Purification and characterization of rat lens pyrroline-5-carboxylate reductase

Takashi Shiono, Peter F. Kador, Jin J. Kinoshita

Research output: Contribution to journalArticlepeer-review

13 Scopus citations


Δ1-Pyrroline-5-carboxylate reductase (L-proline: NAD(P)+ 5-oxidoreductase, EC has been purified from the rat lens anf biochemically characterized. Purification steps included ammonium sulface fractionation, affinity chromatography on Amicon Matrex Orange A, and gel filtration with Sephadex G-200. These steps were carried out at ambient temperature (22°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 Δ1-purroline-5-carboxylate reductase may be regulated by the energy level and redox state of the lens.

Original languageEnglish (US)
Pages (from-to)72-78
Number of pages7
JournalBBA - General Subjects
Issue number1
StatePublished - Mar 19 1986
Externally publishedYes


  • (Rat lens)
  • Enzyme kinetics
  • Pyrroline-5-carboxylate reductase

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology


Dive into the research topics of 'Purification and characterization of rat lens pyrroline-5-carboxylate reductase'. Together they form a unique fingerprint.

Cite this