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

Takashi Shiono, Peter F Kador, Jin J. Kinoshita

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

Δ 1 -Pyrroline-5-carboxylate reductase (L-proline: NAD(P) + 5-oxidoreductase, EC 1.5.1.2) 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 K m 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 K m 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, K m 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 Δ 1 -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
Volume881
Issue number1
DOIs
StatePublished - Mar 19 1986

Fingerprint

Pyrroline Carboxylate Reductases
NADP
NAD
Lenses
Purification
Rats
Oxidoreductases
Enzymes
Gel Chromatography
Substrates
Gels
Chloromercuribenzoates
Affinity chromatography
Heavy Ions
Mercaptoethanol
Enzyme activity
Fractionation
Heavy Metals
Chromatography
Electrophoresis

Keywords

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

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology

Cite this

Purification and characterization of rat lens pyrroline-5-carboxylate reductase. / Shiono, Takashi; Kador, Peter F; Kinoshita, Jin J.

In: BBA - General Subjects, Vol. 881, No. 1, 19.03.1986, p. 72-78.

Research output: Contribution to journalArticle

Shiono, Takashi ; Kador, Peter F ; Kinoshita, Jin J. / Purification and characterization of rat lens pyrroline-5-carboxylate reductase. In: BBA - General Subjects. 1986 ; Vol. 881, No. 1. pp. 72-78.
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AB - Δ 1 -Pyrroline-5-carboxylate reductase (L-proline: NAD(P) + 5-oxidoreductase, EC 1.5.1.2) 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 K m 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 K m 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, K m 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 Δ 1 -purroline-5-carboxylate reductase may be regulated by the energy level and redox state of the lens.

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