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...

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Bibliographic Details
Published in:Analytical biochemistry 1995-07, Vol.229 (1), p.92-98
Main Authors: Shingles, R., Mccarty, R.E.
Format: Article
Language:English
Subjects:
Adenosine Triphosphatases - metabolism
Biological Transport, Active
Cell Fractionation - methods
Cell Membrane - enzymology
Cell Membrane - ultrastructure
Chloroplasts - enzymology
Chloroplasts - ultrastructure
Intracellular Membranes - enzymology
Intracellular Membranes - ultrastructure
Particle Size
Phosphates - metabolism
Pisum sativum - enzymology
Pisum sativum - ultrastructure
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Summary: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.
ISSN:0003-2697
1096-0309
DOI:10.1006/abio.1995.1383