Journal Article

Purification and Characterization of Δ<sup>1</sup>-Pyrroline-5-Carboxylate Reductase Isoenzymes, Indicating Differential Distribution in Spinach (<i>Spinacia oleracea </i>L.) Leaves

Minoru Murahama, Takayuki Yoshida, Fumio Hayashi, Takuya Ichino, Yukika Sanada and Keishiro Wada

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 42, issue 7, pages 742-750
Published in print July 2001 | ISSN: 0032-0781
Published online July 2001 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/pcp/pce093
Purification and Characterization of Δ1-Pyrroline-5-Carboxylate Reductase Isoenzymes, Indicating Differential Distribution in Spinach (Spinacia oleracea L.) Leaves

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Δ1-Pyrroline-5-carboxylate reductase (P5CR) (EC 1.5.1.2. l-proline: NAD(P)-5-oxidoreductase), the second enzyme in the proline biosynthetic pathway, was purified from spinach (Spinacia oleracea L.) leaves. Following ammonium sulfate fractionation, purification was performed by several chromatographic methods: Blue Cellulofine, DEAE-TOYOPEARL, Sephacryl S-300 HR, and POROS QE/M. Two isoenzymes resolved by anion exchange chromatography were designated P5CR-1 and P5CR-2. Only P5CR-2 was purified from the intact chloroplasts, indicating differential distribution of the isoenzymes. P5CR isoenzymes, P5CR-1 and P5CR-2, are a homopolymer with an apparent molecular mass of 310 kDa, consisting of 10 to 12 subunits of about 28.5 kDa. P5CR-1 and P5CR-2 showed Km values of 9 and 19 µM for NADPH and values of 0.122 and 0.162 mM for Δ1-pyrroline-5-carboxylate (P5C), respectively. We decided partial amino acid sequences of P5CR-1 which showed the 70 to 80% homology to the deduced amino acid sequences of several plant P5CR cDNAs. Both isoenzymes had much lower affinity for NADH than for NADPH and were inhibited by free ATP and Mg2+ ion. The inhibition was partially mitigated when ATP and Mg2+ were added simultaneously to the reaction mixture. Cations at high concentration were inhibitory to P5CR activity. Interestingly, P5CR-2 was more stable to heat treatment at 40°C than P5CR-1.

Keywords: Key words: Chloroplast — Isoenzemes — Partial amino acid sequence — Proline biosynthesis — Δ1-Pyrroline-5-carboxylate (P5C) — Spinach (Spinacia oleracea) — Water stress.; Abbreviations: G6PDH, glucose-6-phosphate dehydrogenase; Glc6P, glucose-6-phosphate; P5C, Δ1-pyrroline-5-carboxylate; P5CR, Δ1-pyrroline-5-carboxylate reductase; P5CS, Δ1-pyrroline-5-carboxylate synthetase; PMSF, phenylmethylsulfonyl fluoride.

Journal Article.  6030 words.  Illustrated.

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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