Use of sulfhydryl reagents to investigate branched chain alpha-keto acid transport in mitochondria

The goal of this paper was to determine the contribution of the mitochondri al branched chain aminotransferase (BCATm) to branched chain alpha-keto aci d transport within rat heart mitochondria. Isolated heart mitochondria were treated with sulfhydryl reagents of varying permeability, and the data sug gest that essential cysteine residues in BCATm are accessible from the cyto solic face of the inner membrane. Treatment with 15 nmol/mg N-ethylmaleimid e (NEM) inhibited initial rates of alpha-ketoisocaproate (KIC) uptake in re constituted mitochondrial detergent extracts by 70% and in the intact organ elle by 50%. KIC protected against inhibition suggesting that NEM labeled a cysteine residue that is inaccessible when substrate is bound to the enzym e. Additionally, the apparent mitochondrial equilibrium KIC concentration w as decreased 50-60% after NEM labeling, and this difference could not be at tributed to effects of NEM on matrix pH or KIC oxidation. In fact, NEM was a better inhibitor of KIC oxidation than rotenone. Measuring matrix asparta te and glutamate levels revealed that the effects of NEM on the steady-stat e KIC concentration resulted from inhibition of BCATm catalyzed transaminat ion of KIC with matrix glutamate to form leucine. Furthermore, circular dic hroism spectra of recombinant human BCATm with liposomes showed that the co mmercial lipids used in the reconstituted transport assay contain BCAT amin o acid substrates. Thus BCATm is distinct from the branched chain alpha-ket o acid carrier but may interact with the inner mitochondrial membrane, and it is necessary to inhibit or remove transaminase activity in both intact a nd reconstituted systems prior to quantifying transport of alpha-keto acids which are transaminase substrates. (C) 2000 Elsevier Science B.V. All righ ts reserved.