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Development of a fast kinetic method for the determination of carboxypeptidase U (TAFIa) using C-terminal arginine containing peptides as substrate.
- Source :
-
Analytical biochemistry [Anal Biochem] 2005 May 01; Vol. 340 (1), pp. 106-12. - Publication Year :
- 2005
-
Abstract
- Carboxypeptidase U (CPU, TAFIa) is a novel determinant of the fibrinolytic rate. It circulates in blood as an inactive zymogen, procarboxypeptidase U, which is activated during the process of coagulation and fibrinolysis. CPU has a very short half-life at 37 degrees C. Its intrinsic instability complicates the determination of kinetic parameters of different substrates using an endpoint method. We developed a fast kinetic assay for measuring continuously the release of the C-terminal arginine by CPU independent of the nature of the substrate peptide used, allowing us to perform substrate specificity studies of CPU. This method uses arginine kinase, pyruvate kinase, and lactate dehydrogenase as auxiliary enzymes. The CPU activities measured using this kinetic assay were in the range of 97-103% of those determined with our HPLC-assisted reference assay, and the obtained K(m) and k(cat) values for hippuryl-l-arginine and bradykinin were in good accordance with those described in the literature. As expected, no arginine cleaving was seen using dipeptides and peptide substrates with a proline in the penultimate position. The presented kinetic assay enables the fast screening of substrates with a C-terminal arginine and is a valuable new tool for the kinetic evaluation of both synthetic and physiological substrates of CPU.
- Subjects :
- Amino Acid Sequence
Arginine Kinase isolation & purification
Arginine Kinase metabolism
Bradykinin metabolism
Carboxypeptidase B2 analysis
Chromatography, High Pressure Liquid
Enzyme Precursors metabolism
Kinetics
L-Lactate Dehydrogenase metabolism
Pyruvate Kinase metabolism
Reproducibility of Results
Sensitivity and Specificity
Substrate Specificity
Time Factors
Tuftsin chemistry
Tuftsin metabolism
Arginine metabolism
Carboxypeptidase B2 metabolism
Peptides chemistry
Peptides metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 0003-2697
- Volume :
- 340
- Issue :
- 1
- Database :
- MEDLINE
- Journal :
- Analytical biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 15802136
- Full Text :
- https://doi.org/10.1016/j.ab.2005.01.039