recombinant enzymes enpp1  (R&D Systems)

Journal: Cardiovascular Diabetology

Article Title: Elevated glucose levels increase vascular calcification risk by disrupting extracellular pyrophosphate metabolism

doi: 10.1186/s12933-024-02502-w

Figure Lengend Snippet: Impact of high glucose on extracellular pyrophosphate metabolism. Aortic smooth muscle cells were cultured for one month in media containing either low (1 g/L) or high (4.5 g/L) glucose. A Measurement of extracellular pyrophosphate levels. B Extracellular pyrophosphate-to-ATP ratio. C , D Analysis of the gene expression of key enzymes involved in extracellular pyrophosphate metabolism, including eNTPD1, eNPP1, and TNAP, from isolated total RNA. (E) Immunoblot analysis of proteins associated with extracellular pyrophosphate metabolism. F , G Quantification of protein levels via ELISA, highlighting significant differences. The data are shown as the mean ± SEM, with data derived from 4 independent experiments, each containing 4 replicate plates. Statistical significance was determined via Student’s t test, with asterisks denoting significance levels: * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: The recombinant enzymes eNPP1 (catalog number 6136-EN) and eNTPD1 (catalog number 4397-EN) were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Cell Culture, Expressing, Isolation, Western Blot, Enzyme-linked Immunosorbent Assay, Derivative Assay

Journal: Cardiovascular Diabetology

Article Title: Elevated glucose levels increase vascular calcification risk by disrupting extracellular pyrophosphate metabolism

doi: 10.1186/s12933-024-02502-w

Figure Lengend Snippet: High glucose levels impair the pyrophosphate-to-phosphate ratio. Aortic smooth muscle cells were incubated for one month in medium containing 1 g/L or 4.5 g/L glucose. A Autoradiograph displaying representative products from the hydrolysis of ATP (1 µmol/L ATP, 10 µCi/mL [γ 32 Pi]ATP) incubated with or without recombinant eNPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1) or eNTPD1 (ectonucleoside triphosphate diphosphohydrolase 1) enzymes. Enzymatic hydrolysis generated radiolabeled 32 PPi (32-pyrophosphate) and 32 Pi (32-phosphate), which, alongside unreacted [γ 32 Pi]ATP, were separated by thin-layer chromatography (TLC), as detailed in the section. B Representative time course of ATP hydrolysis showing the products released over time. C Synthesis of the pyrophosphate 32 PPi via hydrolysis of [γ 32 Pi]ATP (10 µCi/mL; 1 µmol/L ATP) in the absence or presence of 100 µmol/L SBI245 (a specific TNAP inhibitor) or inorganic pyrophosphatase (PPase). D The pyrophosphate-to-phosphate ( 32 PPi/ 32 Pi) ratio was quantified following hydrolysis of [γ 32 Pi]ATP (10 µCi/mL; 1 µmol/L ATP) under various conditions: in the absence of inhibitors (Control), in the presence of an ectonucleoside triphosphate diphosphohydrolase (eNTPD) inhibitor (INH, 200 µmol/L), or with the recombinant enzymes eNPP1 and eNTPD1 (100 ng/mL). Experiments were conducted in media containing either physiological (1 g/L) or elevated (4.5 g/L) glucose concentrations. D) Synthesis of 32 PPi by hydrolysis of [γ 32 Pi]ATP (10 µCi/mL and 1 µmol/L ATP). E Hydrolysis of 32 PPi (10 µCi/mL and 5 µmol/L PPi). The results are shown as the mean ± SEM (4 independent experiments with 4 plates per experiment). Student’s t test ( E, F ) or one-way ANOVA with Tukey’s post hoc test ( C, D ) was used for statistical analysis. Asterisks indicate a statistically significant difference compared with the control group: * P < 0.05; *** P < 0.001. ### Indicates a value of P < 0.001 compared with the control group (1 g/L)

