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.

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.