OXYGEN RADICAL-INDUCED INHIBITION OF ALKALINE-PHOSPHATASE ACTIVITY INRECONSTITUTED MEMBRANES

The effects of lipid peroxidation on membrane-bound enzyme activity we re examined using reconstituted membranes consisting of intestinal alk aline phosphatase (ALP) and phosphatidylcholine (PC) or dipalmitoylpho sphatidylcholine (DPPC). When the PC-reconstituted membranes were incu bated with ascorbic acid/Fe2+, the ALP activity decreased with increas es in the thiobarbituric acid-reactive substances and conjugated diene values in a time-dependent manner. The kinetic studies on the ALP act ivity with varying the p-nitrophenyl phosphate or beta-glycerophosphat e concentrations showed that the inhibition of the enzyme activity by treatment with these oxidizing agents is mainly due to a decrease in t he V-max value rather than a change in the K-m value. The results with several antioxidants suggested that ascorbic acid/Fe2+-induced inhibi tion of the ALP activity is related to generation of . OH radicals. Mo dification of the reconstituted membranes with malondialdehyde, trans- 2-nonenal, or n-heptaldehyde did not affect the ALP activity, suggesti ng that the secondary degraded products of lipid hydroperoxides had no influence on the enzyme activity. Increasing bityrosine production in the membrane constituents was observed by ascorbic acid/Fe2+ treatmen t, depending on the incubation time. This finding suggests the possibi lity that amino acid modifications in the protein molecule are induced by the treatment. Furthermore, the contribution of the lipid organiza tion in ascorbic acid/Fe2+-induced inhibition of the ALP activity in t he reconstituted membranes was examined by measurements of the fluores cence anisotropy of diphenylhexatriene-labeled membranes. In addition, it was found that the ALP activity in DPPC-reconstituted membranes wa s also inhibited by treatment with ascorbic acid/Fe2+, similar to the case in PC-reconstituted ones. On the basis of these results, a possib le mechanism of ascorbic acid/Fe2+-induced inhibition of membrane-boun d ALP activity is discussed. (C) 1994 Academic Press, Inc.