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The transbilayer cholesterol distribution of human erythrocytes was examined by two independent techniques, quenching of dehydroergosterol fluorescence and fluorescence photobleaching of NBD-cholesterol. Dehydroergosterol in conjunction with leaflet selective quenching showed that, at equilibrium, 75% of the sterol was localized to the inner leaflet of(More)
A relatively rapid transbilayer motion of phospholipids in the microsomal membrane seems to be required due to their asymmetric synthesis in the cytoplasmic leaflet. Marked discrepancies exist with regard to the rate and specificity of this flip-flop process. To reinvestigate this problem, we have used both spin-labeled and radioactively labeled long chain(More)
The outside-inside translocation rate and transmembrane equilibrium distribution, at 37 degrees C, of 16 different amphiphilic spin-labeled phospholipids have been determined in human erythrocytes. The transmembrane distribution was assessed by bovine serum albumin extraction of the spin-labels present in the outer monolayer. Within 15 min, more than 90% of(More)
The fluorescent phospholipid 1-acyl-2-[12-(7-nitrobenz-2-oxa-1,3-diazol-4- yl)aminododecanoyl]phosphatidylcholine (NBD-phosphatidylcholine) and the corresponding aminophospholipid derivatives (NBD-phosphatidylethanolamine and NBD-phosphatidylserine) were introduced in the human erythrocyte membrane by a nonspecific phospholipid exchange protein purified(More)
A Mg2(+)-ATPase-enriched fraction was obtained from solubilized human erythrocyte membranes by ammonium sulphate precipitation and anion-exchange chromatography. The solubilized enzyme, of apparent molecular weight 120 kDa, requires phosphatidylserine to be fully active. Phosphatidylethanolamine but not other anionic phospholipids can only partially restore(More)
We have investigated by electron spin resonance, at 37 degrees C, the outside-inside passage and the equilibrium distribution of spin-labeled phospholipids, respectively, in ATP-containing ghosts, in heat-treated erythrocytes, and in heat-induced vesicles. The heat-treated vesicles were spectrin depleted to approximately 25% of the original content and had(More)
The lipid bilayer of a membrane is sometimes seen as an inert hydrophobic phase allowing the 'solubility' of transmembrane proteins and acting as a barrier between two compartments. However, the bilayer is, in fact, a highly organized system subjected to many movements leading to a dynamically equilibrated structure. A lipid within a membrane experiences(More)
The aminophospholipid translocase is a plasma membrane Mg2(+)-ATPase which selectively pumps the aminophospholipids (phosphatidylserine and phosphatidylethanolamine) from the outer to the inner monolayer in eukaryotic cells and is predominantly responsible for the asymmetric phospholipid distribution of the plasma membrane. Similar ATP-dependent transport(More)
We have used ESR and NMR linewidth broadening by spin-labels to determine the overall orientation of spin-labeled analogues of cholesterol and androstanol in egg lecithin bilayers. While the cholesterol analogues were found to have a single orientation in each monolayer, with the acyl chain pointing towards the center of the bilayer, the androstanol(More)
We have used a spin label analog of cholesterol bearing a nitroxide on the alkyl chain (26-nor-25-doxylcholestanol) to study cholesterol-protein interactions in the human erythrocyte membrane. As judged from the ESR spectrum, the spin label is readily incorporated into the membrane when added from a concentrated ethanolic solution to a cell or ghost(More)
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