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Gly-Pro-pNA (hydrochloride) Sale

(Synonyms: N-甘氨酰脯氨酰-对硝基苯胺盐酸盐) 目录号 : GC18201

A substrate for DPP IV

Gly-Pro-pNA (hydrochloride) Chemical Structure

Cas No.:103213-34-9

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Sample solution is provided at 25 µL, 10mM.

产品文档

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实验参考方法

Cell experiment [1]:

Cell lines

Mare whey protein hydrolysates

Preparation Method

20 uL of sample solution and 100 uL of substrate solution (H- Gly-Pro-pNA (hydrochloride), final concentration 0.5 mM) were added. Enzyme reaction was started by adding DPP-IV. The absorbance at 405 nm was monitored after the microplate was incubated at 37°C for 0.5 h.

Reaction Conditions

Final concentration 0.5 mM ,37 °C for 0.5 h.

Applications

The degradation rate of the substrate Gly-Pro-pNA (hydrochloride) of DPP-IV was slowed by the addition of Mare Whey proteins.

References:

[1]. Song JJ, Wang Q, et,al. Identification of dipeptidyl peptidase-IV inhibitory peptides from mare whey protein hydrolysates. J Dairy Sci. 2017 Sep;100(9):6885-6894. doi: 10.3168/jds.2016-11828. Epub 2017 Jul 12. PMID: 28711271.

产品描述

Gly-Pro-pNA (hydrochloride) is a chromogenic substrate that can be cleaved by the circulating enzyme, dipeptidyl peptidase IV (DPP IV). Enzyme activity can be quantified by colorimetric detection of free p-nitroanilide at 405 nm. This substrate can be used to screen for DPP IV inhibitors.[5].

Mare whey proteins show good DPP-IV inhibitory activity in vitro,have the able to inhibit DPP-IV from degrading Gly-Pro-pNA (hydrochloride) Gly-Pro-pNA (hydrochloride) [1]. The in vitro DPP-IV inhibitory activities of quinoa protein hydrolysates can determined using Gly-Pro-pNA (hydrochloride) as a substrate[2]. Gly-Pro-pNA (hydrochloride) can also be used to evaluate the activity of DPP-IV in human serum[3]. Demonstrating that DPP IV in diabetic patients is not very different from that in normal conditions[4]. Gly-pro p-nitroanilide hydrochloride was used as a substrate to detect DPPIV level in patients with Polycystic syndrome (PCOS) ,a deregulation of DPP4 serum levels could be an additional characteristic of the metabolic imbalances associated with PCOS[5]

Using Gly-Pro-pNA (hydrochloride) as substrate can assess the enzymatic activity and biochemical status of dipeptidyl peptidase IV (DPP IV)in patients with rheumatoid arthritis[6]

References:
[1]. Song JJ, Wang Q, et,al. Identification of dipeptidyl peptidase-IV inhibitory peptides from mare whey protein hydrolysates. J Dairy Sci. 2017 Sep;100(9):6885-6894. doi: 10.3168/jds.2016-11828. Epub 2017 Jul 12. PMID: 28711271.
[2]. You H, Wu T, et,al. Preparation and identification of dipeptidyl peptidase IV inhibitory peptides from quinoa protein. Food Res Int. 2022 Jun;156:111176. doi: 10.1016/j.foodres.2022.111176. Epub 2022 Mar 19. PMID: 35651037.
[3]. Yeganeh F, Mousavi SMJ, et,al. Association of CD26/dipeptidyl peptidase IV mRNA level in peripheral blood mononuclear cells with disease activity and bone erosion in rheumatoid arthritis. Clin Rheumatol. 2018 Dec;37(12):3183-3190. doi: 10.1007/s10067-018-4268-y. Epub 2018 Aug 22. PMID: 30136129.
[4]. Sun AL, Deng JT, et,al. Dipeptidyl peptidase-IV is a potential molecular biomarker in diabetic kidney disease. Diab Vasc Dis Res. 2012 Oct;9(4):301-8. doi: 10.1177/1479164111434318. Epub 2012 Mar 2. PMID: 22388283.
[5]. Blauschmidt S, Greither T, et,al. Dipeptidyl peptidase 4 serum activity and concentration are increased in women with polycystic ovary syndrome. Clin Endocrinol (Oxf). 2017 Dec;87(6):741-747. doi: 10.1111/cen.13444. Epub 2017 Sep 13. PMID: 28799235.
[6]. Mavropoulos JC, Cuchacovich M, et,al. Anti-tumor necrosis factor-alpha therapy augments dipeptidyl peptidase IV activity and decreases autoantibodies to GRP78/BIP and phosphoglucose isomerase in patients with rheumatoid arthritis. J Rheumatol. 2005 Nov;32(11):2116-24. PMID: 16265688.

