Purified human erythrocyte pyrroline-5-carboxylate reductase. Preferential oxidation of NADPH

J Biol Chem. 1989 Jun 5;264(16):9352-8.

Abstract

Pyrroline-5-carboxylate reductase catalyzes the final step in proline synthesis by NAD(P)H-dependent reduction of pyrroline-5-carboxylate. We have purified and characterized this enzyme from human erythrocytes. Purification to homogeneity (approximately 600,000-fold) was accomplished by sonication, ultracentrifugation, 2',5'-ADP-Sepharose affinity chromatography, and DEAE-Sephacel ion exchange chromatography. The enzyme runs as a single band of 30,000 Mr on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Sizing chromatography under nondenaturating conditions demonstrates activity in the 300,000-350,000 Mr range, suggesting that the native enzyme exists as a 10- to 12-mer. The purified enzyme exhibits kinetic characteristics similar to those previously described for whole red cell homogenates. The Vmax is 10-fold higher and the Km for pyrroline-5-carboxylate is 7-fold higher with NADH versus NADPH as cofactor. The affinity for NADPH is 15-fold higher than that for NADH. Erythrocyte pyrroline-5-carboxylate reductase is competitively inhibited by NADP+. Unlike the enzyme from some other sources, erythrocyte pyrroline-5-carboxylate reductase is not inhibited by proline or ATP. Double label studies using [14C]pyrroline-5-carboxylate and [3H]exNADPH in the presence of both NADH and NADPH were performed to determine the preferred source of reducing equivalents. In the presence of physiologic concentrations of pyrroline-5-carboxylate and both pyridine nucleotides, all of the reducing equivalents came from NADPH. We suggest that, in some cell types including human erythrocytes, a physiologic function of pyrroline-5-carboxylate reductase is the generation of NADP+.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 1-Pyrroline-5-Carboxylate Dehydrogenase
  • Enzyme Activation
  • Erythrocytes / enzymology*
  • Humans
  • Kinetics
  • Molecular Weight
  • NAD / metabolism
  • NAD / physiology
  • NADP / metabolism*
  • NADP / physiology
  • Oxidation-Reduction
  • Oxidoreductases Acting on CH-NH Group Donors / blood*
  • Oxidoreductases Acting on CH-NH Group Donors / isolation & purification
  • Oxidoreductases Acting on CH-NH Group Donors / physiology
  • Proline / biosynthesis
  • Proline / isolation & purification

Substances

  • NAD
  • NADP
  • Proline
  • 1-Pyrroline-5-Carboxylate Dehydrogenase
  • ALDH4A1 protein, human
  • Oxidoreductases Acting on CH-NH Group Donors