Abstract
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.
Highlights
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The PM fractionwas examined by electron microscopy and found to consist primarfiy of single compartment membrane vesicles, 3000-5000 8, in diameter
The PM formwesre modified by sialic acid, fraction, while 22% was in the PM fraction
Summary
The PM fractionwas examined by electron microscopy and found to consist primarfiy of single compartment membrane vesicles, 3000-5000 8, in diameter. The membrane pellet from the sucrose density purification step exhibited glucose-6-phosphataseactivity as expected from the original characterization of the pellet as ER by Crumpton 8z Snary (1974) (Table I). This membrane pellet exhibited little contamination by 5"nucleotidase (EC 3.1.3.5) activity, an enzyme characteristic ofPM. The sucrose density interface fraction, in contrast, exhibited a relatively high 5'-nucleotidase activity indicative of PM (Table I). This interface fraction, enriched in PM, contained some glucose-6-phosphatase activity characteristic of contamination by ER membranes. The best fit to that line by linear regressions is used to generate the indicated curves and thecalculated equivalents required for 50%inhibition of cytotoxicity
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