Physical separation and biochemical characterization of H-2b-encoded proteins on target cell plasma membranes and...

Plasma membranes and endoplasmic reticulum were purified on the basis of their physical properties, and their identity was confirmed by membrane marker activities. Comparative adsorption analysis indicated that plasma membrane and endoplasmic reticulum vesicle fractions adsorbed 25% and 5%, respectively, as much anti-D6 alloantiserum as intact cells. Two-dimensional gel analyses of immunoprecipitates of the H-2b-encoded proteins from the EL4 tumor cell line indicated a series of proteins containing complex carbohydrates in the plasma membrane and two additional N-asparagine-linked proteins in the endoplasmic reticulum. Both neuraminidase and protein phosphatase treatments were required to convert the mobilities of the plasma membrane forms of the H-2Kb and H-2Db antigens in two-dimensional gel electrophoresis to the mobilities of the endoplasmic reticulum forms. The endoplasmic reticulum form of H-2Db was shown to contain three N-asparagine-linked carbohydrates by endoglycosidase H treatment.