Evidence for a proton-dependent regulation of mitochondrial nicotinamide-nucleotide transhydrogenase.

Abstract

The relationship between energy requirement, [NAD+] [NADPH]/[NADH] [NADP]+ ratio and velocity of the nicotinamide‐nucleotide transhydrogenase reaction, catalyzed by submitochondrial particles from beef heart, was investigated under conditions where the pH and the energy level were varied. At low energy levels, obtained by decreasing the rate of oxidation of respiratory substrates, energy‐dependent anilinonaphthalene sulfanote fluorescence is a sensitive tool for estimating the relative energy requirement by the transhydrogenase reaction. The velocity of the energy‐linked transhydrogenase reaction is directly proportional to the anilinonaphthalene sulfonate fluorescence. The energy requirement is related to the total rate of the transhydrogenase reaction rather than to the degree of activation of the reaction by energy. Under conditions where the energy requirement and the total rate of the transhydrogenase reaction are constant, a decreased pH is accompanied by a decrease in the activation by energy. It is concluded that the kinetic effect and the thermodynamic effect of energy on the transhydrogenase reaction may be separated by varying the pH of the reaction medium. Acidification in the absence of an energy source promotes the conversion of the transhydrogenase into an active form, presumably a protonated species of the enzyme, the kinetic properties of which are similar to those of the energy‐activated enzyme. In the presence of an energy source acidification may replace energization as the means for activation of the transhydrogenase. The relationship between the proton‐activated and the energy‐activated forms of the transhydrogenase with regard to the coupling mechanism is discussed.