Identification and purification of the ornithine/citrulline carrier from rat liver mitochondira

Abstract

The ornithine/citrulline carrier from rat liver mitochondria, solubilized with Triton X-1 00 and partially purified on hydroxyapatite, was identified and completely purified by PD-10, DEAESephacel and celite chromatography. On SDS/polyacrylamide gel electrophoresis, the purified ornithine/citrulline carrier consisted of a single protein band with an apparent molecular mass of 33.5 kDa. When reconstituted into liposomes the ornithine carrier protein catalyzed an active mersalyl sensitive ornithine/ornithine exchange. It was purified 438-fold with a recovery of 38% and a protein yield of 0.09% with respect to the extract derived from mitoplasts. The purified and reconstituted protein did not catalyze a significant unidirectional transport of ornithine. Citrulline was found to be the best countersubstrate for the transport of ornithine, followed by lysine and arginine. The exchange activity was inhibited by several sulphydryl reagents.In liver, two enzymes of the urea cycle, ornithine transcarbamylase and carbamyl phosphate synthetase I, are located in the mitochondrial matrix, while the other enzymes are cytoplasmic. Thus, for the operation of the urea cycle, ornithine must pass from the cytosol to the mitochondrial matrix where it is carbamylated to citrulline, and citrulline must leave the mitochondria in order to regenerate ornithine.The presence of an ornithine/citrulline carrier in the inner mitochondrial membrane has already been predicted in 1972 from theoretical and metabolic considerations [l]. However, the experimental evidence supporting the existence of this carrier in intact mitochondria is only circumstantial and not as definitive as for other mitochondrial metabolite carriers (for a review see [2 and 31). Based on the osmotic swelling of rat liver mitochondria, Gamble and Lehninger [4] [7], using rat liver mitochondria loaded with citrulline, presented evidence for the existence of an ornithine/citrulline-exchange system. These authors observed the uptake of ornithine in citrulline-loaded mitochondria and citrulline efflux from these mitochondria upon addition of ornithine or lysine. In a subsequent study 181, no evidence could be obtained for an ornithine/citrulline or lysine/citrulline exchange, whereas it was concluded that ornithine and lysine are taken up by rat liver mitochondria in exchange for H'. Recently, in rat kidney mitochondria, an The final proof for the existence of a particular carrier protein is its isolation. So far, an ornithine-transport protein has only been reconstituted into liposomes from a Lubrol WX extract of rat liver mitochondria [lo]. In this paper, we describe the isolation and purification of the ornithine/citrulline carrier from rat liver mitochondria using functional reconstitution as a monitor of the carrier activity during the isolation procedure. MATERIALS AND METHODS MaterialsHydroxyapatite (Bio-Gel HTP) was purchased from BioRad, Celite 535 from Roth, Amberlite XAD-2 from Fluka, DEAE-Sephacel, Sephadex PD-10, Sephadex G-50 and Sephadex G-75 from Pharmacia, L-[ ...

The Mitochondrial Ornithine Transporter

“…: 44(0)1223252703; Fax: 44(0)1223252705; E-mail: walker@mrc-dunn.cam.ac.uk. 1 The abbreviations used are: ORC, ornithine carrier protein; nt, nucleotide(s); HHH, hyperornithinemia-hyperammonemia-homocitrullinuria.…”

“…Rat liver mitochondria contain an ornithine-citrulline transport protein, often known as the ornithine carrier (ORC) 1 (1)(2)(3)(4)(5). The reconstituted purified protein is highly active in ornithinecitrulline exchange and is somewhat less active in unidirectional transport of ornithine.…”

“…The exchange reaction operates by a simultaneous (sequential) mechanism, and the unidirectional transport of ornithine or lysine (but not of citrulline) is compensated by a proton. The exchange of cytosolic ornithine for matrix citrulline is part of the urea cycle (1,3,6), and the ornithine-H ϩ exchange also has an important role in the catabolism of excess arginine and the biosynthesis of polyamines (4).…”

Intragenic complementation and the structure and function of argininosuccinate lyase

“…The second enzyme, ornithine transcarbamylase, is a trimer of identical 38-kDa subunits [11,12]. Citrulline, the product of the OCT reaction, is exported out of the mitochondria to the cytosol [13][14][15] by facilitated diffusion through an ornithine/citrulline antiporter. Enzyme localization experiments and experiments with labeled substrates and intermediates indicate that the urea cycle operates as a metabolon spanning the two compartments with considerable channeling of intermediates from one enzyme to the next [16][17][18].…”

Mitochondrial transporters for ornithine and related amino acids: a review

“…Although a transporter for ornithine in mitochondria had already been postulated in 1972 (Chappell et al 1972), a carrier that counter exchanges ornithine, citrulline, lysine, and arginine was first purified from rat liver mitochondria in 1992 (Indiveri et al 1992). Based on its substrate specificity, it was suggested to play a role in the urea cycle.…”