Just before the amine oxidase activity assays, human ScAT samples were thawed, then 1–2 g was homogenized for 30 seconds with a homogenizer (Tissue Master-125, Omni International, Kennesaw, GA, USA) and sonicated for 10 s (Branson sonifier-150, Danbury, CT, USA) at room temperature in 10 volumes of 200 mM phosphate buffer (pH 7.5). Homogenization at room temperature was mandatory to avoid the formation of a fat cake, which at colder temperatures becomes solid and thereby hampers sample distribution and enzymatic reactions.
Homogenates were immediately distributed into 96-well dark microplates (at approximately 50 µg protein/well) and pre-incubated for 10 min without (control) or with 1 mM semicarbazide (abolishing the SSAO activity), or with the tested methylxanthines and their solubilizing vehicles. Incubation was initiated by the addition of 50 µL of substrate at the indicated final concentrations. A fluorimetric method developed for the MAO assay [31 (link)] was slightly adapted as previously described [32 (link)] to monitor hydrogen peroxide released by the oxidation of 0.05–1 mM benzylamine by ScAT homogenates. Briefly, hydrogen peroxide release was detected by the addition at 50 µL/well of a chromogenic mixture (4 U/mL horseradish peroxidase plus 40 µM of the fluorescent probe Amplex Red), which generates fluorescent resorufin (ex/em: 540/590 nm). Activity of human PrAO was assessed by measuring the fluorescence during a 30-min incubation at 37 °C in 200 mM sodium phosphate buffer (pH 7.5) at a final volume of 200 µL/well. Raw data were collected in a Fluoroskan Ascent microplate reader (Thermo Labsystems, Turku, Finland) and were normalized with reference to a hydrogen peroxide standard curve (from 0.05 to 5 µM H2O2). As previously reported [33 (link)], maximal velocity of native PrAO present in ScAT was reached at 1 mM benylamine. As this was equivalent to an increase of more than four times the baseline (1568 ± 188 vs 337 ± 76 relative units of fluorescence, n = 16, p << 0.001), the majority of the inhibition studies were performed at this benzylamine concentration unless otherwise stated.
Homogenates were immediately distributed into 96-well dark microplates (at approximately 50 µg protein/well) and pre-incubated for 10 min without (control) or with 1 mM semicarbazide (abolishing the SSAO activity), or with the tested methylxanthines and their solubilizing vehicles. Incubation was initiated by the addition of 50 µL of substrate at the indicated final concentrations. A fluorimetric method developed for the MAO assay [31 (link)] was slightly adapted as previously described [32 (link)] to monitor hydrogen peroxide released by the oxidation of 0.05–1 mM benzylamine by ScAT homogenates. Briefly, hydrogen peroxide release was detected by the addition at 50 µL/well of a chromogenic mixture (4 U/mL horseradish peroxidase plus 40 µM of the fluorescent probe Amplex Red), which generates fluorescent resorufin (ex/em: 540/590 nm). Activity of human PrAO was assessed by measuring the fluorescence during a 30-min incubation at 37 °C in 200 mM sodium phosphate buffer (pH 7.5) at a final volume of 200 µL/well. Raw data were collected in a Fluoroskan Ascent microplate reader (Thermo Labsystems, Turku, Finland) and were normalized with reference to a hydrogen peroxide standard curve (from 0.05 to 5 µM H2O2). As previously reported [33 (link)], maximal velocity of native PrAO present in ScAT was reached at 1 mM benylamine. As this was equivalent to an increase of more than four times the baseline (1568 ± 188 vs 337 ± 76 relative units of fluorescence, n = 16, p << 0.001), the majority of the inhibition studies were performed at this benzylamine concentration unless otherwise stated.
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