Preparation of inside-out vesicles of pig lymphocyte plasma membrane

Abstract

Between 30 and 50% of pig lymphocyte plasma membrane vesicles were not bound by concanavalin A(Con A)-Sepharose. Various results suggest that the Con A-unretarded fraction represents inside-out membrane vesicles. An alternative cell surface ligand, anti-lymphocytic serum, gave a similar fractionation to Con A. Lack of binding by Con A was not due to lack of carbohydrate or to masking of carbohydrate by extraneous protein, because the unfractionated membrane and the unretarded fraction had similar carbohydrate and polypeptide compositions. Although the carbohydrate of the unretarded membrane vesicles was accessible to 125I-labeled Con A and to release by soluble trypsin, it was not accessible to ferritin-Con A or trypsin-Sepharose. Antisera against the external surface of the Con A-unretarded vesicles strongly agglutinated the unretarded membrane, but caused negligible agglutination of whole lymphocytes. When attached to Sepharose these antisera bound all of the Con A-unretarded fraction, but failed to bind the membrane that adhered to Con A-Sepharose.