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Studies on the Pyruvate Dehydrogenase Complex in Brain with the Arylamine Acetyltransferase‐Coupled Assay
Author(s) -
KsiezakReding Hanna,
Blass John P.,
Gibson Gary E.
Publication year - 1982
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1982.tb06643.x
Subject(s) - pyruvate dehydrogenase complex , thiamine pyrophosphate , biochemistry , pyruvate dehydrogenase phosphatase , acetyl coa , monoamine oxidase , dihydrolipoyl transacetylase , enzyme , arylamine n acetyltransferase , pyruvate decarboxylation , pyruvate carboxylase , pyruvate dehydrogenase kinase , fumarase , chemistry , biology , cofactor , acetylation , gene
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 MgCI 2 , 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 the maximum recorded rates of pyruvate flux for brain in vivo . Activities as low as 0.6 nmol/min could be measured. Use of ArAT of different purities (1–2‐fold and 11–%‐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 rat brain, and 10% in mouse liver. Total PDHC activity was unchanged postmortem during storage of mouse brain in situ at +4°C or at ‐20°C for 3 days or at +20°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.