Isolation of plasma and nuclear membranes of thymocytes. II. Biochemical composition

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Abstract
Thymocyte plasma and nuclear membranes were analyzed for their biochemical composition. Plasma membranes were very rich in phospholipid, cholesterol and sialic acid; they did not contain nucleic acids. Nuclear membranes had a lower phospholipid to protein ratio and contained much less sialic acid and cholesterol. Fifty percent of the cellular cholesterol and of the membrane-bound sialic acid was found in the plasma membranes and 14% in the nuclear membranes. Live cells were labeled with 131I, and the acid-insoluble radioactivity was followed in the subfractions. A good correlation with the distribution and enrichment of plasma membrane marker-enzymes was obtained. Label enrichment was about 50-fold in the 2 lightest of the 3 plasma membrane fractions. Of the label, 60% was contained in the plasma membranes, only 4% in the nuclear membranes. Cross-contamination of these 2 types of membranes was negligible. Sodium dodecyl sulfate-gel electrophoresis revealed 3 different patterns specific for, respectively, plasma membranes, the microsomal-mitochondrial fraction and nuclear membranes. Each pattern was characterized by a set of proteins and glycoproteins, among which high MW glycoproteins could be considered as marker-proteins of, respectively, 280,000, 260,000 and 230,000 daltons. 131I-labeling of live cells tagged (with a very high specific activity) 3 glycoproteins of MW 280,000, 200,000 and 135,000 daltons. Nuclear membranes prepared from labeled isolated nuclei had a set of labeled proteins completely different from plasma membranes.