Journal: Glycobiology

Article Title: Universal phosphatase-coupled glycosyltransferase assay.

doi: 10.1093/glycob/cwq187

Figure Lengend Snippet: Fig. 1. Phosphatase-coupled glycosyltransferase assay. This strategy can be applied to any glycosyltransferase reaction where the leaving group contains a removable phosphate. (A) Glycosyltransferase reaction with a diphosphonucleotide leaving group can be coupled to an ENTPD, such as CD39L3. (B) Glycosyltransferase reaction with a monophosphonucleotide leaving group can be coupled to a 5′-nucleotidase, such as CD73. The inorganic phosphate released by the coupling phosphatase may be detected using various phosphate detection reagents.

Article Snippet: Biantennary N-linked core pentasaccharide was from V-LABS, Inc. Recombinant human KTELC1, ST6GAL1, CD73, CD39L3, TNAP, MUC-1, TcdB containing the glucosyltransferase domain, other recombinant glycosyltransferases from Table II and Malachite Green Phosphate Detection Kit were from R&D Systems.

Techniques:

Journal: Glycobiology

Article Title: Universal phosphatase-coupled glycosyltransferase assay.

doi: 10.1093/glycob/cwq187

Figure Lengend Snippet: Fig. 2. Common leaving nucleotides treated with different phosphatases/nucleotidases. Common leaving nucleotides of glycosyltransferase reactions, CMP (green), GDP (purple) and UDP (blue), were treated with CD73, CD39L3 and TNAP. All reactions were done in 50 µL of 25 mM Tris, 150 mM NaCl, 5 mM MgCl2 and 5 mM MnCl2 at pH 7.5 in a 96-well plate at room temperature for 5 min. Released phosphate was detected using the Malachite Green Phosphate Detection kit. Reactions or standards with phosphate content >4 nmol were diluted to keep the absorbance within the linear range of the detection kit. The absorbances (ODs) were obtained by multiplying the observed ODs by the dilution factors. In each case, ODs were plotted against the nucleotide inputs. Phosphate standard curves were plotted with a dashed red line for comparison. Treatment with 0.1 µg of CD73 (A), 0.1 µg of CD39L3 (B), 0.1 µg of TNAP (C) or 1 µg of TNAP (D).

Article Snippet: Biantennary N-linked core pentasaccharide was from V-LABS, Inc. Recombinant human KTELC1, ST6GAL1, CD73, CD39L3, TNAP, MUC-1, TcdB containing the glucosyltransferase domain, other recombinant glycosyltransferases from Table II and Malachite Green Phosphate Detection Kit were from R&D Systems.

Techniques: Comparison

Journal: Glycobiology

Article Title: Universal phosphatase-coupled glycosyltransferase assay.

doi: 10.1093/glycob/cwq187

Figure Lengend Snippet: Fig. 5. ST6GAL1 assayed using CD73. Each reaction was coupled to 0.1 µg of CD73. The phosphate content of each well was calculated based on a phosphate standard curve determined side by side. Km and apparent Vmax (V 0 max) were obtained by fitting the data to the Michaelis–Menten equation. (A) Specific activity (SA) against donor substrate CMP-NeuAc in the presence of 1 mM acceptor LN. (B) Specific activity vs. acceptor substrate LN in the presence of 0.2 mM CMP-NeuAc. (C) Activity vs. enzyme dose in the presence of 2 mM CMP-NeuAc and 8 mM LN. The dashed line represents the linear regression line of the data points. The slope of the line represents the specific activity and was taken as the measured Vmax. R, correlation coefficient.

Article Snippet: Biantennary N-linked core pentasaccharide was from V-LABS, Inc. Recombinant human KTELC1, ST6GAL1, CD73, CD39L3, TNAP, MUC-1, TcdB containing the glucosyltransferase domain, other recombinant glycosyltransferases from Table II and Malachite Green Phosphate Detection Kit were from R&D Systems.

Techniques: Activity Assay

Journal: Purinergic Signalling

Article Title: Characterization of the N 6 -etheno-bridge method to assess extracellular metabolism of adenine nucleotides: detection of a possible role for purine nucleoside phosphorylase in adenosine metabolism

doi: 10.1007/s11302-020-09699-x

Figure Lengend Snippet: When N6-etheno-ATP (a) and N6-etheno-AMP (b) were incubated for 30 min at 30 °C in the absence of ecto-nucleotidases, the only chromatographic peaks observed were intact N6-etheno-ATP and intact N6-etheno-AMP, respectively, thus indicating that N6-etheno-ATP and N6-etheno-AMP were chemically stable under these test conditions. When N6-etheno-ATP was incubated for 30 min at 30 °C in the presence of either 20 ng of rhCD39 (c), 80 ng of rhENPP-1 (d), 40 ng of rhENTPD2 (e), or 11 ng of rhENTPD3 (f), N6-etheno-ATP was essentially quantitatively converted to N6-etheno-AMP. When N6-etheno-AMP (g) was incubated for 30 min at 30 °C in the presence of rhCD73 (40 ng), all of the N6-etheno-AMP was recovered as N6-etheno-adenosine (ADO)

