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

Journal: bioRxiv

Article Title: Requirement of sequential hydrolysis by CD73 and ALP for uptake of vitamin B 2 into cells

doi: 10.1101/2022.07.20.500786

Figure Lengend Snippet: A. Structures of FAD and metabolites of FAD B. FMN and FAD hydrolysis by human bone alkaline phosphatase (ALP) and recombinant human CD73 Riboflavin (RF) and FMN concentrations were measured 15 min after incubation of 10 μM FMN or FAD, respectively, with ALP (10 μg/mL) or CD73 (10 ng/mL). Data represent means ± S.D. ( n = 3). N.D.: Not detectable.

Article Snippet: Human CD73 His-tag was purchased from BPS Bioscience (San Diego, CA).

Techniques: Recombinant, Incubation

Journal: bioRxiv

Article Title: Requirement of sequential hydrolysis by CD73 and ALP for uptake of vitamin B 2 into cells

doi: 10.1101/2022.07.20.500786

Figure Lengend Snippet: A. Time course of RF production from FMN by ALP. The RF concentration was measured after incubation of 10 μM FMN with ALP (10 μg/mL). Data represent means ± S.D. ( n = 3). B. ALP concentration-dependence of RF production from FMN. The RF concentration was measured 5 min after incubation of 10 μM FMN with ALP (2.5–10 μg/mL). Data represent means ± S.D. ( n = 3). C. Michaelis-Menten plot of RF production from FMN by ALP. The RF concentration was measured 5 min after incubation of FMN (0.5–5 μM) with ALP (5 μg/mL). Data represent means ± S.D. ( n = 3). D, G. Time course of FMN production (D) or adenosine production (G) from FAD by CD73. The FMN or adenosine concentration was measured after incubation of 10 μM FAD with CD73 (10 ng/mL). Data represent means ± S.D. ( n = 3). E, H. CD73 concentration-dependence of FMN production (E) or adenosine production (H) from FAD by CD73. The FMN or adenosine concentration was measured 15 min after incubation of 10 μM FAD with CD73 (5–20 ng/mL). Data represent means ± S.D. ( n = 3). F, I. Michaelis-Menten plot of FMN production (F) or adenosine production (I) from FAD by CD73. The FMN or adenosine concentration was measured 15 min after incubation of 10 μM FAD with CD73 (10 ng/mL). Data represent means ± S.D. ( n = 3). When the S.D. is smaller than the symbols, it is not shown.

Article Snippet: Human CD73 His-tag was purchased from BPS Bioscience (San Diego, CA).

Techniques: Concentration Assay, Incubation

Journal: bioRxiv

Article Title: Requirement of sequential hydrolysis by CD73 and ALP for uptake of vitamin B 2 into cells

doi: 10.1101/2022.07.20.500786

Figure Lengend Snippet: A. Comparison of inhibitory effect of various compounds on hydrolysis of 1 μM FMN and 1 μM AMP by ALP. FMN hydrolysis activities were measured by RF production from FMN; AMP hydrolysis activities were measured by adenosine production from AMP. The concentration of the inhibitors was 10 μM, except for α,β-methylene adenosine 5′-diphosphate (APCP; 2 μM) and levamisole (1 mM). Data represent means ± S.D. ( n = 3). When the S.D. is smaller than the symbols, it is not shown. B. Comparison of inhibitory effects of various compounds on hydrolysis of 10 μM FAD and 4 μM AMP by CD73. FAD hydrolysis activities were measured by FMN production from FAD; AMP hydrolysis activities were measured by adenosine production from AMP. The concentration of the inhibitors was 10 μM, except for APCP (2 μM) and levamisole (1 mM). Data represent means ± S.D. ( n = 3). When the S.D. is smaller than the symbols, it is not shown. C. Comparison of inhibitory effects of various compounds on hydrolysis of 1 and 4 μM AMP by ALP and CD73, respectively. AMP hydrolysis activities were measured by adenosine production from AMP. The concentration of the inhibitors was 10 μM, except for APCP (2 μM) and levamisole (1 mM). Data represent means ± S.D. ( n = 3). When the S.D. is smaller than the symbols, it is not shown.

Article Snippet: Human CD73 His-tag was purchased from BPS Bioscience (San Diego, CA).

