Influence of the redox state of pyridine nucleotides on mitochondrial sulfhydryl groups and permeability transition

This work addresses a correlation between the redox state of pyridine nucleotides and that of sulfhydryl groups of the mitochondrial membranes. Several major observations emerge: (1) Conditions leading to an oxidation of the pyridine nucleotides such as incubation with tert-butyl hydroperoxide or acetoacetate determine a decrease of total mitochondrial sulfhydryl groups. Glutathione does not follow the same pattern since it decreases in the presence of tert-butyl hydroperoxide but not in the presence of acetoacetate. In addition, only in the presence of tert-butyl hydroperoxide is the decrease of sulfhydryl groups concomitant with a membrane protein polymerization, observed by polyacrylamide gel electrophoresis. (2) Under all conditions tested, the oxidation of sulfhydryl groups is further stimulated by the presence of calcium and phosphate ions. (3) Respiratory substrates, which prevent the swelling of mitochondria, also partially prevent the decrease of sulfhydryl groups

Thallium induces apoptosis in Jurkat Cells

Thallium induces swelling in mitochondria and apoptosis in Jurkat cells. Both the swelling and the apoptosis are inhibited by means of cyclosporine A (CsA), thus suggesting that these phenomena result from the opening of the membrane transition pore (MTP) in mitochondria. Therefore, apoptosis can be explained as due to the opening of the MTP in mitochondria

Purification of mitochondrial thioredoxin reductase and its involvement in the redox regulation of membrane permeability

The isolation to purity of a rat liver mitochondrial thioredoxin reductase is reported. The mitochondrial enzyme shows a chromatographic behavior different from that of the cytosolic enzyme. The purified enzyme, after sodium dodecylsulfate-polyacrylamide gel electrophoresis, yields a single band with a molecular weight of approximately 54 kDa. The apparent Km for E. coli thioredoxin is about 13 microM, while the apparent Km for 5,5'-dithiobis (2-nitrobenzoic acid) is 530 microM, values comparable to those reported for the cytosolic enzyme. Mitochondrial thioredoxin reductase, in addition to its natural substrate thioredoxin, is also able to reduce chemically unrelated compounds such as 5,5 '-dithiobis (2-nitrobenzoic acid), selenite, and alloxan; the enzyme is inhibited by classical inhibitors of the cytosolic enzyme such as 1-chloro-2,4-dinitrobenzene and 13-cis-retinoic acid. A strong inhibitory action is also elicited by Mn2+ and Zn2+ ions. Thiol status appears critically involved in the control of membrane permeability and, therefore, a thiol/disulfide transition involving reduced pyridine nucleotides, matrix soluble thiols, and inner membrane thiols appears to play a fundamental role. The potential role of thioredoxin/thioredoxin reductase system in the control and redox regulation of the mitochondrial membrane permeability, is discussed

Conversion of rat xanthine dehydrogenase to xanthine oxidase during oxidative stress

The enzyme “xanthine oxidase” is one of the best characterized sources of superoxide anion and hydrogen peroxide. “in vivo” it mainly acts as a dehydrogenase which, by reducing NAD+, appears to be the physiologic form. Nevertheless under variety of conditions the enzyme can undergo a conversion to an oxidase which delivers the electrons to oxygen to form superoxide and hydrogen peroxide. The transformation xanthine dehydrogenase to xanthine oxidase is irreversible when it is induced by a proteolitic attack or reversible when the SH groups of the enzyme have been oxidized

The mitochondrial anti-oxidant defence system and its response to oxidative stress

The antioxidant systems of mitochondria are not well known. Using a proteomics-based approach, we defined these mitochondrial antioxidant systems and analyzed their response to oxidative stress. It appears that the major mitochondrial antioxidant system is made of manganese superoxide dismutase on the one hand, and of peroxiredoxin III, mitochondrial thioredoxin and mitochondrial thioredoxin reductase on the other hand. With the exception of thioredoxin reductase, all these proteins are induced by oxidative stress. In addition, a change in the peroxiredoxin III pattern can also be observed

The interaction of zinc pyrithione with mitochondria from rat liver and a study of the mechanism of inhibition of ATP synthesis

