Q000648:ultrastructure*; D007425:Intracellular Membranes / Q000201:enzymology / Q000648:ultrastructure; D010316:Particle Size; D018532:Pisum sativum / Q000201:enzymology / Q000648:ultrastructure; D010710:Phosphates / Q000378:metabolism
D010710:Phosphates; D000251:Adenosine Triphosphatases