Abstract
This chapter discusses an assay method, purification, and properties of pyruvate dehydrogenase kinase from bovine kidney. Kinase activity in preparations of the pyruvate dehydrogenase complex can be detected by monitoring inactivation of the complex in the presence of adenosine triphosphate (ATP) and Mg2+ or incorporation of 32P-labeled phosphoryl groups from [γ-32P]ATP into the complex. Quantitative assay of kinase activity is based on measurement of the initial rate of incorporation of 32P-labeled phosphoryl groups into crystalline pyruvate dehydrogenase in the presence of dihydrolipoyl transacetylase (E2). The purification procedure involves resolution of pyruvate dehydrogenase complex at pH 9.0, ammonium sulfate precipitation, treatment with p-hydroxymercuriphenyl sulfonate, and purification and crystallization of pyruvate dehydrogenase. The purified kinase consists of two subunits with molecular weights of about 48,000 and 45,000 as estimated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Pyruvate dehydrogenase kinase requires a divalent cation (Mg2+ or Mn2+) and its activity is not affected by cyclic adenosine monophosphate (AMP) or cyclic guanosine monophosphate (cGMP). Pyruvate dehydrogenase kinase is highly specific for pyruvate dehydrogenase.
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