Gly-Pro-pNA(盐酸盐)是一种显色底物,可被循环酶二肽基肽酶 IV (DPP IV) 裂解。酶活性可以通过在 405 nm 处游离对硝基苯胺的比色检测来量化。该底物可用于筛选 DPP IV 抑制剂。[5]

马乳清蛋白在体外表现出良好的DPP-IV抑制活性,能够抑制DPP-IV降解Gly-Pro-pNA (hydrochloride) Gly-Pro-pNA (hydrochloride) [1]。以 Gly-Pro-pNA(盐酸盐)为底物,可以测定藜麦蛋白水解物的体外 DPP-IV 抑制活性[2]。 Gly-Pro-pNA(盐酸盐)也可用于评价 DPP-IV 在人血清中的活性[3]。证明糖尿病患者的 DPP IV 与正常情况下的差异不大[4]。以甘氨酰对硝基苯胺盐酸盐为底物检测多囊综合征(PCOS)患者的DPPIV水平,DPP4血清水平失调可能是PCOS相关代谢失衡的另一个特征[5]< /sup>

以Gly-Pro-pNA(盐酸盐)为底物可评估类风湿性关节炎患者二肽基肽酶IV(DPP IV)的酶活性和生化状态[6]

Chemical Properties

Cas No. 103213-34-9 SDF
别名 N-甘氨酰脯氨酰-对硝基苯胺盐酸盐
化学名 glycyl-N-(4-nitrophenyl)-L-prolinamide, monohydrochloride
Canonical SMILES O=C(CN)N1CCC[C@H]1C(NC2=CC=C([N+]([O-])=O)C=C2)=O.Cl
分子式 C13H16N4O4.HCl 分子量 328.8
溶解度 30mg/mL in DMSO, 30mg/mL in DMF, 30mg/mL in Ethanol 储存条件 Store at -20°C,stored under nitrogen
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1 mM 3.0414 mL 15.2068 mL 30.4136 mL
5 mM 0.6083 mL 3.0414 mL 6.0827 mL
10 mM 0.3041 mL 1.5207 mL 3.0414 mL
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Research Update

Mechanism of Gly-Pro-pNA cleavage catalyzed by dipeptidyl peptidase-IV and its inhibition by saxagliptin (BMS-477118)

Arch Biochem Biophys2006 Jan 1;445(1):9-18.PMID: 16364232DOI: 10.1016/j.abb.2005.11.010

Dipeptidyl peptidase-IV (DPP-IV) is a serine protease with a signature Asp-His-Ser motif at the active site. Our pH data suggest that Gly-Pro-pNA cleavage catalyzed by DPP-IV is facilitated by an ionization of a residue with a pK of 7.2 +/- 0.1. By analogy to other serine proteases this pK is suggestive of His-Asp assisted Ser addition to the P1 carbonyl carbon of the substrate to form a tetrahedral intermediate. Solvent kinetic isotope effect studies yielded a D2Okcat/Km=2.9+/-0.2 and a D2Okcat=1.7+/-0.2 suggesting that kinetically significant proton transfers contribute to rate limitation during acyl intermediate formation (leaving group release) and hydrolysis. A "burst" of product release during pre steady-state Gly-Pro-pNA cleavage indicated rate limitation in the deacylation half-reaction. Nevertheless, the amplitude of the burst exceeded the enzyme concentration significantly (approximately 15-fold), which is consistent with a branching deacylation step. All of these data allowed us to better understand DPP-IV inhibition by saxagliptin (BMS-477118). We propose a two-step inhibition mechanism wherein an initial encounter complex is followed by covalent intermediate formation. Final inhibitory complex assembly (kon) depends upon the ionization of an enzyme residue with a pK of 6.2 +/- 0.1, and we assigned it to the catalytic His-Asp pair which enhances Ser nucleophilicity for covalent addition. An ionization with a pK of 7.9 +/- 0.2 likely reflects the P2 terminal amine of the inhibitor hydrogen bonding to Glu205/Glu206 in the enzyme active site. The formation of the covalent enzyme-inhibitor complex was reversible and dissociated with a koff of (5.5 +/- 0.4) x 10(-5) s(-1), thus yielding a Ki* (as koff/kon) of 0.35 nM, which is in good agreement with the value of 0.6 nM obtained from steady-state inhibition studies. Proton NMR spectra of DPP-IV showed a downfield resonance at 16.1 ppm. Two additional peaks in the 1H NMR spectra at 17.4 and 14.1 ppm were observed upon mixing the enzyme with saxagliptin. Fractionation factors (phi) of 0.6 and 0.5 for the 17.4 and 14.1 ppm peaks, respectively, are suggestive of short strong hydrogen bonds in the enzyme-inhibitor complex.

