The camitine transporter was solubilized from rat liver microsomes with Triton X-100 and reconstituted into liposomes, after addition of Triton X-114, by removing the detergent from mixed micelles by hydrophobic chromatography on Amberlite (Bio-Beads SM 2). The reconstitution was optimized with respect to the detergent/phospholipid ratio, the protein concentration, and the number of passages through a single Amberlite column. The reconstituted camitine transporter catalyzed a first-order uniport reaction inhibited by HgCl, and DIDS. The IC50 for HgCl2 was 0.16 +/- 0.03 mM. The reconstituted transporter also catalyzed camitine efflux from the proteoliposomes; the efflux was stimulated by externally added long-chain acylcamitines. Besides camitine, ornithine, arginine, glutamine and lysine were taken up by the reconstituted liposomes with lower efficiency respect to camitine. Optimal activity was found at pH 8.0. The Km for camitine on the external side of the transporter was 10.9 +/- 0.16 mM. The activation energy of the camitine transport derived by Arrhenius plot was 16.1 kJ/mol. (c) 2006 Elsevier B.V. All rights reserved.
Functional reconstitution into liposomes and characterization of the carnitine transporter from rat liver microsomes
GALLUCCIO, Michele;INDIVERI, Cesare
2006-01-01
Abstract
The camitine transporter was solubilized from rat liver microsomes with Triton X-100 and reconstituted into liposomes, after addition of Triton X-114, by removing the detergent from mixed micelles by hydrophobic chromatography on Amberlite (Bio-Beads SM 2). The reconstitution was optimized with respect to the detergent/phospholipid ratio, the protein concentration, and the number of passages through a single Amberlite column. The reconstituted camitine transporter catalyzed a first-order uniport reaction inhibited by HgCl, and DIDS. The IC50 for HgCl2 was 0.16 +/- 0.03 mM. The reconstituted transporter also catalyzed camitine efflux from the proteoliposomes; the efflux was stimulated by externally added long-chain acylcamitines. Besides camitine, ornithine, arginine, glutamine and lysine were taken up by the reconstituted liposomes with lower efficiency respect to camitine. Optimal activity was found at pH 8.0. The Km for camitine on the external side of the transporter was 10.9 +/- 0.16 mM. The activation energy of the camitine transport derived by Arrhenius plot was 16.1 kJ/mol. (c) 2006 Elsevier B.V. All rights reserved.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
