Premium
Pyruvate Dehydrogenase Activity in Osmotically Shocked Rat Brain Mitochondria: Stimulation by Oxaloacetate
Author(s) -
Haas Richard H.,
Thompson Geoffrey,
Morris Bernard,
Conright Kelly,
Andrews Torre
Publication year - 1988
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.1471-4159.1988.tb02966.x
Subject(s) - citrate synthase , pyruvate dehydrogenase complex , thiamine pyrophosphate , biochemistry , pyruvate decarboxylation , pyruvate dehydrogenase phosphatase , mitochondrion , oxoglutarate dehydrogenase complex , chemistry , coenzyme a , biology , enzyme , cofactor , reductase
Pyruvate dehydrogenase complex activity (PDHC) measured by CO 2 release isotopic assay has generally been much lower than activity measured by the spec‐trophotometric arylamine acetyltransferase assay (ArAT). Decarboxylation of [l‐ 14 C]pyruvate was measured in osmotically shocked rat brain cortical mitochondria. Activity is dependent on the concentration of the substrate pyruvate. Activity of 74.6 units ± 12.3 SD (n = 22) was observed at 4 m M pyruvate (1 unit = 1 nmol pyruvate decarboxy‐lated/min/mg protein). Activity was dependent on added NAD, CoA, and thiamine pyrophosphate, implying increased mitochondrial permeability after osmotic shock. Freeze/thaw with sonication of the mitochondrial preparation reduced PDHC activity to 11.5 units ± 3.0 SD (n = 4). Oxaloacetate produced a marked stimulation of activity. The optimal assay contained 3 m M oxaloacetate, and without oxaloacetate activity fell to 15.4 units ± 9.9 SD (n = 8). These studies highlight the importance of optimal substrate concentrations in the CO 2 release isotopic PDHC method. Higher PDHC activity is found with intact mitochondria and thus activity values should be interpreted in the light of the presence or absence of intact mitochondria in individual preparations.