OXIDATIVE MODIFICATION OF NICOTINAMIDE NUCLEOTIDE TRANSHYDROGENASE INSUBMITOCHONDRIAL PARTICLES - EFFECT OF ENDOGENOUS UBIQUINOL

The present paper describes the sensitivity of the mitochondrial nicot inamide nucleotide transhydrogenase (EC1.6.1.1) to oxidative modificat ion, and the effects of endogenous ubiquinol on this modification. A c omparison is made between the effects of treatment with ADP-Fe3+ and a scorbate and with peroxynitrite, using kinetic, electrophoretic, and i mmunological analyses, together with Lipid peroxidation measurements. The transhydrogenase was inactivated by both types of oxidative modifi cation, but apparently through different mechanisms. Ubiquinol protect ed the enzyme against inactivation only when the modification was caus ed by ADP-Fe3+ and ascorbate treatment. Kinetic measurements revealed a threefold increase of the K-m value of the enzyme for NADPH after ex posure to ADP-Fe3+ and ascorbate, and a twofold increase of the K-m va lues for both NADH and NADPH after exposure to peroxynitrite. NAD(H) e xerted a protection against trans-hydrogenase inactivation when added to the preincubation in the case of peroxynitrite, but neither NAD(H) or NADP(H) protected in the case of ADP-Fe3+ and ascorbate. Using immu noblotting it was shown that the enzyme became both aggregated and fra gmented, although to different extents, depending on the oxidative sys tem used. Again, ubiquinol prevented these effects only in the case of ADP-Fe3+ and ascorbate treatment. Furthermore, there occurred a strik ing decrease in the 66-kDa trypsin fragment after exposure of the enzy me to ADP-Fe3+ and ascorbate, and of the 48-kDa trypsin fragment after exposure to peroxynitrite. It is concluded that the mitochondrial nic otinamide nucleotide transhydrogenase is sensitive to oxidative stress and that the mechanism underlying this can vary according to the chal lenge to which the enzyme is exposed Endogenous ubiquinol may play a r ole in protecting the enzyme against agents perturbing the lipid phase of the membrane. (C) 1996 Academic Press, Inc.