Article Snippet: The recombinant enzymes eNPP1 (catalog number 6136-EN) and eNTPD1 (catalog number 4397-EN) were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Incubation, Autoradiography, Recombinant, Generated, Thin Layer Chromatography, Control

Journal: Cardiovascular Diabetology

Article Title: Elevated glucose levels increase vascular calcification risk by disrupting extracellular pyrophosphate metabolism

doi: 10.1186/s12933-024-02502-w

Figure Lengend Snippet: STZ-treated rats exhibit impaired extracellular pyrophosphate metabolism in the aortic wall. A A representative time course of ATP hydrolysis was conducted using a 1 µmol/L ATP solution containing 10 µCi/mL [γ- 32 P]ATP as a radiotracer. The products of hydrolysis, 32 PPi (32-pyrophosphate), 32 Pi (32-phosphate), and [γ- 32 P]ATP-, were separated and quantified via thin layer chromatography, as outlined in the section. B The synthesis of pyrophosphate (PPi) was analyzed by hydrolyzing 1 µmol/L ATP containing 10 µCi/mL [γ- 32 P]ATP as a radiotracer. The reactions were carried out in the absence or presence of either a specific TNAP inhibitor (SBI-425) or inorganic pyrophosphatase (PPase). C The ratio of 32 PPi to 32 Pi generated by ATP hydrolysis was calculated to assess the efficiency and specificity of pyrophosphate synthesis. D The synthesis of 32 PPi was evaluated by hydrolyzing 1 µmol/L ATP containing 10 µCi/mL [γ- 32 P]ATP. E The release of 32 Pi was measured following the hydrolysis of 5 µmol/L pyrophosphate, which contained 10 µCi/mL 32 PPi as a radiotracer. F Quantification of protein levels via ELISA. G , H Total RNA was isolated from rat aortas to evaluate the expression levels of key enzymes involved in extracellular pyrophosphate metabolism, including eNTPD1 (ectonucleoside triphosphate diphosphohydrolase 1), eNPP1 (ectonucleotide pyrophosphatase/phosphodiesterase 1), and tissue-nonspecific alkaline phosphatase (TNAP) (panel G . Additionally, the expression of calcification-related proteins, such as matrix Gla protein (MGP) and osteopontin (OPN), was assessed (panel H). The data are shown as the mean ± SEM and represent data from 12–16 independent aortas. Statistical analyses were performed via Student’s t test. Asterisks indicate a significant difference with *** P < 0.001

Article Snippet: The recombinant enzymes eNPP1 (catalog number 6136-EN) and eNTPD1 (catalog number 4397-EN) were obtained from R&D Systems (Minneapolis, MN, USA).

Techniques: Thin Layer Chromatography, Generated, Enzyme-linked Immunosorbent Assay, Isolation, Expressing

Journal: Journal of enzyme inhibition and medicinal chemistry

Article Title: Identification of novel pyrrolopyrimidine and pyrrolopyridine derivatives as potent ENPP1 inhibitors.

doi: 10.1080/14756366.2022.2119566

Figure Lengend Snippet: Figure 2. (a) Docking model of 18p bound to ENPP1. 18p (light gold) is represented in stick model and other protein domains are briefly shown in cartoon model. Two zinc (gray) and their coordinating Histidines (cyan) are shown. Several key residues interacting with 18p are shown in blue. (b) The ligand (purple) and protein (brown) were represented in ball (C: black; N: blue; O: red; Zn: cyan) and stick (bond) model, green dash shows hydrogen bonding or metal coordination. Hydrophobic intermolecular interactions are described by half whisker shapes with its amino acid residues.

Article Snippet: Serially diluted ENPP1 inhibitors (usually range from 10 lM to 0.5 nM) are pre-incubated with human recombinant ENPP1 enzyme (R&D systems) at 3 ng/reaction for 5–10min at room temperature (RT).

Techniques: Whisker Assay