ABSTRACT
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.
Subject(s)
Calcium-Transporting ATPases/metabolism , Lens, Crystalline/metabolism , Membrane Lipids/metabolism , Microsomes/metabolism , Animals , Cattle , Cell Membrane/metabolism , Epithelium , Glutathione , Iodoacetates , Kinetics , Mercaptoethanol , Oxidation-Reduction , RabbitsABSTRACT
Na,K-ATPase was studied in the two cell types that make up the lens of the eye. Membrane material was isolated from lens fibre cells, which make up the bulk of the lens cell mass, and also from lens epithelial cells, which are present only as a monolayer on the anterior lens surface. Judged by immunoblotting, greater amounts of Na,K-ATPase alpha1 and beta1 polypeptides were found in fibre cell membrane material than in epithelial cell membrane material. However, the NA,K-ATPase activity in epithelial cell membrane material was 20 times that measured in fibre cell membrane material. In 86Rb uptake experiments with intact lenses, ouabain-inhibitable 86Rb uptake was observed for lens epithelium but not for lens fibres. These findings are consistent with a low Na,K-ATPase activity in lens fibre cells even though these cells express a considerable amount of Na,K-ATPase alpha1 and beta1 polypeptides. The lipid composition of lens fibre cell membranes causes them to be more ordered than epithelial cell membranes; this was confirmed by measurements of the infrared CH2 symmetric stretching band frequency. Because lipid composition can influence Na,K-ATPase activity, experiments were conducted to determine whether the activity of Na,K-ATPase alpha1 beta1 is inhibited by lens fibre lipid. However, no significant difference in Na,K-ATPase activity was detected when Na,K-ATPase alpha1 beta1 was purified from rabbit kidney and then reconstituted with lipid that had been isolated from either lens epithelium or lens fibre cells. These studies indicate that lens fibre cells contain both Na,K-ATPase alpha1 and beta1 polypeptides but have low Na,K-ATPase activity. However, the results do not support the notion that this is due to the lipid composition of lens fibre cell membranes.
Subject(s)
Lens, Crystalline/cytology , Lens, Crystalline/enzymology , Sodium-Potassium-Exchanging ATPase/metabolism , Animals , Cell Membrane/enzymology , Centrifugation, Density Gradient , Epithelial Cells , Epithelium/enzymology , Immunoblotting , Kidney/enzymology , Kidney Medulla/enzymology , Phospholipids/isolation & purification , Phospholipids/metabolism , Rabbits , Rubidium/metabolism , Rubidium Radioisotopes , Sodium-Potassium-Exchanging ATPase/isolation & purification , Spectrophotometry, InfraredABSTRACT
Case records of 78 patients of acute lymphoblastic leukemia have been reviewed. Complete remission occurred in seven cases following an episode of septicemia and supportive care.
