Inhibition of the oxidation of hydroxyl radical scavenging agents after alkaline phosphatase treatment of rat liver microsomes

Susana Puntarulo, Arthur I. Cederbaum

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Treatment of rat liver microsomes with alkaline phosphatase results in a loss in the FMN but not the FAD flavin prosthetic group of NADPH-cytochrome P-450 reductase (Taniguchi, H. and Pyerin, W. (1987) Biochim. Biophys. Acta 912, 295-307) Experiments were carried out to evaluate the effect of preventing electron transfer from the FADH2 to FMN component of the reductase, and subsequent mixed function oxidase activity, on reduction of ferric chelates, production of H2O2, and the generation of ·OH-like species by microsomes. Treatment with alkaline phosphatase was confirmed to decrease NADPH-cytochrome c, but not NADPH-ferricyanide, reductase activity by microsomes and by purified NADPH cytochrome P-450 reductase. The oxidation of hydroxyl radical scavenging agents by microsomes and reductase was decreased by the alkaline phosphatase treatment in accordance with the decline in cytochrome c reductase activity. This decrease in hydroxyl radical production occurred in the presence of various ferric chelate catalysts. Rates of microsomal reduction of the ferric chelates were also inhibited after alkaline phosphatase treatment. Production of H2O2 was decreased in accordance to the fall in cytochrome c reductase activity and ·OH production. Rates of H2O2 production appeared to be rate-limiting for the overall generation of ·OH as the addition of an external H2O2-generating system stimulated ·OH production as well as prevented the decline in ·OH production caused by the alkaline phosphatase treatment. These results suggest that both the FAD and FMN flavin prosthetic groups of the reductase contribute towards the reduction of various ferric chelates. However, loss of the FMN component and activities dependent on electron transfer from this prosthetic group result in a decrease in H2O2 production, which appears to be responsible for the decline in the generation of ·OH-like species by microsomes after treatment with alkaline phosphatase.

Original languageEnglish
Pages (from-to)12-18
Number of pages7
JournalBiochimica et Biophysica Acta - General Subjects
Issue number1
StatePublished - 24 May 1991


  • (Rat liver)
  • Cytochrome P-450
  • Microsome
  • NADPH-cytochrome-c reductase
  • Phosphatase


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