Rapid detection of DPP-IV activity in porcine serum: A fluorospectrometric assay

Anal Biochem2020 Mar 1;592:113557.PMID: 31866290DOI: 10.1016/j.ab.2019.113557

Dipeptidyl peptidase IV (DPP-IV) is an aminopeptidase that cleaves the N-terminal dipeptide from peptides bearing proline or alanine residues. Currently, DPP-IV activity is quantified by spectrophotometric or fluorometric methods, which employ Gly-Pro-pNA and Gly-Pro-AMC respectively, as substrate. However, these methods require high enzyme and substrate concentrations. In this study, we adapted the DPP-IV fluorospectrometric assay using NanoDrop 3300, which requires only nanogram levels of the enzyme (30 ng crude DPP-IV) and considerably low substrate concentrations (100 μM). Fluorescence measurement required a reaction mixture of only 2 μL, thus eliminating the need for microtiter plates or cuvettes.We employed this assay to demonstrate DPP-IV activity in porcine serum for the first time. The enzymatic activity peaked at pH 8.0 in porcine (84 nM/min), human (87 nM/min) and bovine (89.1 nM/min) sera, with the optimum temperature of 37 °C. The enzyme showed maximum activity upon incubation for 40 min at 37 °C. In contrast, activity in the porcine serum was the highest after incubation for 30 min at the same optimized parameters. The IC50 values of diprotin A against DPP-IV from human, porcine, and bovine sera were 7.83, 8.62, 9.17 μM, respectively. The present assay procedure is a convenient, sensitive, accurate and high-throughput method suitable for primary screening of DPP-IV inhibitors.

An endo-acting proline-specific oligopeptidase from Treponema denticola ATCC 35405: evidence of hydrolysis of human bioactive peptides

Infect Immun1994 Nov;62(11):4938-47.PMID: 7523301DOI: 10.1128/iai.62.11.4938-4947.1994

An endo-acting proline-specific oligopeptidase (prolyl oligopeptidase [POPase], EC 3.4.21.26) was purified to homogeneity from the Triton X-100 extracts of cells of Treponema denticola ATCC 35405 (a human oral spirochete) by a procedure that comprised five successive fast protein liquid chromatography steps. The POPase is a cell-associated 75- to 77-kDa protein with an isoelectric point of ca. 6.5. The enzyme hydrolyzed (optimum pH 6.5) the Pro-pNA bond in carbobenzoxy-Gly-Pro-p-nitroanilide (Z-Gly-Pro-pNA) and bonds at the carboxyl side of proline in several human bioactive peptides, such as bradykinin, substance P, neurotensin, angiotensins, oxytocin, vasopressin, and human endothelin fragment 22-38. The minimum hydrolyzable peptide size was tetrapeptide P3P2P1P'1, while the maximum substrate size was ca. 3 kDa. An imino acid residue in position P1 was absolutely necessary. The hydrolysis of Z-Gly-Pro-pNA was potently inhibited by the following, with the Ki(app) (in micromolar) in parentheses: insulin B-chain (0.7), human endothelin-1 (0.5), neuropeptide Y (1.7), substance P (32.0), T-kinin (4.0), neurotensin (5.0), and bradykinin (16.0). Chemical modification and inhibition studies suggest that the POPase is a serine endopeptidase whose activity depends on the catalytic triad of COOH ... Ser ... His but not on a metal. The amino acid sequence around the putative active-site serine is Gly-Gly-Ser-Asn-Pro-Gly. The enzyme is suggested to contain a reactive cysteinyl residue near the active site. Amino acid residues 4 to 24 of the first 24 N-terminal residues showed a homology of 71% with the POPase precursor from Flavobacterium meningosepticum and considerable homology with the Aeromonas hydrophila POPase. The ready hydrolysis of human bioactive peptides at bonds involving an imino acid residue suggests that enzymes like POPase may contribute to the chronicity of periodontal infections by participating in the peptidolytic processing of those peptides.

