Isolation and characterization of mel cell plasma membranes
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Plasma membranes can be isolated from Friend erythroleukemia cells strain GM86 in high yield and purity, with out resorting to ultra high speed centrifugation. The isolation procedure involves (1) coating intact washed cells with Conconavalin A (Con A) to protect against subsequent membrane fragmentation and vesiculation after cell lysis thereby avoiding intracelluar constituent entrapment (2) separating Con A coated plasma membranes from other celluar constituents by low speed centrifugation and (3) converting open plasma membrane sheets into vesicles by removing more than 90% of the Con A with methyl-D-mannopyranoside.The DNA content of isolated plasma membranes sheets is less than 20% of that found in whole cells on a mg/mg protein basis; DNA content of vesicles is below the level of detection. RNA is not detectable in plasma membrane sheets or vesicles. Plasma membranes have a high sterol to phospholipid ratio characteristic of mammalian plasma membranes; however this ratio is uncharacteristically high in whole cells compared to eucaryotic cells in general. Membrane preparations were enriched 19 fold in Mg dependent ATPase, 19 fold in (Na^+,K^+) stimulated Mg ATPase, 35 fold in (Ca^2+, Mg^2+) calmodulin stimulated ATPase and 6 fold in 5'nucleotidase activities. Succinate dehydrogenase, a mitochondrial marker, was present in cells, but below the level of detection in plasma membrane preperations. Calcium transport experiments show that vesicles export Ca^2+ energized, thus compared to the direction of Ca^2+ movement in cells, vesicles are largely 'right side' out. Of all energy sources tested-adenosine triphosphate (ATP), acetate, D-glucose, pyruvate, lactate, phosphoenol pyruvate (PEP), succinate, NADPH (nicotinamide-adenine dinucleotide phosphate), NADH (nicotinamide-adenine dinucleotide), guanosine triphosphate (GTP)-only ATP was effective in energizing 45Ca export from vesicles. Experiments with CCCP (carbonyl cyanide chlorophenol hydrozone), nigerisin, valinomycin, DMO (5,5-dimethyloxazolidine-2,4-dione) and TPP (tetraphenylphosphonium bromide) show that at pH 7.5 [delta][psi] is the driving force for the maintance of vesicular Ca^2+ concentration.
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