Determination of cholesterol asymmetry by rapid kinetics of filipin-cholesterol association: effect of modification in lipids and proteins

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Abstract
The rapid kinetic behavior of filipin association with cholesterol was unaffected by binding of water-soluble proteins to vesicles and mycoplasma membranes and by proteolytic digestion of mycoplasma membrane proteins. The kinetic properties were, however, dependent on the membrane phospholipids, in that the initial rate of filipin association with cholesterol was enhanced by phospholipase A2 treatment, by the incorporation of lysophosphatidylcholine, and by increasing the degree of unsaturation in phospholipid vesicles and mycoplasma membranes. The 2nd-order rate constant was also dependent on the mole percent of cholesterol in small unilamellar vesicles but not in large unilamellar vesicles. The ratio of rate constants in intact mycoplasma cells relative to isolated membranes provides an estimate of cholesterol distribution in membranes. This ratio was unaffected by proteolytic digestion of intact cells and by the incorporation of exogenous phospholipids into the Mycoplasma capricolum cell membrane. On cross-linking of surface proteins of M. capricolum by dimethylsuberimidate, cholesterol was localized predominantly in the outer half of the bilayer. On aging of mycoplasma cultures, the cholesterol distribution remained constant in membranes of M. capricolum cells but was enriched in the outer leaflet of the M. gallisepticum cell membrane. The results of these experiments are discussed in relation to the use of the rapid kinetics of filipin binding as a probe of cholesterol distribution.