Modification of bovine heart mitochondrial transhydrogenase with tetranitromethane |
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Authors: | Licia N.Y. Wu Ronald R. Fisher |
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Affiliation: | Department of Chemistry, University of South Carolina, Columbia, SC 29208 U.S.A. |
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Abstract: | Modification of pyridine dinucleotide transhydrogenase with tetranitromethane resulted in inhibition of its activity. Development of a membrane potential in submitochondrial particles during the reduction of 3-acetylpyridine adenine dinucleotide (AcPyAD+) by NADPH decreased to nearly the same extent as the transhydrogenase rate on tetranitromethane treatment of the membrane. Kinetics of the inactivation of homogeneous transhydrogenase and the enzyme reconstituted into phosphatidylcholine liposomes indicate that a single essential residue was modified per active monomer. NADP+, NADPH and NADH gave substantial protection against tetranitromethane inactivation of both the nonenergy-linked and energy-linked transhydrogenase reactions of submitochondrial particles and the NADPH → AcPyAD+ reaction of reconstituted enzyme. NAD+ had no effect on inactivation. Tetranitromethane modification of reconstituted transhydrogenase resulted in a decrease in the rate of coupled H+ translocation that was comparable to the decrease in the rate of NADPH → AcPyAD+ transhydrogenation. It is concluded that tetranitromethane modification controls the H+ translocation process solely through its effect on catalytic activity, rather than through alteration of a separate H+-binding domain. Nitrotyrosine was not found in tetranitromethane-treated transhydrogenase. Both 5,5′-dithiobis(2-nitrobenzoate)-accessible and buried sulfhydryl groups were modified with tetranitromethane. NADH and NADPH prevented sulfhydryl reactivity toward tetranitromethane. These data indicate that the inhibition seen with tetranitromethane results from the modification of a cysteine residue. |
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Keywords: | Transhydrogenase Proton translocation Tetranitromethane Enzyme modification (Bovine heart mitochondria) oxidized 3-acetylpyridine adenine dinucleotide oxidized thionicotinamide adenine dinucleotide FCCP ANS 8-anilino-1-naphthalenesulfonic acid DTNB 5,5′-dithiobis-(2-nitrobenzoic acid) Tricine DCCD To whom correspondence should be addressed. |
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