The behaviour of pyridine nucleotides of mitochondria in a ‘saline medium’

Abstract

The amounts and behavior of the pyridine nucleotides of rat-liver mitochondria depend on the method of preparation and the constituents of the suspending medium. In the present work, the tissue was fractionated in a sucrose-nicotinamide medium and the mitochondria were incubated in a "saline medium". The rate of loss of the mito-chondrial pyridine nucleotide was about 1/10 of that of the coenzymes of the mince from which the particles were isolated. Nicotinamide increased the initial amounts and stabilized the pyridine nucleotides of the isolated mitochondria during incubation at 25[degree]. The endogenous respiration and the capacity of the particles to maintain reduced coenzyme were both decreased. With nicotinamide the proportions of reduced coenzyme produced by succinate and pyruvate were lowered, but the respiration rates were unaltered. Ethylenediaminetetraacetic acid (EDTA) decreased by about 7% the initial proportions of reduced coenzyme and diminished the reduction of the nucleotides by both endogenous substrate and added pyruvate. The rate of destruction was increased about 4-fold by EDTA, and the respiration rate, though initially lower, was also more stable. Mg was required for the oxidation of reduced pyridine nucleotide during incubation without added substrate and for the reduction of the coenzymes by added pyruvate. The initial rate of respiration without added substrate and with pyruvate were lower by about 25% when Mg was present. Analyses of freshly isolated mitochondria at 0[degree] showed that the ratio of reduced diphosphopyridine nucleotide/oxidized diphosphopyridine nucleotide ranged from 0.4 to 1.1 (mean 0.62) and that of reduced triphosphopyridine nucleotide/ oxidized triphosphopyridine nucleotide ranged from 1.8 to 7.5 (mean 4.12). The levels of reduced diphosphopyridine nucleotide and oxidized triphosphopyridine nucleotide were least variable. The "fluffy layer" of these preparations contained less total pyridine nucleotide/mg dry weight but the same proportion of each individual nucleotide as the rest of the particles. The rate of leakage of coenzymes from the mitochondria at 25[degree] was usually low, about 10% in 1 hr.; it was increased (up to 9-fold) when there was considerable oxidation of nucleotides. Added pyruvate and succinate decreased the leakage occurring in 1 hr. but amytal and EDTA increased it. The released pyridine nucleotide was reduced by pyruvate plus malate and B-hydroxybutyrate. Rat-brain mitochondria had lower levels of all the coenzymes, particularly of the triphosphopyridine nucleotides. The initial ratio of reduced coenzyme to oxidized coenzyme was relatively low (0.11-0.17) and was increased by incubation with succinate and glutamate (up to 6-fold), but not with pyruvate or a-oxoglutarate.