PMID: 3753884Mar 19, 1986Paper

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

Biochimica Et Biophysica Acta
T ShionoJ H Kinoshita

Abstract

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 NA...Continue Reading

References

Mar 30, 1979·Biochemical and Biophysical Research Communications·J M PhangJ A Williams
Jul 15, 1977·The Biochemical Journal·A HerzfeldW E Knox
Sep 1, 1977·Analytical Biochemistry·R J SmithJ M Phang
Mar 1, 1985·Experimental Eye Research·T ShionoJ H Kinoshita
May 30, 1980·Biochemical and Biophysical Research Communications·G C Yeh, J M Phang
Apr 1, 1981·The Journal of Clinical Investigation·G C YehJ M Phang
Apr 1, 1982·Experimental Eye Research·S HayasakaI Ishiguro
Nov 1, 1980·Experimental Eye Research·F J Giblin, V N Reddy
Jan 29, 2011·Proceedings of the National Academy of Sciences of the United States of America·Diego U FerreiroPeter G Wolynes

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Citations

May 30, 2006·Journal of Molecular Biology·Zhaohui MengZihe Rao
Apr 8, 2006·Protein Expression and Purification·Zhaohui MengZihe Rao
Aug 1, 1996·Journal of Dairy Science·J J BaschH M Farrell
Aug 1, 1991·Archives of Biochemistry and Biophysics·O P ChilsonN R Siegel
Apr 13, 2018·Biochemistry·John J TannerDonald F Becker

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