Transport of ornithine and citrulline across the mitochondrial membrane.

Assay of ornithine transcarbamylase and carbamyl phosphate synthetase I in submitochondrial fractions of rat liver mitochondria shows they are located primarily in the mitochondrial matrix. Therefore during the urea cycle ornithine must pass from the cytoplasm, where it is formed, to the mitochondrial matrix, where it is carbamylated. The resulting citrulline must then pass from the matrix to the cytoplasm before it can be converted to arginine. Osmotic swelling tests show that nonrespiring rat liver mitochondria do not allow the ornithine+ cation, the ornithine zwitterion, or N-acetylornithine to pass into the matrix, regardless of the presence of permeant anions, uncoupling agents, or valinomycin plus K+. However, when state 4 respiration is instituted with succinate as substrate, ornithine+ readily enters the mitochondria if a permeant proton-yielding anion such as phosphate, acetate, or bicarbonate is present. However, the permeant anions nitrate and thiocyanate, which pass the membrane without carrying protons, do not support ornithine+ entry. The respiration-energized unidirectional entry of [W]ornithine+ is inhibited by respiratory inhibitors, by uncoupling agents, and by valinomycin plus K+, but not by oligomycin. ADP also inhibits ornithine+ entry, presumably by competing for respiratory energy. The driving force for entry of ornithine+ is concluded to be a negativeinside transmembrane potential produced when proton-conducting anions enter mitochondria to relieve the alkalineinside pH gradient generated by electron transport. It is postulated that the L-ornithine+ cation is transported by a specific electrogenic uniport carrier. This view is supported by (a) the apparent specificity of the system, which transports ornithine+ but not the closely related arginine+‘or lysine+, (b) the stereospecificity for the L stereoisomer of ornithine+, and (c) the tissue specificity of ornithine+ transport, which occurs in liver mitochondria but not in those from heart, which cannot synthesize urea. The influx and efliux of citrulline in rat liver mitochondria does not depend on respiratory energy or the presence of permeant anions or cations. Because penetration of citrufine occurs into liver but not