Supernatants of precipitated samples were used for the analysis of histamine and 1-methylhistamne as described below. The supernatant (100 μL) from non-precipitated samples was transferred to Amicon ultra
10K analytical filters (Millipore, Carrightwohill, Ireland), and centrifuged for 30 min at 14 000 g. Then, 200 μL of the homogenization solution was added to the concentrated samples and centrifuged selleckchem twice (14 000 g, 30 min) to remove residual histamine and 1-methylhistamine before enzyme activity assays were performed. Concentrated samples were adjusted to 200 μL by addition of homogenization solution, and gently mixed. These samples were divided into 100-μL aliquots for either HDC or HNMT assays. The 100-μL sample prepared for the HDC or HNMT assay (see above) was further divided into two halves: one part served as a negative control (without substrate), and the other part was mixed with 50 μL of the HDC reaction mixture, consisting of (final concentrations) 5 μm aminoguanidine, 10 μm pyridoxal phosphate, 1% polyethylene
glycol 300, and 1 mm histidine, diluted in the homogenization solution to initiate the reaction. The reaction mixture was incubated at 37 °C for 60 min, and the reaction was then terminated by the addition of 10 μL of 2 m HClO4; the reaction mixture was centrifuged for 5 min at 15 000 g, and the supernatant was analysed for histamine with high-performance liquid chromatography (HPLC). The pellet was used for the protein Ponatinib in vivo measurement assay. The procedure for HNMT activity measurement was analogous to the HDC activity assay, with a few exceptions. The HNMT reaction mixture consisted of (final concentrations) 100 μm pargyline, 25 μm S-adenosyl-methionine, and 20 μm histamine, diluted
in the homogenization solution to initiate the reaction, and incubated for 30 min at Sclareol 37 °C. After the reaction had been terminated by the addition of 10 μL of 2 m HClO4, the supernatant was analysed for 1-methylhistamine. The pellet was used for the protein measurement assay. Protein pellets were resuspended in 0.1 m phosphate buffer (pH 7.0) by sonication. The total protein concentration was then measured with the bicinchoninic acid protein assay, according to the manufacturer’s instructions (ThermoFisher, Waltham, MS, USA). On the basis of this data, the activity was expressed as mole of product per hour per milligram of protein. The analytical HPLC system consisted of four Shimadzu LC20AD pumps, a Shimadzu SIL-20AC autosampler, a Shimadzu RF-10Axl fluorescence detector, a Shimazdu CBM-20A controller, and lcsolution 1.21 software (Shimadzu, Kyoto, Japan). The dialysis samples were analysed without prior purification. The histamine analysis method was based on online post-column derivatization with o-phthalaldehyde, as described originally in Yamatodani et al. (1985). Briefly, samples were separated on a 4.