Title
Validation of a specific prolylcarboxypeptidase activity assay and its suitability for plasma and serum measurements
Author
Faculty/Department
Faculty of Pharmaceutical, Biomedical and Veterinary Sciences. Pharmacy
Publication type
article
Publication
New York, N.Y. ,
Subject
Chemistry
Biology
Source (journal)
Analytical biochemistry. - New York, N.Y.
Volume/pages
443(2013) :2 , p. 232-239
ISSN
0003-2697
ISI
000327279700018
Carrier
E
Target language
English (eng)
Full text (Publishers DOI)
Affiliation
University of Antwerp
Abstract
Prolylcarboxypeptidase (PRCP, EC 3.4.16.2), a lysosomal carboxypeptidase, was discovered 45 years ago. However, research has been hampered by a lack of well-validated assays that are needed to measure low activities in biological samples. Two reversed-phase high-performance liquid chromatography (RP-HPLC) methods for quantifying PRCP activity in crude homogenates and plasma samples were optimized and validated. PRCP activity was determined by measuring the hydrolysis of N-benzyloxycarbonyl-L-proline (Z-Pro)-Phe. The enzymatically formed Z-Pro and Phe were measured independently under different HPLC conditions. The in-house methods showed good precision, linearity, accuracy, and specificity. Based on Michaelis-Menten constants, Z-Pro-Phe was chosen over Z-Pro-Ala as the substrate of preference. Cross-reactivity studies with dipeptidyl peptidases (DPPs) 2, 4, and 9 and prolyl oligopeptidase (PREP) confirmed the specificity of the PRCP activity assay. The average PRCP activity in plasma and serum of 32 healthy individuals was found to be 0.65 +/- 0.02 and 0.72 +/- 0.03 U/L, respectively. Both methods can be used to measure PRCP activity specifically in different biological samples and are well suited to evaluate PRCP inhibitors. These well-validated methods are valuable tools for studying PRCP's role in cardiovascular diseases, stroke, inflammation, and metabolic syndrome. (C) 2013 Elsevier Inc. All rights reserved.
E-info
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