Abstract
Shearing of ghosts in a French pressure cell produces three classes of microvesicles that differ from endocytic vacuoles, exocytic vacuoles, and insideโout vesicles. It was thought that an analysis of these vesicles might provide some clues about the assembly of proteins within the human erythrocyte membrane. The microvesicles were separated into three visible bands, labeled top, middle, and bottom, and assayed for activity of Mg^++^โATPase, Na^+^, K^+^โATPase, acetylcholinesterase, glyceraldehydeโphosphate dehydrogense, and NADH oxidoreductase. Their proteins were also characterized by polyacrylamide gel electrophoresis with both Coomassie blue staining, to assess total protein content and distribution, and PASโstaining, to characterize sialoglycopeptides. In order to minimize problems inherent in ghost preparation, Dodge or hypotonic ghosts and glycol or isotonic ghosts were used in all studies. Middle membrane vesicles most resembled intact ghosts. Top vesicles had reduced levels of NADH oxidoreductase and more PASโ2 at the expense of PASโ1. The bottom vesicle class was very much enriched with PASโ1 at the expense of PASโ2, and PASโ3 was completely absent. In addition bottom vesicles had highest NADH oxidoreductase activity but lowest activity of all the other enzymes measured. These vesicle classes could not have been produced by tangential shearing through the membrane, nor could radial shearing through a membrane in which all proteins were free to move laterally have accounted for the three discrete vesicle classes or for their different patterns of enzymes and proteins. The analysis of the microvesicles produced by shearing is most consistent with radial shearing through membranes where there may be fixed domains superimposed on the basic fluidโmosaic structure.