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. 1990 Feb;87(3):1028–1031. doi: 10.1073/pnas.87.3.1028

Solubilization and functional reconstitution of the branched-chain alpha-keto acid transporter from rat heart mitochondria.

S M Hutson 1, S Roten 1, R S Kaplan 1
PMCID: PMC53403  PMID: 2300569

Abstract

The mitochondrial branched-chain alpha-keto acid transporter was solubilized from rat heart mitochondria and its function was reconstituted in phospholipid vesicles. The transporter was extracted from mitoplasts with Triton X-114 in the presence of exogenous cardiolipin and alpha-ketoisocaproate. Upon incorporation of this extract into asolectin vesicles by the freeze-thaw-sonication technique, a p-chloromercuribenzoate-sensitive, protein-dependent transport of alpha-ketoisocaproate into the proteoliposomes was observed. Significant inhibition of alpha-ketoisocaproate transport was observed in the reconstituted system with branched-chain alpha-keto acids (64-83%) and the related carboxylates alpha-ketocaproate (58%) and alpha-ketovalerate (49%), but not with substrates for the pyruvate carrier (less than 5%). The reconstituted carrier was substantially inhibited by sulfhydryl reagents, by the histidine-specific reagent diethyl pyrocarbonate, and by the tyrosine-specific reagent N-acetylimidazole. The extraction and functional reconstitution of the branched-chain alpha-keto acid transporter represents an important first step towards purification and molecular characterization of this anion carrier.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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