Application and evaluation of an NADH-linked spectrophotometric assay for pyruvate dehydrogenase complex using chicken liver homogenates

Abstract

1. A method for measuring the activity of the pyruvate dehydrogenase complex [EC 1.2.4.1] (Hinman and Blass, 1981) has been adapted for use with crude chicken liver homogenates. 2. It was found necessary to remove cytoplasmic lactate dehydrogenase by centrifugation before releasing the complex from the mitochondria by treatment with sodium deoxycholate. 3. Lipoamide dehydrogenase was preferable to phenazine methosulphate as the electron carrier between the complex and NADH. 4. The assay was not suitable for use at 40 degrees C (approximate body temperature) but was satisfactory at 25 degrees C. 5. The assay exhibited the properties expected of pyruvate dehydrogenase complex i.e. a complete dependence on the presence of NAD, coenzyme A and pyruvate and a partial dependence on MgCl2, dithiothreitol and thiamine pyrophosphate. The pH optimum was between 7.8 and 8.0. 6. The mean activity for a group of normally fed young chickens was 654 mumol/g dry weight h and 88 for birds starved overnight.