Cholesterol oxidase susceptibility of the red cell membrane.
We have used the highly variable and conditional susceptibility of cholesterol oxidase to probe molecular rearrangements in the human red cell membrane. Cholesterol in the intact erythrocyte normally is not a substrate for this enzyme. Susceptibility was induced however, by these pretreatments: mild enrichment in membrane cholesterol, exposure to greater than or equal to 0.03% (3 mM) glutaraldehyde and warming in dilute salt solutions (mu approx. 0.001). Cholesterol reactivity in dilute salt solutions emerged only following a lag of 30 min or more. The lag time was shortened by raising the temperature, by reducing the salt concentration or by treating with glutaraldehyde. The induced sensitivity to the enzyme was inhibited by restoring physiologic ionic strength or by introducing 0.1 mol lysophosphatidylcholine per mol cholesterol into the membrane. (In striking contrast, lysophosphatidylethanolamine and lysophosphatidylserine did not inhibit oxidation). The various effectors of cholesterol oxidase sensitivity strongly influenced the impact of the others, suggesting that each shifted cholesterol toward or away from an enzyme-sensitive disposition. None of these effects was observed in pure cholesterol or red cell membrane lipids dissolved in detergent, which were uniformly highly reactive with the enzyme. We conclude that the observed variation in cholesterol oxidase sensitivity reflects changes in the organization of the bilayer, perhaps a lateral redistribution of lipids which creates cholesterol-rich phases or domains in which cholesterol is more or less accessible to the enzyme. If so, the time-dependent increase in cholesterol susceptibility during warming at low ionic strength might be a novel indicator of the kinetics of phase changes in the bilayer of the red cell.
D015096:3-Hydroxysteroid Dehydrogenases / Q000097:blood*; D002784:Cholesterol / Q000097:blood; D002789:Cholesterol Oxidase / Q000097:blood*; D004910:Erythrocyte Membrane / Q000378:metabolism*; D005976:Glutaral / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008244:Lysophosphatidylcholines / Q000494:pharmacology; D008563:Membrane Lipids / Q000378:metabolism; D009994:Osmolar Concentration
D008244:Lysophosphatidylcholines; D008563:Membrane Lipids; D002784:Cholesterol; D015096:3-Hydroxysteroid Dehydrogenases; D002789:Cholesterol Oxidase; D005976:Glutaral