Studies on the pyruvate dehydrogenase complex in brain with the arylamine acetyltransferase-coupled assay

A spectrophotometric assay for the brain pyruvate dehydrogenase complex (PDHC) with arylamine acetyltransferase (ArAT; EC 2.3.1.5) to follow the production of acetyl-CoA has been standardized. Activity was proportional to time and protein. It depended completely on added pyruvate, CoA, NAD, and MgCl2, and partially on thiamine pyrophosphate. Triton X-100, and a sulfhydryl compound. The activities are the highest in the literature for brain PDHC (50 nmol/min/mg protein) and equal to maximum recorded rates of pyruvate flux for brain in vivo. Activities as low as 0.6 nmol/min could be measured. Use of ArAT at different purities (I--2-fold and II--55-fold) allowed convenient measurement of total PDHC (ArAT-I) and of the active form of PDHC (ArAT-II). The proportion of PDHC in the active form was 50% in mouse brain, 30% in brain, and 10% in mouse liver. Total PDHC activity was unchanged postmortem during storage of mouse brain in situ at +4 degrees C or at -20 degrees C for 3 days or at +20 degrees C for 24 h. The relative specific activity of PDHC in cytoplasmic or synaptoplasmic fractions was less than that of two other mitochondrial enzymes, fumarase (EC 4.2.1.2) and monoamine oxidase (EC 1.4.3.4), which argues strongly against the hypothesis of a cytoplasmic PDHC in cholinergic nerve endings.