The reactivity of human erythrocyte membrane cholesterol with a cholesterol oxidase

Cholesterol oxidase (EC 1.1.3.6, Brevibacterium sp.), which catalyzes the reaction: cholesterol + O2 leads to delta4-cholestenone + H2O2, has no effect on the cholesterol of intact (human) erythrocytes and of "resealed" ghosts, when it is present only outside these ghosts. The cholesterol of "leaky" ghosts, of "resealed" ghosts with enzyme trapped within, and of "inside-out" vesicles, was completely oxidized. This pattern indicates that the inner ((cytoplasmic) membrane surface must be exposed to the enzyme for the reaction to occur, and that outer surface cholesterol only becomes reactive after the membrane has been degraded by the oxidation of inner surface cholesterol. The enzymatic oxidations followed monotonic first-order kinetics, and hence gave no evidence to support the two states of cholesterol in the membrane that had been postulated earlier from studies on the plasma lipoprotein extraction of cholesterol from the membrane.