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Bacterial overexpression, purification, and reconstitution of the carnitine/acylcarnitine carrier from rat liver mitochondria.

Authors: Indiveri, C  Iacobazzi, V  Giangregorio, N  Palmieri, F 
Citation: Indiveri C, etal., Biochem Biophys Res Commun. 1998 Aug 28;249(3):589-94. doi: 10.1006/bbrc.1998.9197.
Pubmed: (View Article at PubMed) PMID:9731180
DOI: Full-text: DOI:10.1006/bbrc.1998.9197

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.


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CRRD Object Information
CRRD ID: 39458032
Created: 2020-10-10
Species: All species
Last Modified: 2020-10-10
Status: ACTIVE


RGD is funded by grant HL64541 from the National Heart, Lung, and Blood Institute on behalf of the NIH.