Kabuli chickpeas were provided by the Hebrew University of Jerusalem, Israel. Pepsin (MEROPS ID: A01.071), pancreatin and stem bromelain (EC 3.4.22.32) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The chromogenic substrate Gly-Pro-pNA and dipeptidyl peptidase IV (EC 3.4.14.5), isolated from porcine kidney, were obtained from Sigma–Aldrich (St. Louis, MO, USA). α-amylase (EC 3.2.1.1), isolated from porcine pancreas, was also obtained from Sigma-Aldrich (St. Louis, MO, USA).
Gly pro pna
Manufactured by Merck Group
Sourced in United States
Gly-Pro-pNA is a synthetic substrate used in biochemical assays to measure the activity of enzymes that cleave proteins or peptides containing the sequence Gly-Pro. It provides a simple and quantitative method for monitoring the catalytic activity of such enzymes.
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Lab products found in correlation
Spectramax m2, by Molecular Devices (2 mentions)
Stem bromelain, by Merck Group (1 mentions)
Dipeptidyl peptidase 4, by Merck Group (1 mentions)
Pepsin, by Merck Group (1 mentions)
α amylase, by Merck Group (1 mentions)
P nitroaniline, by Merck Group (1 mentions)
Leu pna, by Merck Group (1 mentions)
Neubauer improved counting chamber, by Marienfeld (1 mentions)
Powerwave xs, by Agilent Technologies (1 mentions)
Synergy 2, by Agilent Technologies (1 mentions)
Dpp 4, by Merck Group (1 mentions)
Bio plex magpix, by Bio-Rad (1 mentions)
Bio plex pro diabetes assay, by Bio-Rad (1 mentions)
12 protocols using gly pro pna
1
Chickpea Enzyme Digestion and Characterization
2
Quantitative Assay of DPP-4 Activity
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DPP-4 activity was determined according to the cleavage rate of p-nitroaniline (pNA; Sigma) using the synthetic substrate H-glycyl-prolyl-p-nitroanilide (Gly-Pro-pNA; Sigma) [14 (link)]. Briefly, 100 µl of assay mixture was prepared containing 50 mM Tris (pH 8.0) and the protein sample from cells or mice tissues, or cell culture medium. The reaction was initiated by the addition of a 5 mM Gly-Pro-pNA substrate solution to a final concentration of 2.5 mM. After incubation for 10~30 min at 37℃, the absorbance of the sample in each well was measured at 405 nm using a plate reader. DPP-4 activity was expressed as the amount of cleaved pNA released per minute per ml (nmol/min/ml) for the culture medium and as the amount of pNA released per minute per weight (nmol/min/mg) for cells/tissues.
3
Comprehensive Peptidase Activity Assay
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Stationary phase cells grown overnight in MR3i broth and counted by a Neubauer improved counting chamber (Marienfeld, Lauda-Königshofe, Germany) to obtain a cell suspension of 109 cell/mL concentration, were harvested by centrifugation at 12,000× g for 5 min, washed twice with sterile 50 mM potassium phosphate buffer, pH 7.0, and re-suspended in the same buffer. Two general aminopeptidase (PepNC), proline iminopeptidase (PepI), glutamyl aminopeptidase (PepA) and X-prolyl dipeptidyl aminopeptidase (PepX) activities were measured according to Macedo et al. [29 (link)] using Lys-p-nitroanilide (pNA), Leu-p-NA, Pro-p-NA, Glu-p-NA and Gly-Pro-p-NA (Sigma Aldrich,) respectively, as synthetic substrates. The assay mixture contained 30 μL of a 20 mM, synthetic substrate methanol solution; 195 μL of 50 mM potassium phosphate buffer, pH 7.0; 95 μL of 0.05% (w/v) sodium azide solution; and 75 μL of cell suspension. After incubation at 30 °C for 4 h, the reaction was stopped by addition of 900 μL of 10% (v/v) acetic acid. The release of p-nitroanilide (p-NA) was measured spectrophotometrically at 410 nm after centrifugation of the reaction mixture at 12,000 × g for 5 min. To determine the peptidase activity, the data obtained were compared to a calibration curve prepared using p-NA at concentrations in the range 0.1–20.0 mM.
4
Measuring CD26 Peptidase Activity
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The CD26 peptidase activity (U/1,000 cells; 1 U=1 pmol ρ-nitroanilide per minute) of the cells was measured in 96-well microplates using the chromogenic substrate Gly-Pro-p-nitroanilide (Gly-Pro-pNA, Sigma)35 (link).
5
Spinal Cord DPP IV Activity Assay
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The rats were euthanized and the spinal cord was dissected and homogenized. The supernatants were collected for DPP IV activity assay. DPP IV activity of the spinal cord was measured by using the chromogenic substrate Gly-Pro-p-nitroanilide (Gly-Pro-pNA; Sigma) as previously reported [24] . 100 µl of supernatant was incubated at 37°C with 1.5 mM Gly-Pro-pNA in 100 µl of phosphate-buffered saline containing 10 mg/ml bovine serum albumin. Absorbance was measured at 405 nm on a microplate reader at 3-min intervals for a total of 60 min, and the number of picomoles of pNA formed was calculated by comparison to a pNA standard curve. The results were plotted as picomoles of pNA versus time, giving a measure of DPP IV activity expressed as picomoles per minute. The peak pNA production rate was used for analysis between groups.
