Simultaneous purification of the histamine degrading enzymes diamine oxidase and histamine N-methyltransferase from the same tissue

Monoclonal antibodies for human and porcine histamine N-methyltransferase (HMT) facilitate protein expression and localization studies

“…Although a cytosolic localisation of HMT could be deduced from the facts that mammalian HMT polypeptide sequences (Fig. 1c) lack sorting signals for other cellular compartments and that HMT activity is usually associated with the soluble fraction obtained by differential centrifugation of cell lysates [15, 24, 25], we here provide direct immunohistochemical evidence for the subcellular localisation of the protein in man and pig. Our findings are in accordance with results of HMT localisation experiments in human astrocytes reported by Yoshikawa and colleagues [26].…”

“…Both constructs were expressed at high levels and produced largely soluble fusion proteins that were recovered from the supernatants of bacterial lysates after centrifugation for 5 min at 5000 g , 4 °C and purified to near homogeneity by chromatography on glutathione sepharose (GSTrap FF, GE Healthcare, Vienna, Austria) according to manufacturer’s instructions. The purified GST fusion proteins, which had a similar HMT enzymatic activity as the enzyme prepared from kidney tissue [15] were dialyzed against PBS buffer (10 mM NaH 2 PO 4 , pH 7.2, 150 mM NaCl) and used for immunizations.…”

Systematic analysis of histamine and N-methylhistamine concentrations in organs from two common laboratory mouse strains: C57Bl/6 and Balb/c

“…In addition, this method allowed the parallel measurement of N-methylhistamine, the main metabolite of histamine [12], which is generated by the activity of the enzyme histamine-N-methyltransferase. Histamine-N-methyltransferase is a cytosolic enzyme that inactivates intracellular histamine that is either synthezised in the cell, but not stored and, therefore, protected from inactivation, or taken up from the extracellular compartment [13]. Thus, we were able to draw quite a complete picture of histamine production and metabolism in mice with good spatio-temporal resolution.…”