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Comparative studies of coupled assays for phosphoenolpyruvate carboxylase
Author(s) -
Wedding Randolph T.,
Kline Katie
Publication year - 1994
Publication title -
physiologia plantarum
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.351
H-Index - 146
eISSN - 1399-3054
pISSN - 0031-9317
DOI - 10.1111/j.1399-3054.1994.tb05326.x
Subject(s) - phosphoenolpyruvate carboxylase , citrate synthase , malate dehydrogenase , pyruvate carboxylase , biochemistry , enzyme , phosphoenolpyruvate carboxykinase , malic enzyme , cofactor , dehydrogenase , citric acid cycle , biology , lactate dehydrogenase , chemistry
Phosphoenolpyruvate carboxylase (PEPC) from higher plants is usually assayed by using malate dehydrogenase (MDH) as a coupling enzyme. To avoid erroneous readings caused by metal ions, which convert oxaloacetate (OAA) to pyruvate, lactic dehydrogenase can be included. Reporting the total NADH used by both coupling enzymes gives the total OAA production. Microbial PEPC has been assayed by employing citrate synthase (CS) as a coupling enzyme which detects the reaction of CoA with Ellman's reagent. Comparable K m values for MgPEP are found with the two assays. When MDH alone is used as the coupling system, the V max value is about 60% larger than the one found with the CS assay. However, when MDH is added to the CS assay without the NADH cofactor, V max is brought back to the same level as that with the NADH‐coupled enzyme. Malate inhibition of PEPC assayed with the CS coupling system is blocked by low concentrations of citrate in the range produced in the assay. High concentrations of citrate inhibit PEPC. Glucose‐6‐phosphate in concentrations higher than 1 m M blocks the response of PEPC to added MDH in the CS assay.