6
CD26 Activity Assay with Gly-Pro-pNA Substrate
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In a flat bottom 96 well plate, (Greiner Bio-One, Kremsmünster, Austria), 5 U/L purified natural human CD26 was incubated with 500 µM of the substrate Gly-Pro-p-nitroanilide (Gly-Pro-pNA; Sigma-Aldrich), with or without GAG, in 0.22 µm-filtered 75 mM Tris-HCl buffer (pH 8.3). Evolution of the optic density (OD) at 405 nm was followed using a spectrophotometer (BioTek PowerWave XS, Winooski, VT, USA) and represented the kinetics of CD26-mediated enzymatic conversion of colorless Gly-Pro-pNA into yellowish pNA. OD measurements were performed at 37 °C every 5 min for 3 h. CD26 activity curves were constructed by plotting OD values against time. Slopes of activity curves reflected the number of converted substrate molecules per min and were used to calculate enzymatic activities in U/L using the Lambert-Beer law. CD26 activity assays were also used to investigate the CD26 activity of PHA-activated T cells. Briefly, 104 to 3 × 106 T cells per mL in 75 mM Tris-HCl buffer (pH 8.3), with or without 20 µM to 2 mM sitagliptin (Merck Sharpe & Dohme (MSD) Whitehouse Station, NJ, USA), were incubated with 500 µM Gly-Pro pNA and the same protocol was followed.
7
Renal DPP4 Activity Assay
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For DPP4 activity, 2 μL of kidney homogenate extracts were diluted with 130 mL of HEPES buffer (10 mM) pH 7.6 and incubated for 10 min at 37°C in 96‐well plates. A total of 100 μL of Gly‐Pro‐pNA (4‐nitroaniline, Sigma Aldrich, Germany) was added as DPP4 substrate (1 mM solution) and kinetics (20 min, every 60 s) were detected at 405 nm. The reaction was observed to be linear up until 11 min; thus, this timeframe was used to assess renal DPP4 activity. Because Gly‐Pro‐pNA is an artificial DPP4 substrate and high protease activity is present in the kidney, we added 10 μM sitagliptin to a parallel set of extracts, to define DPP4‐specific activity baseline. Values were given as specific DPP4 activity in (δ OD405 nm·min−1) by subtracting sitagliptin‐inhibited baseline. For graphic display and statistical analysis, treatment group‐specific AUC of the linear kinetic period (0–11 min) were calculated (minutes were converted to seconds).
8
Inhibition of DPP4 Enzyme Activity
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FAPI-04, FAPI alkyne 11, and FGlc-FAPI were incubated in concentrations between 1 nM and 1 mM together with 125 mU purified DPP4 (Sigma Aldrich) in 130 mL of HEPES buffer (pH 7.6) for 10 min at 37°C in 96-well microtiter plates. Purified DPP4 with activity between 3.9 and 125 mU was used as standards. After addition of Gly-Pro-pNA (4-nitroaniline, Sigma Aldrich) as DPP4 substrate (100 mL of 1 mM solution) kinetics of enzymatic pNA release (25 cycles in 20 min) were monitored at 405 nm in a microplate ELISAreader (Synergy II, BioTek Instruments Inc.). IC 50 values were calculated using Prism 6.0 software (GraphPad, One-Site Fit logIC 50 ). All measurements were carried out in duplicate.
9
Quantifying DPP-4 Enzymatic Activity
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DPP-4 activity was measured in peripheral plasma by quantification of the cleavage of para-nitroanilide (PNA) from Gly-Pro-PNA (Sigma, St. Louis, MO). Samples (15 μL) were diluted in TRIS-base buffer (50 mM, pH 8.3) and incubated with Gly-Pro-PNA. The enzymatic activity was measured in a kinetic analysis of 30 min at 37 C (380 nm) (SpectraMax M2; Molecular Devices, San Jose, CA) and quantified using a standard curve of free PNA.
10
Spectrophotometric Quantification of DPPIV Enzyme Activity
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DPPIV enzyme activity was measured spectrophotometrically using Gly-L-Pro p-nitroanilide (Gly-Pro-pNA; Sigma-Aldrich) as the DPPIV substrate [32 (link)]. To measure the cellular capacity for ecto-ADA binding, HT-29 cells in 48-well plates were treated with 10 μg/mL calf spleen ADA1 (Worthington Biochemical) in medium for 60 min at 37 °C and then assayed for bound ADA using 1 μg/mL rabbit anti-bovine ADA antibody (Alpha Diagnostic International) and 0.5 μCi/mL 125I-labeled donkey anti-rabbit secondary antibody F(abʹ)2 fragment (Amersham Biosciences), using the procedures previously described [32 (link)].
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