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A Simple and Accurate Spectrophotometric Assay for Phosphoenolpyruvate Carboxylase Activity

A Simple and Accurate Spectrophotometric Assay for Phosphoenolpyruvate Carboxylase Activity Abstract The rate of phosphoenolpyruvate carboxylase activity measured through the conventional coupled assay with malate dehydrogenase is underestimated due to the instability of oxaloacetate, which undergoes partial decarboxylation into pyruvate in the presence of metal ions. The addition of lactate dehydrogenase to the conventional assay allows the reduction of pyruvate formed from oxaloacetate to lactate with the simultaneous oxidation of NADH. Then, the enzymic determination of substrate and products shows that the combined activities of malate dehydrogenase and lactate dehydrogenase account for all the phosphoenolpyruvate consumed. The net result of the improved assay is a higher V max with no apparent effect on K m. The free divalent cation concentration appears to be the major factor in the control of the rate of oxaloacetate decarboxylation. 2 Permanent address: Laboratoire de Biologie Vegetale IV (CNRS, UA 1180), Universite Pierre et Marie Curie, Paris 75005, France. Recipient of a CNAM/CAB/NATO grant. 1 Supported in part by National Science Foundation grant (DMB 85-15181). This content is only available as a PDF. © 1988 American Society of Plant Biologists This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Plant Physiology Oxford University Press

A Simple and Accurate Spectrophotometric Assay for Phosphoenolpyruvate Carboxylase Activity

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References (23)

Publisher
Oxford University Press
Copyright
Copyright © 2021 American Society of Plant Biologists
ISSN
0032-0889
eISSN
1532-2548
DOI
10.1104/pp.86.2.325
Publisher site
See Article on Publisher Site

Abstract

Abstract The rate of phosphoenolpyruvate carboxylase activity measured through the conventional coupled assay with malate dehydrogenase is underestimated due to the instability of oxaloacetate, which undergoes partial decarboxylation into pyruvate in the presence of metal ions. The addition of lactate dehydrogenase to the conventional assay allows the reduction of pyruvate formed from oxaloacetate to lactate with the simultaneous oxidation of NADH. Then, the enzymic determination of substrate and products shows that the combined activities of malate dehydrogenase and lactate dehydrogenase account for all the phosphoenolpyruvate consumed. The net result of the improved assay is a higher V max with no apparent effect on K m. The free divalent cation concentration appears to be the major factor in the control of the rate of oxaloacetate decarboxylation. 2 Permanent address: Laboratoire de Biologie Vegetale IV (CNRS, UA 1180), Universite Pierre et Marie Curie, Paris 75005, France. Recipient of a CNAM/CAB/NATO grant. 1 Supported in part by National Science Foundation grant (DMB 85-15181). This content is only available as a PDF. © 1988 American Society of Plant Biologists This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Journal

Plant Physiology – Oxford University Press

Published: Feb 1, 1988

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