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J. Biochem, 1995, Vol. 118, No. 3 508-514
© 1995 Japanese Biochemical Society


other

Contribution of Peroxidation Products to Oxidative Inactivation of Rat Liver Microsomal Glucose-6-Phosphatase

Takao Ohyashiki, Kanae Kamata, Miyako Takeuchi and Katsuhiko Matsui

Department of Biochemistry, Faculty of Pharmaceutical Sciences, Hokuriku University Kanagawa-machi, Kanazawa, Ishikawa 920-11

Exposure of rat liver microsomes to ascorbic acid/Fe2+ caused decreases in the membranebound glucose-6-phosphatase (G-6-Pase) activity and the protein thiols after a short lag period (4 min). Under the same conditions, the production of thiobarbituric acid-reactive substances and fluorescent products was also initiated from 4 min after the start of the treatment, although conjugated diene was formed immediately on incubation of the microsomes with ascorbic acid/Fe2+. After centrifugation of the treated microsomes, the fluorescent products and the enzyme activity remained in the membrane fraction. The results of kinetic studies of the enzyme activity indicated that ascorbic acid/Fe2+ induced inhibition of the enzyme activity is mainly due to an increased Km value for the substrate. A decreased activity of the microsomal G-6-Pase was also observed when the microsomes were incubated with aldehydes such as malondialdehyde, n-heptaldehyde, acetaldehyde, and trans-2-nonenal. However, loss of protein thiols was detected only upon treatment of the microsomes with trans-2-nonenal. Glucose–6–phosphate (G-6-P) effectively prevented ascorbic acid/Fe2+- or trans–2–nonenal-induced inhibition of the enzyme activity, but the substrate failed to protect the protein thiols in both systems. The results of fluorescence anisotropy measurements of diphenylhexatriene-labeled microsomes suggested that changes in the lipid dynamics are not directly related to peroxidation-mediated inhibition of the enzyme activity. Based on these results, a possible reason for the inhibition of the microsomal G-6-Pase activity associated with ascorbic acid/Fe2+ treatment is discussed


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