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Ozone was applied to sealed red cell ghost membranes at the rate of 95 nmol/min for periods up to 20 min. Acetylcholine esterase, on the outer face of the membrane, was inhibited up to 20%. Glyceraldehyde-3-phosphate dehydrogenase, on the inner surface of the membrane, was inhibited up to 87%. These differences reflected the inherent susceptibilities of the two enzymes and the presence or absence of the membrane barrier. Analysis of the total lipids of the ozone-treated ghosts showed no significant change in the distribution of lipid classes and no significant change in the fatty acid composition. There was no significant change in the fatty acid composition of the phosphatidylcholine fraction. There was a slight increase in 18:0 and 20:2 + 20:3 in the phosphatidylethanolamine fraction. There was no change in the molecular species distribution of the phosphatidylcholine or the phosphatidylethanolamine fraction. There was no evidence for the formation of the phospholipid ozonolysis product, 1-acyl-2-(9-oxo-nonanyl) derivatives of glyceryl-phosphoryl choline. There was no decline in the amount of cholesterol in the lipids derived from ozone-treated red cell membranes. Treatment of red cell ghost membranes and, by implication, the plasma membrane of cells by ozone therefore oxidizes peripheral proteins before it oxidizes lipids.

Ozone is a widespread component of polluted air. It is the cause of many adverse effects on the lung such as decreased athletic performance and exacerbation of asthma. Ozone inactivated acetylcholine esterase (AChE) both in intact washed human erythrocytes and in ghosts prepared from the erythrocytes. This is consistent (a) with the location of AChE on the outer face of the membrane and (b) with the change in structure of AChE when amino acids were oxidized. The glyceraldehyde-3-phosphate dehydrogenase (G3PDH) of intact washed erythrocytes was unaffected by ozone. However, ozone severely inactivated G3PDH of ghosts, much more severely than AChE in ghosts. This result raised questions about the relative permeability of intact erythrocytes and ghosts and also about the inherent susceptibility of the two enzymes. Inhibition of the ozone-treated erythrocyte AChE with the competitive inhibitor trimethyl-(p-aminophenyl) ammonium chloride was measured. The inhibited enzyme had a higher K(M) and slightly lower Vmax than the control. Ozone did not affect the K(M) of the uninhibited enzyme but decreased the K(M) of the inhibited enzyme. Ozone decreased the Vmax of both the inhibited and the uninhibited enzyme. The K(I) was unchanged by the treatment with ozone. This suggested that the active site of the enzyme was not affected by ozone, but other features of the protein were changed by ozone. The effects of products of lipid ozonolysis [hydrogen peroxide, nonanal, and 1-palmitoyl-2-(9-oxononanyl)-sn-3-glycerophosphorylcholine (PN1PC)] were tested on the ghost preparations. The ozonolysis products were tested at concentrations equivalent to calculated amounts that could have been produced by ozone. Hydrogen peroxide had no effect on the G3PDH and AChE. Nonanal slightly increased the permeability of the ghost membrane, as judged by the increase in rate of G3PDH in the absence of Triton X-100, but did not inhibit enzyme activity. PN1PC increased the permeability of the ghosts, as judged by the increase in rate of G3PDH in the absence of Triton X-100. There was also an increase in the activity of G3PDH in the presence of Triton X-100. AChE was not inhibited by ozone in the presence or absence of Triton X-100.