Masanori Yonaha

Mechanism of cobalt (II) ion inhibition of iron-supported phospholipid peroxidation

Co2+ inhibited nonenzymatic iron chelate-dependent lipid peroxidation in dispersed lipids, such as ascorbate-supported lipid peroxidation, but not iron-independent lipid peroxidation. Histidine partially abolished the Co2+ inhibition of the iron-dependent lipid peroxidation. The affinity of iron for phosphatidylcholine liposomes in Fe(2+)-PPi-supported systems was enhanced by the addition of an anionic lipid, phosphatidylserine, and Co2+ competitively inhibited the peroxidation, while the inhibiting ability of Co2+ as well as the peroxidizing ability of Fe(2+)-PPi on liposomes to which other phospholipids, phosphatidylethanolamine, or phosphatidylinositol had been added was reduced. Co2+ inhibited microsomal NADPH-supported lipid peroxidation monitored in terms of malondialdehyde production and the peroxidation monitored in terms of oxygen consumption. The inhibitory action of Co2+ was not associated with iron reduction or NADPH oxidation in microsomes, suggesting that Co2+ does not affect the microsomal electron transport system responsible for lipid peroxidation. Fe(2+)-PPi-supported peroxidation of microsomal lipid liposomes was markedly inhibited by Co2+.

Mechanism of the biphasic effect of ethylenediaminetetraacetate on lipid peroxidation in iron-supported and reconstituted enzymatic system

The biphasic action of ethylenediaminetetraacetate (EDTA), depending on its concentration, on lipid peroxidation was examined in an iron-supported and reconstituted enzymatic system. In the presence of NADPH-cytochrome P450 reductase and NADPH, Fe(3+)-PPi or Fe(3+)-ADP, though not reducible in the absence of EDTA, was markedly reduced with increasing concentration of EDTA. Lipid peroxidation, in the reconstituted system containing negatively charged liposomes, showed the maximal rate at 0.5 molar ratio of EDTA/iron, but no peroxidation occurred in positively charged liposomes, suggesting production of a positively charged iron complex as the prooxidant. Isotachophoresis indicated production of net-negative charge, EDTA-Fe(3+)-PPi complex, from Fe(3+)-PPi and EDTA at 1.1 ratio of EDTA/iron. The complex quenched Fe(2+)-PPi-supported lipid peroxidation. We suggest that EDTA-iron complexes of different charges are generated, depending on the amount of EDTA in the enzymatic system and, consequently, there is a switch between prooxidant and inhibitory effect at some critical ratio of EDTA/iron.

A microsomal membrane component associated with iron reduction in NADPH-supported lipid peroxidation

This study was conducted to determine whether a factor responsible for reduced nicotinamide adenine dinucleotide phosphate (NADPH)-supported lipid peroxidation in rat liver microsomes is involved in iron reduction by cooperation with NADPH-cytochrome P450 reductase. Under anaerobic conditions, NADPH-dependent reduction of ferric pyrophosphate in microsomes was not dependent on cytochrome P450 levels and was not inhibited by carbon monoxide (CO). All of the iron complexes with chelators such as adenosine 5′-diphosphate, pyrophosphate, nitrilotriacetate, oxalate or citrate were reduced in microsomes, although in the reconstituted system containing purified NADPH-cytochrome P450 reductase little or no iron reduction was found. A cytochrome P450-free fraction from a cholate-solubilized preparation of microsomes after passage through a laurate sepharose column was required for reduction of iron pyrophosphate in the reconstituted system leading to lipid peroxidation. The iron reduction was not inhibited by CO and was destroyed by heat treatment or trypsin digestion of the fraction. All iron complexes were reduced in the presence of the fraction, using a reducing equivalent of NADPHvia NADPH-cytochrome P450 reductase. The results indicate that a heat-labile component, which is probably a protein distinct from cytochrome P450, is associated with iron reduction responsible for lipid peroxidation in microsomes.