5-Oxoprolinase from rat kidney, I. Assay, purification, and determination of kinetic parameters.

Hoppe-Seyler's Zeitschrift für physiologische Chemie
A Wendel, U I Flügge

Abstract

A new assay for the determination of 5-oxoprolinase activity is described. The enzyme 5-oxoprolinase was purified from rat kidney 285-fold to apparent homogeneity, as judged by analytical disc electrophoresis and discontinous polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate. The specific activity of the preparation was 122 mU/mg of protein. A complete initial rate kinetic analysis of the forward reaction catalyzed by 5-oxoprolinase was carried out using 5-oxo-L-proline and MgATP2theta as substrates. The computer-fitted double reciprocal plots showed intersecting patterns indicating a sequential mechanism. The data were fitted by weighted linear regression analysis using the complete equation for bisubstrate reactions. The limiting Michaelis constants for 5-oxoproline and MgATP2theta were calculated to be 31.6 +/- 2.3 muM and 172.7 +/- 11.5muM, respectively. The maximum forward rate is 1.2 +/- 0.02 mumol X min-1; the turnover number 7.0 min-1.

Citations

Mar 17, 1999·Journal of Biochemical and Biophysical Methods·P WeberS Wolf
May 15, 1982·Experientia·J PuenteM Sapag-Hagar
Jan 1, 1988·Archives of Oral Biology·H A WatermanJ Mellema
Mar 15, 1982·Biochemical and Biophysical Research Communications·V GanapathyF H Leibach

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