The interactions of zinc pyrithione (ZnPT) with rat liver mitochondria were investigated. Since most of the organometals, principally the triorganotin compounds, induce the inhibition of ATP synthesis in rat liver mitochondria, the efficiency of the ATP synthesis was measured in the presence of ZnPT. The results indicate that ZnPT inhibits ATP synthesis. In order to individuate the molecular mechanism responsible for a failure in ATP synthesis, all of the steps involved in ATP synthesis or in its inhibition were investigated separately, i.e. the respiratory chain, the uncoupling effect, the ATPase and the opening of a permeability pore. All of the steps are inhibited by ZnPT, but the crucial one, the one responsible for the inhibition of ATP synthesis, seems to be the opening of a small-size cyclosporine-sensitive pore. The results are different from those obtained using other organometallic compounds, but are similar to those obtained when using methylmercury and Zn2+, both of which also induce the opening of a cyclosporine-sensitive pore. However, although Hg2+ and Zn2+ would seem to induce the opening of large-size pores, in the case of ZnPT the pores involved are of a small size. This action mechanism seems to exclude the possibility that ZnPT is a deliverer of Zn2+

The interaction of zinc pyrithione with mitochondria from rat liver and a study of the mechanism of inhibition of ATP synthesis

The interactions of zinc pyrithione (ZnPT) with rat liver mitochondria were investigated. Since most of the organometals, principally the triorganotin compounds, induce the inhibition of ATP synthesis in rat liver mitochondria, the efficiency of the ATP synthesis was measured in the presence of ZnPT. The results indicate that ZnPT inhibits ATP synthesis. In order to individuate the molecular mechanism responsible for a failure in ATP synthesis, all of the steps involved in ATP synthesis or in its inhibition were investigated separately, i.e. the respiratory chain, the uncoupling effect, the ATPase and the opening of a permeability pore. All of the steps are inhibited by ZnPT, but the crucial one, the one responsible for the inhibition of ATP synthesis, seems to be the opening of a small-size cyclosporine-sensitive pore. The results are different from those obtained using other organometallic compounds, but are similar to those obtained when using methylmercury and Zn2+, both of which also induce the opening of a cyclosporine-sensitive pore. However, although Hg2+ and Zn2+ would seem to induce the opening of large-size pores, in the case of ZnPT the pores involved are of a small size. This action mechanism seems to exclude the possibility that ZnPT is a deliverer of Zn2+

Mitochondrial effects of L- ropivacaine, a new local anesthetic.

The effects of the local anesthetics ropivacaine and bupivacaine were investigated on isolated rat liver mitochondria. The efficiency of oxidative phosphorylation was evaluated by measuring the rates of respiration and ATP synthesis and the magnitude of the transmembrane electrical potential (deltapsi). Bupivacaine did not alter the ADP-stimulated respiration but strongly affected the resting respiration, which was more than doubled at 0.6 mM. In addition, it decreased the transmembrane electrical potential, and the ATP synthesis rate (deltapsi was less than 100 mV at 0.6 mM). Ropivacaine did not alter the ADP-stimulated respiration, and the resting respiration seemed to be substantially unaffected up to 1.2 mM; a slight increase was observed at 1.8 and 2.4 mM. The transmembrane potential was decreased by anesthetic concentrations higher than 1.2 mM and ATP synthesis was consequently affected. The findings suggest that ropivacaine is less toxic than bupivacaine, in rat liver mitochondri

Horseradish peroxidase-catalyzed sulfoxidation of promethazine and properties of promethazine sulfoxide.

Promethazine sulfoxide was obtained with a quantitative yield in a horse radish peroxidase-catalyzed reaction of promethazine and hydrogen peroxide and was also prepared by direct chemical synthesis. The enzymatic sulfoxidation of promethazine was studied in vitro as a function of pH, promethazine, and hydrogen peroxide concentration. Promethazine sulfoxide inhibits with an apparent Ki of 59.7 #M at pH 5.5 the enzymatic reaction, followed spectrophotometrically, polarographically, potentiometrically, and luminometrically. The reaction was also inhibited by ascorbic acid (Ki 26.8 #M) and glutathione (K~ 41,8 #M). The spectrophotometric techniques employed, together with ESR spectrometry, allowed the identification of at least three radical species formed in the course of the reaction. Promethazine sulfoxide is devoid of the antioxidant effect exhibited by promethazine on rat brain synaptosomes. The sulfoxide also lacks photosensitizing action, while retaining the neuroleptic effect of the parent compound