Secondary binding site of dipeptidyl peptidase IV (DPIV)
a dipeptidyl peptidase and binding site technology, applied in the field of secondary binding site of dipeptidyl peptidase iv, can solve the problems of low relative insulin level, gradual loss of insulin secretory capacity, and disease, and achieve the effect of increasing substrate dependent dp iv-selectivity and minimizing side reactions
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example 1
Determination of the Half-Life (t1 / 2)
[0456] Matrix-assisted laser-desorption ionization time of flight mass spectrometry (MALDI-TOF MS) experiments were carried out at 30° C. at pH 7.6 in 0.1 M Tris / HCl (Sigma-Aldrich, Deisenhofen, Germany) buffer with 25 μM peptide solution. The degradation fate of peptides was measured by monitoring the signal intensity of the pseudomolecular ion peaks of parent peptides and N-terminal shorted peptides versus time when incubated with 40 mU procine DP IV, recombinant human DP IV or serum DP IV activity. The enzyme was preincubated with hexapeptide TFTSDY or TFTDDY or the heptapeptide H-Ser-D-Glu-Thr-Gly-D-Val-D-Lys-D-Val-OH (15 min, 30° C., 0,016M, 1:1 with DP IV, the concentration of the hexapeptide or the heptapeptide in the reaction mixture was 160 μM). As control served the preincubation of DP IV with 0.01M Tris-buffer (Sigma-Aldrich, Deisenhofen, Germany). The mass spectrometer employed was a Hewlett-Packard G2025 model with a linear time of...
example 2
Determination of Ki:
[0467] In order to measure the inhibition constant Ki a photometric assay was used The peptides were measured as competitors of the standard substrate GP-4-Nitroanilide. Three different substrate concentrations (0.4 mM to 0.05 mM) were combined with 8 different competitor concentrations (0.5 mM to 2 μM). The reaction was started by addition of 3.5 nM DP IV. Experiments were carried out under standard conditions: 30° C. in pH 7.6 40 mM HEPES (Sigma-Aldrich) buffer. Nitroaniline production was monitored using a HTS 7000+ microplate reader (PerkinElmer, Uberlingen, Germany). The Ki-values were calculated via non-linear regression using the enzyme kinetic program Grafit 4.016 (Erithacus Ltd, UK).
[0468] For a reversible competitive inhibition is to assumed: vi=Vmax*Km[S]+Km(1+[I]Ki)
[0469] Legend. [0470] [I] inhibitor concentration [0471] Ki inhibition constant
example 3
MALDI-TOF Approach
[0472] In order to investigate directly the influence of the test compounds TFTSDY, TFTDDY and H-Ser-D-Glu-Thr-Gly-D-Val-D-Lys-D-Val-OH on the DP IV-catalyzed peptide hydrolysis the MALDI-TOF assay was used.
[0473] As described before (determination of t1 / 2) DP IV and the test compounds were preincubated and the reaction was started by adding the enzyme / hexapeptide mixture to substrate / buffer mix. The control reaction mixture consisted of buffer, enzyme and substrate. From the curves of the first order exponential the initial rate (vi) for the control and the reversible inhibited reaction was calculated.
[0474] For the uninfluenced reaction the Michaelis-Menten-equation was used. [0475] Vi was calculated from plotting the relative substrate concentration versus time. [0476] Km is given, also the substrate concentration. vi=Vmax*KmKm+[S]
[0477] For the reversible inhibited reaction the following reaction was used to calculate Ki: vi=Vmax*Km[S]+Km(1+[I]Ki)
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