Characterization of microvesicles produced by shearing of human erythrocyte membranes

Abstract

Human erythrocyte membranes were sheared into microvesicles with substantial loss of activity of the ATPases but not the nitrophenyl phosphatases. This loss of ATPase activity was not accompanied by alteration in lipid content, sialic acid content or orientation, or sulfhydryl group reactivity. Centrifugation in a linear sucrose gradient, resolved the microsvesicles into distinct classes which differed with respect to their lipid content, the reactivity of their sulfhydryl groups, and the activity of their ATPases and nitrophenyl phosphatases. Vesicles with low sulfhydryl reactivity had low activity of all enzymes tested. Vesicles containing relatively high lipid to protein and lipid to sialic acid ratios had low ATPase and K +-dependent nitrophenyl phosphatase activities. We have found that membranes could be sheared into readily separable membrane classes containing varying amounts of lipid relative to sialic acid and protein, and this observation supports a segmental concept of membrane structure. The parallelism of (Na +, K +)-ATPase and K +-dependent nitrophenylphosphatase activities in the separated microvesicle classes stands in contrast to their divergent behavior following shearing where the K +-dependent nitrophenyl phosphatase is not inactivated. This pattern suggested that K +-dependent nitrophenyl phosphatase probably accurately reflected the K +-dependent dephosphorylation phase of (Na +, K +)-ATPase; however, the prior step required a higher degree of structural integrity and was very susceptible to inactivation by chemical or mechanical perturbation.