Article Snippet: Metabolism of N 6 -etheno-purines by recombinant ecto-nucleotidases Recombinant human CD39 (rhCD39), recombinant human CD73 (rhCD73), recombinant human ecto-nucleotide pyrophosphatase/phosphodiesterase family member 1 (rhENPP-1), recombinant human ectonucleoside triphosphate diphosphohydrolase family member 2 (rhENTPD2), and recombinant human ectonucleoside triphosphate diphosphohydrolase family member 3 (rhENTPD3) were obtained from R&D Systems (Minneapolis, MN; catalog numbers 4397-EN-010, 5795-EN-010, 6136-EN-010, 6087-EN-010, and 4400-EN-010, respectively).

Techniques: Incubation

Journal: Purinergic Signalling

Article Title: Characterization of the N 6 -etheno-bridge method to assess extracellular metabolism of adenine nucleotides: detection of a possible role for purine nucleoside phosphorylase in adenosine metabolism

doi: 10.1007/s11302-020-09699-x

Figure Lengend Snippet: Scatter plots show the percentage (%) of applied substrate (either the natural adenine nucleotide substrate or the corresponding etheno-bridged adenine nucleotide substrate, both at 1 μmol/L) that remained or was recovered as product (either the natural product or corresponding etheno-bridged product) after incubation (5 min at 30 °C) with recombinant human (rh) ecto-nucleotidases a rhENPP-1, b rhENTPD2, c rhENTPD3, d rhCD73, or e rhCD39. For each ecto-nucleotidase, the amount of enzyme incubated with substrate was selected to only partially metabolize the natural adenine nucleotide substrate. eATP = N6-etheno-ATP; eADP = N6-etheno-ADP; eAMP = N6-etheno-AMP; eADO = N6-etheno-adenosine (eADO). *P < 0.05 versus corresponding natural substrate. All individual data points are provided along with the means and SDs

Article Snippet: Metabolism of N 6 -etheno-purines by recombinant ecto-nucleotidases Recombinant human CD39 (rhCD39), recombinant human CD73 (rhCD73), recombinant human ecto-nucleotide pyrophosphatase/phosphodiesterase family member 1 (rhENPP-1), recombinant human ectonucleoside triphosphate diphosphohydrolase family member 2 (rhENTPD2), and recombinant human ectonucleoside triphosphate diphosphohydrolase family member 3 (rhENTPD3) were obtained from R&D Systems (Minneapolis, MN; catalog numbers 4397-EN-010, 5795-EN-010, 6136-EN-010, 6087-EN-010, and 4400-EN-010, respectively).

Techniques: Incubation, Recombinant

Journal: Purinergic Signalling

Article Title: Characterization of the N 6 -etheno-bridge method to assess extracellular metabolism of adenine nucleotides: detection of a possible role for purine nucleoside phosphorylase in adenosine metabolism

doi: 10.1007/s11302-020-09699-x

Figure Lengend Snippet: To determine initial reaction velocities, CD39 (10 ng) was incubated with high concentrations of substrates (25 to 200 μmol/L) for 10 min at 30 °C. In panel a, substrates were either ATP or N6-etheno-ATP and the downstream products (ADP + AMP or N6-etheno-ADP + N6-etheno-AMP) were measured. In panel b, substrates were either ADP or N6-etheno-ADP and the downstream products (AMP or N6-etheno-AMP) were measured. The experiment in panel c was similar to that described for panel b with the exception that the substrates were AMP or N6-etheno-AMP, the enzyme was CD73 (0.25 ng), and the measured products were adenosine and N6-etheno-ADO. eATP = N6-etheno-ATP; eADP = N6-etheno-ADP; eAMP = N6-etheno-AMP. Values represent means ± SDs

Article Snippet: Metabolism of N 6 -etheno-purines by recombinant ecto-nucleotidases Recombinant human CD39 (rhCD39), recombinant human CD73 (rhCD73), recombinant human ecto-nucleotide pyrophosphatase/phosphodiesterase family member 1 (rhENPP-1), recombinant human ectonucleoside triphosphate diphosphohydrolase family member 2 (rhENTPD2), and recombinant human ectonucleoside triphosphate diphosphohydrolase family member 3 (rhENTPD3) were obtained from R&D Systems (Minneapolis, MN; catalog numbers 4397-EN-010, 5795-EN-010, 6136-EN-010, 6087-EN-010, and 4400-EN-010, respectively).

Techniques: Incubation