Effect of oxidation on Ca2+-ATPase activity and membrane lipids in lens epithelial microsomes

Membrane oxidation may contribute to cataractogenesis. In our pursuit to un derstand the etiology of cataracts, we assessed the effect of membrane oxid ation products on the activity of the lens epithelium calcium pump. Microso me preparations from bovine lens epithelium were oxidized to varying degree s with a ferrous and ferric ascorbate system to generate hydrogen peroxide and superoxide. Ca2+-ATPase activity was measured using a colorometric assa y. Lipid oxidation was quantified by infrared spectroscopy. Ca2+-ATPase act ivity decreased as a function of ascorbate concentration between 0 and 200 mu M. The level of Ca2+-ATPase inhibition was correlated to both the level of lipid oxidation and the degree of lipid hydrocarbon chain order. At 25 d egrees C when lipids are more ordered, the Ca2+-ATPase activity was similar to that observed in the oxidized system measured at 37 degrees C. Glutathi one, mercaptoethanol, and iodoacetate were able to reverse the oxidative in hibition of the calcium pump, suggesting that the ascorbate/iron oxidant di rectly oxidized the protein sulfhydryl moieties. To further probe the mecha nism of Ca2+-ATPase inhibition, hydrogen peroxide was used to oxidize muscl e sarcoplasmic reticulum Ca2+-ATPase reconstituted in its native lipid vesi cles, egg phosphatidylcholine, and dihydrosphingomyelin, with saturated hyd rocarbon chains. In these systems, oxidation inhibited the Ca2+-ATPase pump by 60-80%. There was no statistical difference between the level of oxidat ive inhibition and the percentage of dihydrosphingomyelin. Because dihydros phingomyelin cannot be oxidized, whereas egg phosphatidylcholine (PC) can, and because the percentage of inhibition was the same for reconstituted sys tems using either lipid, the mechanism of inhibition is likely not via a se condary process involving oxidation-induced lipid structural changes or pro ducts of lipid oxidation. (C) 1999 Elsevier Science Inc.