Purification and characterization of rat lens pyrroline-5-carboxylate reductase.
delta 1-Pyrroline-5-carboxylate reductase (L-proline:NAD(P)+ 5-oxidoreductase, EC 1.5.1.2) has been purified from rat lens and biochemically characterized. Purification steps included ammonium sulfate fractionation, affinity chromatography on Amicon Matrex Orange A, and gel filtration with Sephadex G-200. These steps were carried out at ambient temperature (22 degrees C) in 20 mM sodium phosphate/potassium phosphate buffer (pH 7.5) containing 10% glycerol, 7 mM mercaptoethanol and 0.5 mM EDTA. The enzyme, purified to apparent homogeneity, displayed a molecular weight of 240 000 by gel chromatography and 30 000 by SDS-polyacrylamide gel electrophoresis. This suggests that the enzyme is composed of eight subunits. The purified enzyme displays a pH optimum between 6.5 and 7.1 and is inhibited by heavy metal ions and p-chloromercuribenzoate. Kinetic studies indicated Km values of 0.62 mM and 0.051 mM for DL-pyrroline-5-carboxylate as substrate when NADH and NADPH respectively were employed as cofactors. The Km values for the cofactors NADH and NADPH with DL-pyrroline-5-carboxylate as substrate were 0.37 mM and 0.006 mM, respectively. With L-pyrroline-5-carboxylate as substrate, Km values of 0.21 mM and 0.022 mM were obtained for NADH and NADPH, respectively. Enzyme activity is potentially inhibited by NADP+ and ATP, suggesting that delta 1-pyrroline-5-carboxylate reductase may be regulated by the energy level and redox state of the lens.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007908:Lens, Crystalline / Q000378:metabolism*; D008297:Male; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000302:isolation & purification*; D011762:Pyrroline Carboxylate Reductases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains
D009243:NAD; D009249:NADP; D000255:Adenosine Triphosphate; D000587:Oxidoreductases Acting on CH-NH Group Donors; D011762:Pyrroline Carboxylate Reductases