Branched-chain α-keto acid dehydrogenase kinase

This chapter presents the procedure for the assay of branched-chain α-keto acid dehydrogenase (BCKDH) kinase. BCKDH kinase catalyzes phosphorylation and inactivation of the BCKDH complex, an intramitochondrial multienzyme complex responsible for regulating branched-chain amino acid catabolism. BCKDH kinase exists bound to the complex and is subject to both short-term and long-term regulation of its activity. Before isolation of the kinase had been accomplished, only its enzyme activity had been identified as a reaction catalyzed by the purified BCKDH complex. Thereafter, it was recognized that dithiothreitol (DTT), which was routinely used in buffers during purification steps, is critically important for binding of the kinase to the complex. Purification of the kinase was further facilitated by the development of a method for isolation of the BCKDH complex based on phenyl-Sepharose chromatography. BCKDH kinase remains tightly associated with the complex during chromatography on this matrix.