title: Isolation of membrane vesicles with inverted topology by osmotic lysis of Azotobacter vinelandii spheroplasts.
abstract: Membrane vesicles were prepared from Azotobacter vinelandii spheroplasts by lysis in either potassium phosphate (pH 7.0) or Tris1-acetate (pH 7.8) buffers. These 2 types of preparations differ considerably in their properties: 1) Examination by scanning electron microscopy reveals that the Pi vesicles consist primarily of closed structures 0.6-0.8 micrometer in diameter with a rough or particulate surface similar to that of spheroplasts. The Tris vesicles are significantly smaller, 0.1-0.3 micrometer in diameter, and have a much smoother surface structure. 2) Antisera from rabbits immunized with A. vinelandii lipopolysaccharide antigen will agglutinate Pi vesicles but not Tris vesicles. 3) Tris vesicles have a fourfold higher specific activity of latent H+-ATPase than Pi vesicles. After exposure to Triton X-100 similar ATPase activities are observed for both types of vesicles. 4) Pi vesicles transport calcium in the presence of ATP or lactate at less than 30% of the rats observed for Tris vesicles. 5) Tris vesicles have less than 22% of the transport capacity of Pi vesicles for accumulation of labeled sucrose and less than 3% of the capacity for valinomycin-induced uptake of rubidium observed during respiration. 6) Quinacrine fluorescence intensity is reduced by 30% during lactate oxidation and 20% during ATP hydrolysis by Tris vesicles. Under similar conditions, fluorescence in Pi vesicles is quenched by only 7% and less than 2%, respectively. These findings suggest that Pi vesicles have the normal orientation of the intact cell whereas Tris vesicles have an inverted topology.
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000372:Agglutination Tests; D001395:Azotobacter / Q000201:enzymology / Q000648:ultrastructure*; D001693:Biological Transport, Active; D002118:Calcium / Q000378:metabolism; D002458:Cell Fractionation / Q000379:methods; D002462:Cell Membrane / Q000201:enzymology / Q000648:ultrastructure*; D008070:Lipopolysaccharides; D008855:Microscopy, Electron, Scanning; D013104:Spheroplasts / Q000648:ultrastructure*
chemical_list: D008070:Lipopolysaccharides; D000251:Adenosine Triphosphatases; D002118:Calcium