23] Pyridoxal phosphate-dependent histidine decarboxylase from Morganella AM-15

Abstract

This chapter illustrates assay, purification, and properties of pyridoxal phosphate (PLP)-dependent histidine decarboxylase (HisDCases) from Morganella AM-15. Either manometric or radiometric determination of CO2 released during decarboxylation can be used. In the manometric assay, the higher pH also requires that two-armed Warburg flasks to be used. An acid tip from the second side arm at the end of the reaction period is made to release dissolved CO2. One unit of activity corresponds to release of 1 μmol of CO2 per minute at 37°. Specific activity represents the number of units per milligram of protein. In impure preparations, protein is determined by the Lowry method following dialysis to remove dithiothreitol, if present. All purification steps of HisDCases are carried out at 4°. The enzyme loses activity in the absence of stabilizing agents, therefore all buffers used during purification generally contain 0.1 mM NaEDTA, 0.1 mM dithiothreitol (DTT), and 5 μM PLP. In the absence of sulfhydryl compounds, HisDCase exhibits absorption bands at 416, 333, and 278 nm. The enzyme is resolved and inactivated by dialysis against cysteine and is reconstituted by combination with one PLP per subunit.