Title : Validation of a specific prolylcarboxypeptidase activity assay and its suitability for plasma and serum measurements - Kehoe_2013_Anal.Biochem_443_232 |
Author(s) : Kehoe K , Verkerk R , Sim Y , Waumans Y , Van der Veken P , Lambeir AM , De Meester I |
Ref : Analytical Biochemistry , 443 :232 , 2013 |
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. |
PubMedSearch : Kehoe_2013_Anal.Biochem_443_232 |
PubMedID: 24036038 |
Kehoe K, Verkerk R, Sim Y, Waumans Y, Van der Veken P, Lambeir AM, De Meester I (2013)
Validation of a specific prolylcarboxypeptidase activity assay and its suitability for plasma and serum measurements
Analytical Biochemistry
443 :232
Kehoe K, Verkerk R, Sim Y, Waumans Y, Van der Veken P, Lambeir AM, De Meester I (2013)
Analytical Biochemistry
443 :232