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.
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Research Article|
July 15 1989
Purification, oligomerization state and malate sensitivity of maize leaf phosphoenolpyruvate carboxylase
G A L McNaughton;
G A L McNaughton
*Department of Biochemistry, University of Glasgow, Glasgow G12 8QQ, U.K.
†Department of Botany, University of Glasgow, Glasgow G12 8QQ, U.K.
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C A Fewson;
C A Fewson
*Department of Biochemistry, University of Glasgow, Glasgow G12 8QQ, U.K.
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M B Wilkins;
M B Wilkins
†Department of Botany, University of Glasgow, Glasgow G12 8QQ, U.K.
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H G Nimmo
H G Nimmo
*Department of Biochemistry, University of Glasgow, Glasgow G12 8QQ, U.K.
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Publisher: Portland Press Ltd
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 1989 London: The Biochemical Society
1989
Biochem J (1989) 261 (2): 349–355.
Citation
G A L McNaughton, C A Fewson, M B Wilkins, H G Nimmo; Purification, oligomerization state and malate sensitivity of maize leaf phosphoenolpyruvate carboxylase. Biochem J 15 July 1989; 261 (2): 349–355. doi: https://doi.org/10.1042/bj2610349
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