Effect of oxidation on Ca2+ -ATPase activity and membrane lipids in lens epithelial microsomes.
Membrane oxidation may contribute to cataractogenesis. In our pursuit to understand the etiology of cataracts, we assessed the effect of membrane oxidation products on the activity of the lens epithelium calcium pump. Microsome preparations from bovine lens epithelium were oxidized to varying degrees with a ferrous and ferric ascorbate system to generate hydrogen peroxide and superoxide. Ca2+ -ATPase activity was measured using a colorometric assay. Lipid oxidation was quantified by infrared spectroscopy. Ca2+ -ATPase activity decreased as a function of ascorbate concentration between 0 and 200 microM. 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 degrees C when lipids are more ordered, the Ca2+ -ATPase activity was similar to that observed in the oxidized system measured at 37 degrees C. Glutathione, mercaptoethanol, and iodoacetate were able to reverse the oxidative inhibition of the calcium pump, suggesting that the ascorbate/iron oxidant directly oxidized the protein sulfhydryl moieties. To further probe the mechanism of Ca2+ ATPase inhibition, hydrogen peroxide was used to oxidize muscle sarcoplasmic reticulum Ca2+ -ATPase reconstituted in its native lipid vesicles, egg phosphatidylcholine, and dihydrosphingomyelin, with saturated hydrocarbon chains. In these systems, oxidation inhibited the Ca2+ -ATPase pump by 60-80%. There was no statistical difference between the level of oxidative inhibition and the percentage of dihydrosphingomyelin. Because dihydrosphingomyelin cannot be oxidized, whereas egg phosphatidylcholine (PC) can, and because the percentage of inhibition was the same for reconstituted systems using either lipid, the mechanism of inhibition is likely not via a secondary process involving oxidation-induced lipid structural changes or products of lipid oxidation.
Ahuja
R P
RP
Department of Biochemistry, University of Louisville, KY, USA.
Borchman
D
D
Dean
W L
WL
Paterson
C A
CA
Zeng
J
J
Zhang
Z
Z
Ferguson-Yankey
S
S
Yappert
M C
MC
D000818:Animals; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D004848:Epithelium; D005978:Glutathione; D007461:Iodoacetates; D007700:Kinetics; D007908:Lens, Crystalline / Q000378:metabolism*; D008563:Membrane Lipids / Q000378:metabolism*; D008623:Mercaptoethanol; D008861:Microsomes / Q000378:metabolism*; D010084:Oxidation-Reduction; D011817:Rabbits
D007461:Iodoacetates; D008563:Membrane Lipids; D008623:Mercaptoethanol; D000252:Calcium-Transporting ATPases; D005978:Glutathione