Development of a fast kinetic method for the determination of carboxypeptidase U (TAFIa) using C-terminal arginine containing peptides as substrate.
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.
Willemse
Johan
J
Laboratory of Medical Biochemistry, University of Antwerp, B-2610 Antwerp, Belgium.
Leurs
Judith
J
Verkerk
Robert
R
Hendriks
Dirk
D
D000595:Amino Acid Sequence; D001120:Arginine / Q000378:metabolism*; D001122:Arginine Kinase / Q000302:isolation & purification / Q000378:metabolism; D001920:Bradykinin / Q000378:metabolism; D025901:Carboxypeptidase B2 / Q000032:analysis / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004792:Enzyme Precursors / Q000378:metabolism; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism*; D011770:Pyruvate Kinase / Q000378:metabolism; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013379:Substrate Specificity; D013997:Time Factors; D014405:Tuftsin / Q000737:chemistry / Q000378:metabolism
D004792:Enzyme Precursors; D010455:Peptides; D001120:Arginine; D007770:L-Lactate Dehydrogenase; D011770:Pyruvate Kinase; D001122:Arginine Kinase; D025901:Carboxypeptidase B2; D014405:Tuftsin; D001920:Bradykinin