Functional reconstitution into liposomes and characterization of the carnitine transporter from rat liver microsomes.
The carnitine 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 carnitine transporter catalyzed a first-order uniport reaction inhibited by HgCl2 and DIDS. The IC50 for HgCl2 was 0.16+/-0.03 mM. The reconstituted transporter also catalyzed carnitine efflux from the proteoliposomes; the efflux was stimulated by externally added long-chain acylcarnitines. Besides carnitine, ornithine, arginine, glutamine and lysine were taken up by the reconstituted liposomes with lower efficiency respect to carnitine. Optimal activity was found at pH 8.0. The Km for carnitine on the external side of the transporter was 10.9+/-0.16 mM. The activation energy of the carnitine transport derived by Arrhenius plot was 16.1 kJ/mol.
National Research Council (CNR) Institute of Biomembranes and Bioenergetics (IBBE), via Amendola 165/A 70126 Bari, Italy.
Annamaria
A
Tonazzi
Michele
M
Galluccio
Francesca
F
Oppedisano
Cesare
C
Indiveri
D000818:Animals; D001692:Biological Transport; D006863:Hydrogen-Ion Concentration; D008081:Liposomes / Q000737:chemistry* / Q000378:metabolism; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism*; D027701:Organic Cation Transport Proteins / Q000737:chemistry* / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D012995:Solubility; D000074058:Solute Carrier Family 22 Member 5; D013997:Time Factors
D008081:Liposomes; D027701:Organic Cation Transport Proteins; C115441:Slc22a5 protein, rat; D000074058:Solute Carrier Family 22 Member 5