The distribution and metabolism of intra- and extra-mitochondrial pyridine nucleotides in suspensions of liver mitochondria

Abstract

The distribution of added and endogenous pyridine nucleotides between rat-liver mitochondria and the medium in which they were suspended has been studied. The distribution of the added coenzymes between the suspending fluid and the mitochondria has been interpreted in terms of the permeability of the mitochondria to the pyridine nucleotides. The amounts of endogenous nucleotides (as [mu]moles/mg. dry wt.) were: DPN+ 2.02; DPNH 0,44; TPN+ 1.26; TPNH 1.08. Of the dry matter of the mitochondria, 66% was protein. Considerable oxidation of the reduced nucleotides occurred when the particles were sedimented. Addition of DPNH and TPNH sometimes produced a shrinkage of the mitochondria, the maximum decrease in the proportion of water to dry matter in the particles being 29% in 15 minutes with DPNH (6.7 m[image]) and 18.5% with TPNH (12.8 m[image]). During shrinkage, there was a selective decrease in the "chloride space" of the mitochondria. Loss of added nucleotides was always slight at 0[degree], the greatest amounting to 18% disappearance of DPNH (originally 2.4 m[image]) in 30 min. The maximum rate of loss was approximately trebled when the temperature was raised to 25[degree]. TPN+ and TPNH were dephosphorylated, the former more rapidly; the products appeared in both the mitochondria and supernatant fluid. DPNH was oxidized slowly (approximately 0.032 [mu]mole/mg dry wt./hr.) in the suspension, but much more rapidly when the mitochondria were sedimented. The rate of oxidation of TPNH under similar conditions was approximately two-thirds of the corresponding values for DPNH. After addition of DPN+, DPNH, TPN+ and TPNH at concentrations greater than 5 m[image] to different suspensions, the mitochondria contained DPN+ and DPNH at concentrations 0-25% and TPN+ and TPNH at concentrations 37-41% of that in the supernatant fluid. This difference in "permeability" was demonstrated over a wide range (2-15 m[image]) of concentrations and at both 0[degree] and 25[degree]. Only DPNH was adsorbed by the mitochondria. TPN+ and TPNH permeating the mitochondria enter the "pool" of functional nucleotides. Degenerative changes in the original mitochondrial preparations (swelling, low level of endogenous reduced nucleotides and inability to maintain reduced nucleotides) resulted in a greatly increased "permeability" to the diphosphopyridine nucleotides.