Acid Reactive Substances Formation Research Articles

A high involvement of [formula omitted] possibly generated in inner membranes for iron-induced microsomal lipid peroxidation

The lipid peroxidation of and the O 2 − generation by rat liver microsomes in the presence of NADPH or both NADPH and Fe 3+ were determined by thiobarbituric acid-reacting substance formation and by chemiluminescence intensities with a cypridina luciferin analog, 2-methyl-6-(p-methoxyphenyl)-3, 7-dihydroimidazo[1,2-a]pyrazin-3-one(MCLA), as a chemiluminescence probe. Judging from the experiments with various inhibitors on the O 2 − generation and the lipid peroxidation, O 2 − generated, at intramembranous site, by cytochrome P-450 system is considered to be highly involved in the iron-induced lipid peroxidation.

THE PHOTODYNAMIC EFFECT OF ROSE BENGAL ON PROTEINS OF THE MITOCHONDRIAL INNER MEMBRANE

Photodynamic action promoted by Rose Bengal was evaluated in solutions of unsaturated fatty acids or histidine, and on beef heart submitochondrial particles. Rose Bengal-promoted photooxidation of histidine was mainly due to the opening up of the imidazole ring by singlet oxygen. Photosensitization of polyunsaturated fatty acids (PUFA) resulted in oxygen consumption and thiobarbituric acid-reactive substances (TBARS) formation, the extent of which was linearly related to the increasing degree of unsaturation. Photosensitization of submitochondrial particles caused oxygen consumption and TBARS production. These processes involved two different reaction components: during the first, most of the mitochondrial proteins were inactivated, the most sensitive being succinate dehydrogenase and cytochrome c. The values for the rate ratios of [TBARS] formation/[O2] consumption for the first and second phase were 0.36 and 1.32%, respectively, pointing to a larger contribution of lipid peroxidation during the second phase. The calculation of the rate constants for reaction of singlet oxygen with mitochondrial proteins suggests that singlet oxygen is more reactive towards proteins than to PUFA. The biological role of this selectivity is discussed in terms of the mitochondria as one of the first targets for photosensitized reactions.

Thiobarbituric acid-reactive substance formation of rat kidney brush border membrane vesicles induced by ferric nitrilotriacetate

An iron chelate, ferric nitrilotriacetate (Fe 3+-NTA), is nephrotoxic and also carcinogenic to the kidney in experimental animals. Iron-promoted lipid peroxidation in the proximal tubules is thought to be responsible for the pathologic process. In the present study, iron-promoted lipid peroxidation, with thiobarbituric acid (TBA) formation as an indication, in the tubular surface was simulated in vitro using rat kidney brush border membrane vesicles and the results were compared with those using linoleate micelles and rat liver microsomal lipid liposomes. Addition of ascorbate, cysteine, or dithiothreitol to the Fe 3+-NTA solution resulted in consumption of dissolved oxygen and promoted the lipid peroxidation in the micelles and in the liposomes. In contrast, addition of glutathione to the Fe 3+-NTA solution caused only sluggish oxygen consumption and far less peroxidation in these lipid systems. When the brush border membrane vesicles were used for the peroxidation substrate, Fe 3+-NTA and glutathione could promote TBA formation at a rate comparable to that elicited by Fe 3+-NTA with cysteine or dithiothreitol. Acivicin, a γ-glutamyl transpeptidase inhibitor, suppressed the peroxidation of the brush border membrane vesicles promoted by Fe 3+-NTA and glutathione. These results suggest the following mechanism of proximal tubular cell lipid peroxidation promoted by Fe-NTA: Fe 3+-NTA filtered through glomeruli is rapidly reduced by cysteine and Fe 2+-NTA starts lipid peroxidation at the site, leading to proximal tubular necrosis. Cysteine is amply supplied by the decomposition of glutathione within the lumen by the action of γ-glutamyl transpeptidase and dipeptidase situated at the proximal tubular brush border membrane.

Increase of the molecular rigidity of the protein conformation in the intestinal brush-border membranes by lipid peroxidation

The effect of lipid peroxidation on the protein conformation of the procine intestinal brush-border membranes was studied using a fluorogenic thiol reagent, N- [7-dimethylamino-4-methylcoumarinyl]maleimide (DACM). By a kinetic analysis of the reaction of the membranes with DACM, it was shown that the reaction rate of the SH groups (SH f) of the membrane proteins, whose reaction with the dye is very fast, decreases in proportion to the extent of thiobarbituric acid-reactive substance formation. The difference in the rate of the reaction of the SH f group for DACM between the control and peroxidized membranes completely disappeared after denaturation of the proteins by treatment with guanidine hydrochloride. The reaction of DACM with the SH f groups of the control membranes acceleration when the temperature was increased with an apparent transition temperature between 25°C and 30 °C. On the other hand, no transition was observed in the peroxidized membranes over the temperature range 20–43°C. These results suggest that the conformation around the SH f groups of the proteins in the peroxidized membranes is apparently different from that in the control membranes. A modification of the conformation around the SH groups in the membrane proteins associated with lipid peroxidation was further demonstrated by finding that the quenching efficiency of the fluorescence of the DACM-labeled membranes Tl + was markedly decreased after lipid peroxidation. Based on these results, changes in the protein conformation of the porcine intestinal brush-border membranes by lipid peroxidation are discussed.