PLANT PHYSIOLOGY , Vol 113, Issue 4 1413-1418, Copyright © 1997 by American Society of Plant Biologists
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WHOLE PLANT, ENVIRONMENTAL, AND STRESS PHYSIOLOGY |
[delta]1-Pyrroline-5-Carboxylate Dehydrogenase from Cultured Cells of Potato (Purification and Properties)
G. Forlani, D. Scainelli and E. Nielsen
Department of Genetics and Microbiology, University of Pavia, I-27100 Pavia, Italy
[delta]1-Pyrroline-5-carboxylate (P5C) dehydrogenase (EC 1.5.1.12), the
second enzyme in the proline catabolic pathway and a catalyst for the
oxidation of P5C to glutamate, was purified from cultured potato (Solanum
tuberosum L. var Desiree) cells. Homogeneous enzyme preparations were
obtained by a three-step procedure that used anion-exchange, adsorption,
and substrate elution chromatography. A 1600-fold purification was
achieved, with a recovery of one-third of the initial activity. The
purified enzyme was characterized with respect to structural, kinetic, and
biochemical properties. It appeared to be an [alpha]-4 tetramer with
subunits of an apparent molecular mass of about 60 kD and had a mildly
acidic isoelectric point value. Potato P5C dehydrogenase had Michaelis
constant values of 0.11 and 0.46 mM for NAD+ and P5C, respectively.
Although NAD+ was the preferred electron acceptor, NADP+ also yielded an
unusually high rate, and thus was found to serve as a substrate. Maximal
activity was observed at pH values in the 7.3 to 8.3 range, and was
progressively inhibited by chloride ions, a finding that strengthens recent
suggestions that hyperosmotic stress negatively modulates in vivo proline
oxidation.
