Distribution of adenine nucleotides between the inner and outer spaces of the mitochondrion as a determinant of phosphorylation pattern.

Abstract

Mitochondrial preparations incubated with 32P1 and 3H-ADP were subjected to rapid filtration through a Millipore filter to study the intramitochondrial distribution of labelled nucleotides. The atractyloside-induced inhibition of the distribution of internally-labelled nucleotides and of externally-added 3H-ADP revealed that the nucleotides in the matrix space are successfully separated by this means from those outside the inner membrane. The addition of Mg2+ to the incubation medium has no effect on the labelling pattern in the matrix space but results in a rapid interconversion of adenine nucleotides outside the inner membrane through the activation of adenylate kinase [EC 2.7.4.3] and nucleosidediphosphate kinase [EC 2.7.4.6], which do not function in an Mg2+-free medium. The localization of GTP-AMP phosphotransferase [EC 2.7.4.10] outside the membrane is questionable, but was not definitely excluded. The translocation of internal ATP outwards in exchange for external ATP or ADP induced by the addition of ATP or ADP, of hexokinase plus glucose or of myokinase (muscle adenylate kinase) plus AMP into the incubation medium appears to be a significant factor in promoting the labelling of ATP, reflecting oxidative phosphorylation. 32P1-Labelling of ADP dependent on substrate-level phosphorylation is greatly suppressed under these conditions. This apparent suppression of substrate-level phosphorylation is at least partly accounted for in terms of the lowered specific radioactivity of the P1 compartment selectively supporting AMP phosphorylation as compared to the specific radioactivity of the major P1 pool serving as the substrate for oxidative phosphorylation. A possible interaction of these two phosphorylation reactions in rat-liver mitochondria is also discussed.