The incretins glucose-dependent insulinotropic polypeptide (GIP 1-42 ) and glucagon-like peptide-1-(7-36)-amide (GLP-1 7-36 ), 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 serumincubated 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 IVcatalyzed incretin hydrolysis to be calculated. The binding constants (K m ) of these incretins to purified porcine kidney-derived enzyme were 1.8 ؎ 0.3 and 3.8 ؎ 0.3 M, whereas the binding constants observed in human serum were 39 ؎ 29 and 13 ؎ 9 M for glucose-dependent-insulinotropic polypeptide and glucagon-like peptide-1-(7-36)-amide respectively. The large range of K m 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.Incretins are hormones of the enteroinsular axis, which potentiate the actions of glucose on the endocrine pancreas (1). The most potent known incretins are glucose-dependent insulinotropic polypeptide (GIP 1 and truncated forms of glucagon-like peptide-1 (GLP-1 7-36 -amide and GLP-1 7-37 ); both are members of the glucagon family of hormones sharing considerable N-terminal sequence homology (2, 3). Both hormones are released from the gut in response to ingested nutrients and were recently shown to be substrates of the circulating exopeptidase dipeptidyl peptidase IV (DP IV, EC 3.4.14.5) (4, 5). This enzyme is a highly specific protease, preferentially hydrolyzing peptides with N-terminal Xaa-Pro and Xaa-Ala motifs (6). Hydrolysis of GIP 1-42 and GLP-1 7-36 by DP IV yields GIP 3-42 and GLP-1 9 -36 and the dipeptides Tyr-Ala and His-Ala, respectively. Activation or inactivation of biologically active peptides is frequently associated with DP IV catalysis. Work by ourselves and others (7,8) has demonstrated that GIP 3-42 and GLP-1 9 -36 are biologically inactive, and it has been hypothesized that serum degradation of GIP 1-42 and GLP-1 7-36 by DP IV is the primary step in the metabolism of these hormones in the circulation (4, 5, 9). In 1993 Mentlein and co-workers (4) reported on the kinetics of enzymatic degradation of GIP 1-42 and GLP-1 7-36 by purified human placental DP IV, as determined by high performance liquid chromatography (HPLC), and suggested that this may be a physiologically important pathway for the degradation of these hormones. This ...

Glutaminyl cyclase (QC, EC 2.3.2.5) catalyzes the formation of pyroglutamate residues from glutamine at the N-terminus of peptides and proteins. In the current study, human QC was functionally expressed in the secretory pathway of Pichia pastoris, yielding milligram quantities after purification from the supernatant of a 5 L fermentation. Initial characterization studies of the recombinant QC using MALDI-TOF mass spectrometry revealed correct proteolytic processing and N-glycosylation at both potential sites with similar 2 kDa extensions. CD spectral analysis indicated a high alpha-helical content, which contrasts with plant QC from Carica papaya. The kinetic parameters for conversion of H-Gln-Tyr-Ala-OH by recombinant human QC were almost identical to those previously reported for purified bovine pituitary QC. However, the results obtained for conversion of H-Gln-Gln-OH, H-Gln-NH2, and H-Gln-AMC were found to be contradictory to previous studies on human QC expressed intracellularly in E. coli. Expression of QC in E. coli showed that approximately 50% of the protein did not contain a disulfide bond that is present in the entire QC expressed in P. pastoris. Further, the enzyme was consistently inactivated by treatment with 15 mM DTT, whereas deglycosylation had no effect on enzymatic activity. Analysis of the fluorescence spectra of the native, reduced, and unfolded human QC point to a conformational change of the protein upon treatment with DTT. In terms of the different enzymatic properties, the consequences of QC expression in different environments are discussed.

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