CONTROL OF REDUCED NICOTINAMIDE-ADENINE DINUCLEO-TIDE OXIDATION BY PIGEON-HEART MITOCHONDRIA

Abstract

The rate of oxidation of 10–35 μM reduced nicotinamide–adenine dinucleotide (NADH) by pigeon-heart mitochondria was increased not only by osmotic swelling and sonic disruption of the organelles but also by milder procedures such as washing or dilution, which had no deleterious effect on the P: O and respiratory control ratios when glutamate was the substrate. In all cases, the enhanced oxidation of 10 μM NADH was suppressed by 5 mM adenosine triphosphate (ATP). From these and other findings it was concluded that the access of extra-mitochondrial NADH to the respiratory chain is controlled at a minimum of two sites. Control of NADH flux through the first site is lost after treatment of the mitochondria by procedures which increase their permeability. A second level of NADH control survives sonic disruption of the mitochondria and is a site at which the oxidation of 10 μM NADH is stimulated by Piand inhibited by ATP, adenosine diphosphate (ADP), and oxidized nicotinamide–adenine dinucleotide (NAD+). The ATP and ADP effects at this level are not blocked by oligomycin. Magnesium releases the adenine nucleotide inhibition of NADH oxidation under certain conditions, but its site and mode of action is not clear as yet. In these experiments, NADH oxidation was determined polarographically and photometrically at 28° in a medium containing 0.23 M mannitol, 0.07 M sucrose, 0.02 M Tris–chloride, and 0.02 mM ethylenediamine tetra-acetic acid (EDTA) at pH 7.2.