SOLUBILIZATION AND FUNCTIONAL RECONSTITUTION OF THE BRANCHED-CHAIN ALPHA-KETO ACID TRANSPORTER FROM RAT-HEART MITOCHONDRIA

The mitochondrial branched-chain α-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 cadiolipin and α-ketoisocaproate. Upon incorporation of this extract into asolectin vesicles by the freeze-thaw-sonication technique, a p-chloromercuribenzoate-sensitive, protein-dependent transport of α-ketoisocaproate into the proteoliposomes was observed. Significant inhibition of α-ketoisocaproate transport was observed in the reconstituted system with branched-chain α-keto acids (64-83%) and the related carboxylates α-ketocaproate (58%) and α-ketovalerate (49%), but not with substrates for the pyruvate carrier (<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 α-keto acid transporter represents an important first step towards purification and molecular characterization of this anion carrier.