Murine thymocytes possess specific cell surface-associated exoaminopeptidase activities: preferential expression by immature CD4-CD8- subpopulation

Eur J Immunol1990 Mar;20(3):459-68.PMID: 2108042DOI: 10.1002/eji.1830200302

Murine thymocytes are shown to possess at least three well-defined exo-N-aminopeptidase activities on their surface. One of them cleaves the prolyl bond in the synthetic dipeptide nitroanilide Gly-Pro-pNA (Km 0.95 mM and Vmax 8 nmol/h at pH 7.4 and 37 degrees C) and is specifically inhibited by phenylmethane sulfonyl fluoride, diprotin A, Gly-Pro-Ala and Gly-Pro-Gly-Gly. These data further support identification of this enzyme with a serine exopeptidase dipeptidyl peptidase IV (DPP IV), previously reported to be specific for collagen. The two other forms of N-exopeptidase activities are detected when Ala-pNA and Leu-pNA are used as substrates. Leu-aminopeptidase activity (Km 1.4 mM, Vmax 15 nmol/h) and Ala-aminopeptidase activity (Km 4.0 mM, Vmax 20 nmol/h) are inhibited by inhibitors for thiol- and trypsin-like proteinases, i.e. tosyl lysyl chloromethyl ketone, leupeptin and N-ethylmaleimide. Addition inhibition of Leu-aminopeptidase activity by peptstatin, a known inhibitor of carboxyl proteases, suggests that aminopeptidase activity detected with Leu-pNA is different in part from Ala-aminopeptidase activity. Among the various lymphoid cell populations tested, the three aminopeptidase activities are increased by three- to fourfold in the immature CD4-CD8- thymocyte subset as well as in the thymoma BW5147. In contrast, cortisone-resistant thymocytes, lymph node and spleen cells exhibit levels of activities almost similar to that of unfractionated thymocytes. During ontogeny, the levels of these activities are increased four- to sevenfold on fetal thymocytes (from days 14 to 16). Finally, when thymocytes or spleen cells are cultured with a mitogenic concentration of concanavalin A, their proliferative responses are correlated with an enhancement of the aminopeptidase activities (1.3- to 5-fold). From these results, a correlation between the presence of these peptidases on the cell surface of immature and mature lymphoid cells and biological responsiveness is suggested.

Identification of two isoforms of Pop in the domestic silkworm, Bombyx mori: Cloning, characterization and expression analysis

Gene2018 Aug 15;667:101-111.PMID: 29753046DOI: 10.1016/j.gene.2018.05.021

Two isoforms, Bmpop-a and Bmpop-b, were cloned and characterized, which were found to encode prolyl oligopeptidase (Pop) of the domestic silkworm Bombyx mori. The full lengths of Bmpop-a and Bmpop-b were 2497 and 2508 bp, deducing 707 and 740 amino acids, respectively. Both of them, possessing the typical characteristics of the Pop family of serine proteinase, were detected to be expressed among different tissues and development stages at the transcription and translation levels. Soluble recombinant BmPop-a (rBmPop-a) had oligopeptidase activity toward the substrates, Z-Gly-Pro-pNA, Z-Gly-Pro-AMC and angiotensin I. An inhibition assay showed that the activity of rBmPop-a was significantly inhibited by KYP-2047 and S17092 in vitro. BmPop-b was identified in the molting fluids at three different stages by Western blotting analysis, showing a predominant expression in the integument. Two isoforms of Bmpop gene and other three genes in the renin-angiotensin system (RAS) in the integument were down-regulated by starvation treatments but up-regulated by refeeding. These results suggested that BmPops may play an important role in balancing the molting fluid pressure to guarantee ecdysis normally. This study provides clues for further elucidating the function and regulation mechanisms of two isoforms of Bmpop gene.