Bacterial overexpression, purification, and reconstitution of the carnitine/acylcarnitine carrier from rat liver mitochondria.
The carnitine/acylcarnitine carrier from rat liver mitochondria was overexpressed in Escherichia coli. The expressed protein, recovered as inclusion bodies, was solubilized with sarkosyl and purified by Sephadex G-200 and celite chromatography. A yield of 15 mg of purified transport protein per liter of cell culture was obtained. Upon reconstitution into liposomes, the purified carrier catalyzed a [3H]carnitine/carnitine exchange inhibited by maleimides, mercurials, and sulfobetaines. Carnitine esters of various lengths were also transported. The Km for carnitine uptake was 0.47 +/- 0.11 mM, the Vmax of the exchange was 0.78 +/- 0.24 mmol/min per gram of protein, and the Ki for octanoylcarnitine was 13.5 +/- 4.3 microM. The transport properties of the recombinant carrier were virtually identical to those of the native transporter. These studies represent the first overexpression of the functionally active mitochondrial carnitine/acylcarnitine carrier, thus enabling structure/function analysis of this protein by site-directed mutagenesis.
Indiveri
C
C
Department of Pharmaco-Biology, University of Bari, Italy.
Iacobazzi
V
V
Giangregorio
N
N
Palmieri
F
F
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D001693:Biological Transport, Active / Q000187:drug effects; D002331:Carnitine / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007700:Kinetics; D008301:Maleimides / Q000494:pharmacology; D008930:Mitochondria, Liver / Q000378:metabolism*; D009941:Organomercury Compounds / Q000494:pharmacology; D011510:Proteolipids; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D002352:Carrier Proteins; D008301:Maleimides; D009941:Organomercury Compounds; D011510:Proteolipids; D011994:Recombinant Proteins; C020485:proteoliposomes; D002331:Carnitine