Isolation and Characterization of Subcellular Membranes

A method is described for the isolation of subcellular membranes of Entamoeba invadens. Plasma membranes were obtained by rate centrifugation followed by isopycnic centrifugation on a sucrose gradient. Intact phagolysosomes floated in a 10% sucrose solution providing a simple technique for isolation. Phagolysosomal membranes were collected by isopycnic centrifugation, after lysis of the phagolysosomes. Microsomes were obtained by differential centrifugation. Membrane fractions were examined by electron microscopy, and the contamination of each fraction was determined with marker enzymes. Mg2+-ATPase is associated with the plasma membrane. Acid phosphatase (/3-glycerophosphate) was associated mainly with phagolysosomal membranes. Plasma membranes also contained acid phosphatase activity which hydrolyzes p-nitrophenylphosphate but not fl-glycerophosphate. The localization of the two phosphatases was confirmed cytochemically. Isolated plasma membranes were contaminated with phagolysosomal membranes (15%) and with microsomes (25%). No more than 5% of the phagolysosomal membrane fraction consisted of plasma membranes. Contamination of the microsomes by plasma and phagolysosomal membranes was 10% and 7%, respectively. Plasma membranes and phagolysosomal membranes had a high ratio of cholesterol to phospholipid (0.93 and 1.05 /xmol/#mol, respectively). Microsomes were relatively poor in cholesterol (0.39 /~mol//zmol). Microsomes, plasma, and phagolysosomal membranes contained increasing amounts of sphingolipids (12%, 17%, and 28%). Phagolysosomal membranes had a high percentage of phosphatidylserine but little phosphatidylcholine. Microsomes were rich in phosphatidylcholine (45%). Differences in phospholipid composition between plasma and phagolysosomal membranes are discussed in view of the phagocytic process. Electron micrographs of Entamoeba reveal the relatively simple structure of these protozoan cells (Fig. 1). The main organelles are the phagolysosomes (31,32) which are scattered throughout the cell. A nucleus is present as well as a Golgi apparatus (9, 30) and endoplasmic reticulum (22, 23, ThE JOURNAL OF CELL BIOLOGY" VOLUME 71, 1976 9 pages 357-369 357 on Jne 4, 2017 D ow nladed fom Published November 1, 1976