Electrophoretic studies of the protein constituents of pig spleen lymphocyte plasma membrane and of a non-ionic detergent extract of intact cells

Abstract

This report describes a study of the protein constituents of pig spleen lymphocyte plasma membrane, separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and analyzed under reducing and non-reducing conditions. The electrophoretic patterns of purified plasma membranes, of the endoplasmic reticulum fraction and of a non-ionic detergent (Nonidet P-40) extract of intact lymphocytes are compared. The lymphocytes were ruptured by the nitrogen cavitation method and the plasma membranes were purified by differential centrifugation in sucrose gradients. Plasma membranes were enriched in marker enzymes and appeared, in electron micrographs, as vesicles of various sizes and as fragmented membranes. The protein constituents were resolved into more than 60 bands which appeared, except in the endoplasmic reticulum fraction, as discrete Coomassie-positive bands. Characteristic bands were present in each of the three fractions when samples were not reduced. However, the majority of Coomassie-stained proteins migrated with the same relative mobility in all three fractions. Interestingly, all Coomassie-positive bands stained (some weakly) for glycoproteins. When samples were reduced and alkylated prior to electrophoresis, the electrophoretogram of each fraction differed from the non-reduced samples. In general, we observed that the fastest migrating portion of each gel was more intensively stained, when compared to non-reduced extracts. In addition, some bands characteristic of each fraction were evident, although most bands were common to each fraction. Coomassie-positive bands also stained with a glycoprotein staining reagent. Activity of alkaline phosphatase was demonstrated in the electrophoretograms of the non-reduced plasma membrane extracts. The results suggest that each of the three fractions analyzed under the conditions described here possesses a characteristic glycoprotein content. Furthermore, the data show that a Nonidet P-40 extract of lymphocytes is effective in solubilizing the glycoproteins from pig lymphocyte plasma membranes. Comparison of electrophoretic patterns with those reported for lymphocytes of other lymphoid organs of the pig suggests close similarities.