Plasma membrane heterogeneity in ascites tumor cells. Isolation of a light and a heavy membrane fraction of the glycogen-free Ehrlich-Lettré substrain

Abstract

In this work we report on the isolation of two plasma membrane fractions of a glycogen-free substrain of Ehrlich-Lettré ascites cells, a light fraction sedimenting in a sucrose gradient at 1.10 g/ml, and a heavy fraction sedimenting at 1.16 g/ml. 95% of the cells were broken without any significant damage to the nuclei by a combination of short-term swelling and mild Dounce homogenization. A 12000 × g postnuclear pellet (PII) containing major portions of the plasma membrane marker enzymes, 5′-nucleotidase, ouabain-sensitive (Na + + K +)-ATPase and the alkaline phosphatase, was prepared by differential centrifugation. The two plasma membrane frations were obtained by centrifugation on a discontinuous sucrose gradient, from which they were further purified on a linear sucrose gradient applying sedimentation velocity conditions only. Enrichment factors for the three marker enzymes were between 5- and 14-fold for the light fraction and between 3- and 7-fold for the heavy fraction with an overall yield of 1–4% and 0.5–1.7%, respectively, of cellular protein. Contamination of both fractions with nuclear material was minor. Mitochondrial contamination was about 8% for the light material and somewhat higher for the heavy material. In the light fraction, co-sedimentation of lysosomal and Golgi marker enzymes was detected. The presence of membrane structures of these organelles could not be confirmed definitely by electron microscopy. Differences in sialic acid content and phospholipid composition within the two fractions, especially in the relative proportion of lecithin to sphingomyelin, suggests differences in membrane fluidity. The light material showed mostly unit membrane vesicles in thin-section and freeze-etch electron microscopy, whereas the heavy fraction mainly consisted of sheet-like membrane fragments.