Pyrophosphate import and synthesis by plant mitochondria

Abstract

The matrix level of pyrophosphate (PPi) in mitochondria isolated from etiolated pea (Pisum sativum L. cv. Alaska) stems was evaluated, on the basis of an enzymatic assay, to be approx. 0.2 mM. Pyrophosphate could enter from the cytoplasm to the mitochondria via adenine nucleotide translocase (ANT), because F– and Ca21 (two penetrating PPiase inhibitors) and atractylate (ANT inhibitor) inhibited PPiase activity in isolated mitochondria supplied with PPi. This result was also confirmed by measuring oxygen consumption and membrane potential (DY) in succinate-energized mitochondria. In a medium free of phosphate (Pi), the addition of PPi before the substrate rendered possible an ADP-stimulated oxygen consumption that was inhibited by F– or Ca21. In a similar experiment, ADP induced the dissipation of DY when it was added after the succinate-generated DY had reached a steady state and, again, F– inhibited this dissipation. These results imply that PPi enters the mitochondria where it is hydrolyzed to 2 Pi which become available for the H1-ATPase (EC 3.6.1.34). In addition, PPi may be synthesized by the H1-PPiase (EC 3.6.1.1), acting as a synthase. This evidence arises from the observation that Pi stimulated an oxygen consumption (respiratory control ratio of 1.7) that was inhibited by F– or Ca21. The physiological role of the mitochondrial H1-PPiase is discussed in the light of the consideration that this enzyme can catalyse a readily reversible reaction