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 μm in diameter with a rough or particulate surface similar to that of spheroplasts. The Tris vesicles are significantly smaller, 0.1–0.3 μm 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 rates 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.