Techniques: Concentration Assay

Journal: bioRxiv

Article Title: Requirement of sequential hydrolysis by CD73 and ALP for uptake of vitamin B 2 into cells

doi: 10.1101/2022.07.20.500786

Figure Lengend Snippet: A. ALP activities in phosphatidylinositol glycan anchor biosynthesis class T (PIGT)-expressing (PIGT+) or vector-transfected (PIGT−) SH-SY5Y cells. ALP activities were measured in lysates of PIGT+ and PIGT− cells. The ALP activity is expressed in terms of the amount of placental ALP (PLAP) in the kit, which was used as a positive control. Data represent means ± S.D. ( n = 4). ** Indicates a significant difference ( p < 0.01) compared with PIGT+ cells. B. CD73 activities in PIGT+ or PIGT− SH-SY5Y cells. CD73 activities were measured by APCP-sensitive adenosine production from AMP in lysate of PIGT+ or PIGT− SH-SY5Y cells. Data represent means ± S.D. ( n = 3). ** Indicates a significant difference ( p < 0.01) compared with PIGT+ cells. C. Surface expression of CD73, ALP, and CD59 on PIGT− and PIGT+ SHSY5Y cells. MFI (mean fluorescent intensity) of PIGT+ vs PIGT-; CD73, 177 vs 82; ALP, 472 vs 102; CD59, 10093 vs 77) The analysis was repeated at least three times. D. Residual amount of vitamin B 2 in medium after 24 h of cultivation of PIGT+ or PIGT− SH-SY5Y cells in medium containing FMN, FAD, RF, or no vitamin B 2 . The residual amount of vitamin B 2 in medium is expressed as the percentage of the total vitamin B 2 concentration (FAD + FMN + RF) in medium incubated without cells. The data represent means for FAD (white), FMN (gray), and RF (black). Error bars represent the S.D. for total vitamin B 2 . ** Indicates a significant difference ( p < 0.01) in the residual amount of total vitamin B 2 in the medium. ND: Not detectable. E. Intracellular vitamin B 2 amount after 24 h of cultivation of PIGT+ or PIGT− SH-SY5Y cells in medium containing FMN, FAD, RF, or no vitamin B 2 . The data represent means for FAD (white), FMN (gray), and RF (black). Error bars represent the S.D. for total vitamin B 2 . ** Indicates a significant difference ( p < 0.01) in the total vitamin B 2 concentration in cells. #, ## indicates a significant difference ( p < 0.05, p < 0.01, respectively) in FMN concentration in cells. †† indicates a significant difference ( p < 0.01) in FAD concentration in cells. ND: Not detectable. F. PLP and PL concentration in medium after 24 h of cultivation of PIGT+ or PIGT− SH-SY5Y cells in medium containing FMN, FAD, RF, or no vitamin B 2 . The data represent means for PLP (gray) and PL (black). Error bars represent the S.D. of the sum of PLP and PL. ** Indicates a significant difference ( p < 0.01) in the sum of PLP and PL in the medium. ND: Not detectable. G. Correlation between intracellular FMN concentration and the sum of PL and PLP concentration after 24 h of cultivation of PIGT+ (closed circles) or PIGT− (open circles) SH-SY5Y cells in medium containing FMN, FAD, RF, or no vitamin B 2 . The data represent means ± S.D. ( n = 3). Statistical analysis: Student’s t-test for (A, B); ANOVA followed by the Tukey–Kramer test for (D, E and F); Pearson correlation for (G).

Article Snippet: Human CD73 His-tag was purchased from BPS Bioscience (San Diego, CA).

Techniques: Expressing, Plasmid Preparation, Transfection, Activity Assay, Positive Control, Concentration Assay, Incubation

Journal: bioRxiv

Article Title: Requirement of sequential hydrolysis by CD73 and ALP for uptake of vitamin B 2 into cells

doi: 10.1101/2022.07.20.500786

Figure Lengend Snippet: A, B. Cells were incubated in the measuring plates with vitamin B 2 -depleted medium for 4 days. The medium was changed to the indicated conditions and the cells were further incubated for 24 h. Then, O 2 consumption was measured. Oligomycin is an ATP synthase inhibitor; FCCP (carbonilcyanide p -triflouromethoxyphenylhydrazone) is an uncoupler; rotenone is a complex I inhibitor; and antimycin A is a complex III inhibitor. The data represent means ± S.D. ( n = 2). Representative data from two independent experiments are shown. C. Scheme of hydrolysis of FAD and FMN by CD73 and alkaline phosphatase for its uptake into cells, vitamin B 6 metabolism and mitochondria function

Article Snippet: Human CD73 His-tag was purchased from BPS Bioscience (San Diego, CA).

Techniques: Incubation