Differential effects of polyamine on the cytosolic and mitochondrial NADP-isocitrate dehydrogenases.

Two isozymes of NADP-dependent isocitrate dehydrogenases (EC 1.1.1.42) exist in mammalian tissues: mitochondrial (ICD1) and cytosolic (ICD2). Effects of polyamines such as spermine, spermidine, and putrescine on the cytosolic and mitochondrial NADP-isocitrate dehydrogenases were analyzed kinetically. Spermine activated ICD2, the cytosolic NADP-isocitrate dehydrogenase from rat liver with the increase in the maximal velocity and the decrease in the affinity for the substrates isocitrate and NADP. The activating action of spermine can be explained by "the uncompetitive effect," and the dissociation constant of spermine for the enzyme-substrate complex was determined to be 1.68 mM. Spermidine and putrescine showed little or no effect. ICD1, the mitochondrial form of NADP-isocitrate dehydrogenase from rat and porcine heart was inhibited by spermine effectively, and by spermidine and putrescine to a lesser extent. Spermine inhibited the enzyme competitively with respect to NADP, and noncompetitively with respect to isocitrate. K(i) value of the enzyme for spermine was 1.3 mM. These results suggest that activation by spermine of cytosolic NADP-isocitrate dehydrogenase can enhance the antioxidant activity by regeneration of GSH, and further is responsible for the stimulation of lipid biosynthesis in cytosol. Spermine may contribute to NADPH supply by enhancing transhydrogenase (EC1.6.1.2) activity through the spermine-dependent activation of Ca(2+) -incorporation to mitochondria.

Effect of hydroxy substituent on the prooxidant action of naphthoquinone compounds.

Prooxidant activity of naphthoquinone compounds was analyzed by lipid peroxidation, and the formation of base adduct in DNA. Naphthoquinones with electron-repelling hydroxyl group in the benzene moiety such as juglone and shikonin of lower concentrations stimulated the microsomal lipid peroxidation, but lawsone and lapachol with hydroxyl group in the quinone moiety did not enhance the formation of lipid peroxides. Naphthoquinone-dependent lipid peroxidation was closely related to the enhancement of Fe(2+) autooxidation. Treatment of DNA with juglone a representative of 5-hydroxylated naphthoquinone stimulated the formation of 8-hydroxy-2'-deoxyguanosine, whereas lawsone and lapachol showed negligible formation of DNA base adduct. ESR spectra showed that juglone can form semiquinone radical in the presence of ferrous ion, but lawsone cannot. Biological toxicity of juglone with the potent electron-repelling group at 5-position may be due to the reactive oxygen species formed by semiquinone radical, but naphthoquinone compounds with an electron-repelling group in the quinone moiety, lawsone shows weak toxicity with only a little ability producing reactive oxygen species by the negligible formation of semiquinone.