CELLULAR MEMBRANES - ISOLATION AND CHARACTERIZATION OF PLASMA AND SMOOTH MEMBRANES OF HELA CELLS

A method is described for preparation of membrane fractions from the 4000g supernatant fluid, obtained from a Dounce homogenate of HeLa cells, using a discontinuous sucrose gradient (20-45% sucrose). Two fractions were obtained, the plasma membranes and the smooth internal membranes that appeared by electron microscopy to be free of nuclei, mitochondria, lysosomes, microsomes, ribosomes, dense bodies, or other recognizable cellular elements. The integrity of the plasma membranes were preserved as shown by electron microscopic examination; ruptured cellular envelopes were seen by phase microscopy. The degree of purity of the plasma membranes was shown by high increase in specific activity of the 5′ nucleotidase over the cellular homogenate of 120 fold. An ATPase, alkaline phosphatase, and phosphodiesterase were also found in the plasma membranes, giving a 30-fold, 21-fold, and 6-fold purification respectively. The 5′ nucleotidase, strongly bound to the plasma membranes, was located exclusively in these membranes. The yield of plasma membranes based on recovery of this enzyme was 40-50%. The absence of mitochondria, microsomes, and smooth membranes from the plasma membrane fraction was revealed by the failure to detect succinic dehydrogenase, esterase, and UDPase, respectively. The plasma membranes were characterized by a high molar ratio of cholesterol/phospholipid (1.05) which is similar to that found in myelin, and erythrocyte stroma. This ratio in the smooth membranes (0.06) illustrates the large difference in composition between these membranes and the plasma membranes. In contrast to the plasma membranes, found as a pellet below the 45% sucrose, the smooth internal membranes were found above the 25% sucrose level. They contained 61% lipid; the plasma membranes had only 29% lipid. Neither fraction contained appreciable amounts of RNA, but both had cholesterol, hexosamine, and sialic acid. The smooth internal membranes had the highest content and specific activity of UDPase, a Golgi enzyme. but the 5′ nucleotidase and ATPase, the plasma membrane enzymes, and succinic dehydrogenase, a mitochondrial enzyme, were not detected. Other enzymes found in the smooth membranes included alkaline phosphatase, esterase, and phosphodiesterase. The amino acid composition of the protein contained in the two membrane fractions was similar; the protein of the smooth internal membranes was characterized by 151 basic amino acid residues per 1000 residues and 221 acidic residues while the plasma membrane protein contained 193 basic and 188 acidic residues per 1000 amino acid residues. The glutamic acid content exceeded the other amino acids in both fractions. © 1968.