Radiometric and LC-MS/MS Assays for INMT Activity

Protein sources for enzyme assays included tissue extracts (rabbit lung, rat brain, rat lung) and recombinant GST-INMTs (rat, rabbit and human). Enzyme assays were conducted in 1.5 mL microcentrifuge tubes and contained approximately 50 µg protein (stock protein concentrations ranged from 4.1 to 6.9 mg/mL for rat brain, 10.6–14.6 mg/mL for rat and rabbit lung, and 0.75–1.05 mg/mL for recombinant GST-INMTs). Radiometric enzyme assays utilized 4 mM tryptamine (Sigma Aldrich) or NMT (Cayman Chemical) along with 2.3 nmol C¹⁴-SAM (specific activity 52.6 mCi/mmol, PerkinElmer Inc.) in a final reaction volume of 150 µL with sodium phosphate buffer. Assay components were combined and incubated at 37 °C for 60 min, and reactions were stopped by adding 1 mL ice cold 0.5 M borate buffer (pH 9.5). To extract reaction products, the reaction mixtures were transferred to 15 mL Falcon tubes with 6 mL of a 97:3 toluene:isoamyl alcohol (T:IAA) solution and tubes were vortexed for three bursts of 5 s each and centrifuged at 1,650 g for 3 min. For quantification, 4 mL of the T:IAA organic layer was added to scintillation vials containing 5 mL scintillation cocktail (Ultima Gold; PerkinElmer Inc.), and radioactivity was counted for 1 min on a Beckman LS Scintillation Counter. Tubes with substrate (tryptamine) omitted served as background controls. Specific activity (SA) was calculated by subtracting the average CPM of 3 background controls for each condition and dividing the resulting CPM value by the amount of protein (µg) used. Results are presented as averages of triplicate experiments in both brain and lung tissues for n = 6 rats per genotype, and as 6 replicate experiments for each GST-INMT condition. Non-radioactive assays were carried out for analysis via uHPLC-MS/MS analysis. These assays utilized 1 mM tryptamine or NMT with 33 µM SAM in a final reaction volume of 150 µL with sodium phosphate buffer. Assay components were combined and incubated at 37 °C for 60 min. Tubes with substrate omitted served as background controls. Reaction products were immediately extracted by adding 1 mL T:IAA and vortexed. 950 µL of the organic phase was transferred to a new 1.5 mL tube, and the organic solvent was evaporated on a bench top VacuFuge™ concentrator (Eppendorf) at room temperature for 2 h. The resulting products were resuspended in 1 mL 30% methanol, vortexed for 3 min, and analyzed via uHPLC-MS/MS (see below for “Methods”). Samples that had NMT as a substrate were further diluted tenfold in 30% methanol. Assays with tissue extracts were run in duplicate for each rat (n = 6 per genotype), and assays with GST-INMT extracts were run in triplicate for each condition, with empty vector GST transformations serving as background controls. Sample identities were blinded by L.C. for uHPLC-MS/MS analysis conducted by N.G.G.