Purification of Mitochondrial Thioredoxin Reductase and Its Involvement in the Redox Regulation of Membrane Permeability

• Published in: Free radical biology & medicine Vol. 24; no. 2; pp. 370 - 376
• Main Authors: Rigobello, Maria Pia, Callegaro, Maria Teresa, Barzon, Elena, Benetti, Monica, Bindoli, Alberto
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 15-01-1998
• Subjects: Alloxan - metabolism; Animals; Calcium - metabolism; Calcium - pharmacology; Dinitrochlorobenzene - pharmacology; Dithionitrobenzoic Acid - metabolism; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors - pharmacology; Intracellular Membranes - drug effects; Isotretinoin - pharmacology; Manganese - pharmacology; Mitochondria, Liver - enzymology; Molecular Weight; Oxidation-Reduction; Permeability - drug effects; Permeability transition; Rat liver mitochondria; Rats; Redox control; Sodium Selenite - metabolism; Thiol groups; Thioredoxin; Thioredoxin reductase; Thioredoxin-Disulfide Reductase - antagonists & inhibitors; Thioredoxin-Disulfide Reductase - isolation & purification; Thioredoxin-Disulfide Reductase - metabolism; Zinc - pharmacology; Thiol groups; Permeability transition; Redox control; CDNB; Rat liver mitochondria; Thioredoxin; Thioredoxin reductase
• ISSN: 0891-5849; 1873-4596
• DOI: 10.1016/S0891-5849(97)00216-5

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
K
m
for
E. coli thioredoxin is about 13 μM, while the apparent
K
m
for 5,5′-dithiobis (2-nitrobenzoic acid) is 530 μM, 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 Mn
2+ and Zn
2+ 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.