Purification, oligomerization state and malate sensitivity of maize leaf phosphoenolpyruvate carboxylase

McNaughton, G.A.L., Fewson, C.A., Wilkins, M.B. and Nimmo, H.G. (1989) Purification, oligomerization state and malate sensitivity of maize leaf phosphoenolpyruvate carboxylase. Biochemical Journal, 261(2), pp. 349-355. (doi: 10.1042/bj2610349) (PMID:2775222) (PMCID:PMC1138832)

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Abstract

A method was developed for the purification of phosphoenolpyruvate carboxylase from darkened maize leaves so that the enzyme retained its sensitivity to inhibition by malate. The procedure depended on the prevention of proteolysis by the inclusion of chymostatin in the buffers used during the purification. The purified enzyme was indistinguishable from that in crude extracts as judged by native polyacrylamide-gel electrophoresis. SDS/polyacrylamide-gel electrophoresis followed by immunoblotting, and Superose 6 gel filtration. Gel-filtration studies showed that the purified enzyme and the enzyme in extracts of darkened or illuminated leaves showed a concentration-dependent dissociation of tetrameric into dimeric forms. Purified phosphoenolpyruvate carboxylase and enzyme in crude extracts from darkened leaves were equally sensitive to inhibition by malate (Ki approx. 0.30 mM) under conditions where it existed in the tetrameric or dimeric forms, but the enzyme in crude extracts from illuminated leaves was less sensitive to malate inhibition (Ki approx. 0.95 mM) whether it was present as a tetramer or as a dimer. It is concluded that changes in the oligomerization state of phosphoenolpyruvate carboxylase are not directly involved in its regulation by light.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Nimmo, Professor Hugh
Authors: McNaughton, G.A.L., Fewson, C.A., Wilkins, M.B., and Nimmo, H.G.
College/School:College of Medical Veterinary and Life Sciences > School of Molecular Biosciences
Journal Name:Biochemical Journal
Publisher:Portland Press
ISSN:0264-6021
ISSN (Online):1470-8728

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