Delta]1-Pyrroline-5-Carboxylate Dehydrogenase from Cultured Cells of Potato (Purification and Properties).

Abstract

[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.