Solubilization and functional reconstitution of the branched-chain alpha-keto acid transporter from rat heart mitochondria.
AUTOR(ES)
Hutson, S M
RESUMO
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.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=53403Documentos Relacionados
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