Abstract
BCKDC is a rate‐limiting enzyme in the branched‐chain amino acid catabolic pathway. This enzyme complex is regulated by a phosphorylation/dephosphorylation cycle. The specific kinase (branched‐ chain α‐ketoacid dehydrogenase kinase (BDK)) is responsible for inactivation of the complex by phosphorylation. We have developed the method for BCKDC purification from rat liver using hydrophobic interaction column chromatography (Shimomura et al., Arch. Biochem. Biophy., 283, 293‐299 (1990)). We here report the modification of the purification method to obtain the highly purified BCKDC associated with BDK. Using the purified enzyme, we analyzed the inhibition of BDK by TPP at two potassium ion (K+) concentrations. IC50 values of 4.6 and 8.0 µM and inhibition constant values of 3.2 and 16.4 µM were obtained in the presence of 20 and 100 mM K+, respectively. These results suggest that BDK is less sensitive to TPP inhibition under physiological TPP and K+ concentrations.
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