Kinetic parameters and tissue distribution of 5-oxo- l-prolinase determined by a fluorimetric assay

• Published in: Journal of biochemical and biophysical methods Vol. 38; no. 1; pp. 71 - 82
• Main Authors: Weber, Peter, Jäger, Martina, Bangsow, Thorsten, Knell, Gunter, Piechaczek, Katharine, Koch, Judith, Wolf, Sabine
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 13.01.1999
• Subjects: 5-Oxo- l-prolinase; Amidohydrolases - metabolism; Animals; Blood–brain barrier; Brain - enzymology; Chromatography, High Pressure Liquid; Cytosol - metabolism; Dose-Response Relationship, Drug; Enzyme assay; Fluorometry - methods; Glutamic Acid - biosynthesis; Glutamic Acid - chemistry; Glutathione; Kidney - enzymology; Kinetics; Liver - enzymology; Myocardium - enzymology; o-Phthalaldehyde - chemistry; Ortho-phthaldialdehyde; Sulfhydryl Compounds - chemistry; Swine; Time Factors; Tissue Distribution; γ-Glutamyl cycle; Ortho-phthaldialdehyde; γ-Glutamyl cycle; 5-Oxo- l-prolinase; Enzyme assay; Glutathione; Blood–brain barrier
• ISSN: 0165-022X; 1872-857X
• DOI: 10.1016/S0165-022X(98)00039-6

5-Oxo- l-prolinase (5-OPase) catalyses the hydrolysis of 5-oxo- l-proline to glutamate with concomitant stoichiometric cleavage of ATP to ADP, a reaction which is known to be part of the γ-glutamyl cycle—an interrelated series of reactions involved in the synthesis and metabolism of glutathione. As recent studies indicate, this cyclic pathway plays a crucial role in the regulation of amino acid transport. Apparently, the intermediate product 5-oxo- l-proline functions as a second messenger molecule that upregulates the activity of certain amino acid transport systems. Thus, the degradation of 5-oxo- l-proline by 5-OPase leads to the downregulation of this stimulus. In this study, a new sensitive fluorimetric assay for 5-OPase activity was established which is based on the derivatization of glutamate with o-phthaldialdehyde in the presence of thiols and subsequent separation of the products by HPLC. The method is suitable for the screening of chromatography fractions as well as for the determination of the kinetic parameters K m and V max of purified 5-OPase. Additionally, it can be used for the measurement of enzyme activity in crude cell extracts and evaluation of tissue distribution.