Investigation of glucose-dependent insulinotropic polypeptide-(1-42) and glucagon-like peptide-1-(7-36) degradation in vitro by dipeptidyl peptidase IV using matrix-assisted laser desorption/ionization-time of flight mass spectrometry. A novel kinetic approach.
The incretins glucose-dependent insulinotropic polypeptide (GIP1-42) and glucagon-like peptide-1-(7-36)-amide (GLP-17-36), hormones that potentiate glucose-induced insulin secretion from the endocrine pancreas, are substrates of the circulating exopeptidase dipeptidyl peptidase IV and are rendered biologically inactive upon cleavage of their N-terminal dipeptides. This study was designed to determine if matrix-assisted laser desorption/ionization-time of flight mass spectrometry is a useful analytical tool to study the hydrolysis of these hormones by dipeptidyl peptidase IV, including kinetic analysis. Spectra indicated that serum-incubated peptides were cleaved by this enzyme with only minor secondary degradation due to other serum protease activity. Quantification of the mass spectrometric signals allowed kinetic constants for both porcine kidney- and human serum dipeptidyl peptidase IV-catalyzed incretin hydrolysis to be calculated. The binding constants (Km) of these incretins to purified porcine kidney-derived enzyme were 1.8 +/- 0.3 and 3.8 +/- 0.3 microM, whereas the binding constants observed in human serum were 39 +/- 29 and 13 +/- 9 microM for glucose-dependent-insulinotropic polypeptide and glucagon-like peptide-1-(7-36)-amide respectively. The large range of Km values found in human serum suggests a heterogeneous pool of enzyme. The close correlation between the reported kinetic constants and those previously described validates this novel approach to kinetic analysis.
Pauly
R P
RP
Department of Physiology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
Rosche
F
F
Wermann
M
M
McIntosh
C H
CH
Pederson
R A
RA
Demuth
H U
HU
D000818:Animals; D018819:Dipeptidyl Peptidase 4 / Q000378:metabolism*; D004798:Enzymes / Q000097:blood; D005749:Gastric Inhibitory Polypeptide / Q000378:metabolism*; D005934:Glucagon; D052216:Glucagon-Like Peptide 1; D004763:Glucagon-Like Peptides; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D010446:Peptide Fragments / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013552:Swine
D004798:Enzymes; D010446:Peptide Fragments; C101760:gastric inhibitory polypeptide (1-42); C055453:glucagon-like peptide 1 (7-36)amide; D005749:Gastric Inhibitory Polypeptide; D004763:Glucagon-Like Peptides; D052216:Glucagon-Like Peptide 1; D005934:Glucagon; D018819:Dipeptidyl Peptidase 4