Purification and characterization of Delta(1)-pyrroline-5-carboxylate reductase isoenzymes, indicating differential distribution in spinach (Spinaciaoleracea L.) leaves

Delta (1)-Pyrroline-5-carboxylate reductase (P5CR) (EC 1.5.1.2. L-proline: NAD(P)-5-oxidoreductase), the second enzyme in the proline biosynthetic pat hway, was purified from spinach (Spinacia oleracea L.) leaves. Following am monium sulfate fractionation, purification was performed by several chromat ographic methods: Blue Cellulofine, DEAE-TOYOPEARL, -Sephacryl S-300 HR, an d 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 chl oroplasts, indicating differential distribution of the isoenzymes, P5CR iso enzymes, P5CR-1 and P5CR-2, are a homopolymer with an apparent molecular ma ss of 310 kDa, consisting of 10 to 12 subunits of about 28.5 kDa, P5CR-1 an d P5CR-2 showed K-m values of 9 and 19 muM for NADPH and values of 0.122 an d 0.162 mM for Delta (1)-pyrroline-5-carboxylate (P5C), respectively We dec ided partial amino acid sequences of PSCR-1 which showed the 70 to 80% homo logy to the deduced amino acid sequences of several plant P5CR cDNAs. Both isoenzymes had much lower affinity for NADH than for NADPH and were inhibit ed by free ATP and Mg2+ ion. The inhibition was partially mitigated when AT P and Mg2+ were added simultaneously to the reaction mixture. Cations at hi gh concentration were inhibitory to P5CR activity, Interestingly, P5CR-2 wa s more stable to heat treatment at 40 degreesC than P5CR-1.