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.
Rigobello
M P
MP
Centro di Studio delle Biomembrane (CNR) and Dipartimento di Chimica Biologica, Università di Padova, Italy.
Callegaro
M T
MT
Barzon
E
E
Benetti
M
M
Bindoli
A
A
D000496:Alloxan / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D004137:Dinitrochlorobenzene / Q000494:pharmacology; D004228:Dithionitrobenzoic Acid / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007425:Intracellular Membranes / Q000187:drug effects*; D015474:Isotretinoin / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008930:Mitochondria, Liver / Q000201:enzymology*; D008970:Molecular Weight; D010084:Oxidation-Reduction; D010539:Permeability / Q000187:drug effects; D051381:Rats; D018038:Sodium Selenite / Q000378:metabolism; D013880:Thioredoxin-Disulfide Reductase / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism*; D015032:Zinc / Q000494:pharmacology
D004137:Dinitrochlorobenzene; D004791:Enzyme Inhibitors; D008345:Manganese; D000496:Alloxan; D004228:Dithionitrobenzoic Acid; D013880:Thioredoxin-Disulfide Reductase; D015474:Isotretinoin; D018038:Sodium Selenite; D015032:Zinc; D002118:Calcium