Abstract
Loss of latency due to membrane lipid peroxidation induced in vitro was studied in highly purified rat liver lysosomes. Enriched fractions of lysosomes were isolated by free flow electrophoresis. Lipid peroxidation of lysosomes, assayed as malondialdehyde formation, was catalyzed by a radical generating system consisting of dihydroxyfumaric acid and Fe3+-ADP. The peroxidation reaction occurred readily at 37 degrees C and reached a plateau at 10 min; however, the loss of lysosomal latency, determined as increased percentage free beta-N-acetylglucosaminidase activity, occurred more gradually and reached a maximum after 30 min. Scavengers of superoxide, hydrogen peroxide, singlet oxygen, and hydroxyl radicals did not inhibit the peroxidation reaction nor prevent the loss of lysosomal latency. However, preincubation of the lysosomes with alpha-tocopherol effectively blocked the induction of peroxidation and substantially reduced the loss of lysosomal latency. These results indicate that the lysosomal membrane is susceptible to free radical-induced lipid peroxidation; further, this process may be the immediate cause of the subsequent disintegration of the lysosome. The nature of the protective effect of alpha-tocopherol is unclear but may be due to its interaction with the unsaturated membrane lipids and the subsequent interruption of the chain-reaction initiated by free radicals.
Highlights
MATERIALS ANDMETHODS a-tocopherol effectively blocked the inductionof per- Chemicals-DHF, 2-thiobarbituric acid, FeC13.6H20, p-nitrooxidation and substantially reduced thleoss of lysosomal latency
A-tocopherol effectively blocked the inductionof per- Chemicals-DHF, 2-thiobarbituric acid, FeC13.6H20, p-nitrooxidation and substantially reduced thleoss of lysosomal latency. These results indicate that the lysosomal membrane is susceptible to free radical-induced lipid peroxidation; further, this process may be the immephenyl-N-acetyl-a-D-glucosaminide, glucose-6-phosphate, cytochrome c, mannitol, triethanolamine, and aTC were purchased from Sigma. 2,5-Dimethylfuran and 1,4-diazabicyclo[2,2,2]
Sprague-Dawley rats (200-300 g) were decapitated and thelivers were perfused with ice-cold 0.25 M sucrose, 0.003 M MgClz, 0.001 M EDTA, Lipid peroxidation hasbeen suggestedto be associatedwith 0.01 M MOPS, pH 7.2.All subsequent steps were performed at 0
Summary
MATERIALS ANDMETHODS a-tocopherol effectively blocked the inductionof per- Chemicals-DHF, 2-thiobarbituric acid, FeC13.6H20, p-nitrooxidation and substantially reduced thleoss of lysosomal latency. The purified lysosomes were resuspended in the reaction buffer (0.12 M KCl, 0.05 M sucrose, 0.01 M potassium phosphate, pH 7.2) and were used immediately for free radical-induced lipid peroxidation studies. Peroxidation of Lysosomes-Aerobic oxidation of DHF generates large steady state levels of superoxide anions (OF), which have been suggested to generate additional active oxygen radicals capable of inducing lipid peroxidation [20, 21].
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