Purification and characterization of l-histidine decarboxylase from mouse mastocytoma P-815 cells.
Histidine decarboxylase was purified from mouse mastocytoma P-815 cells to electrophoretic homogeneity by ammonium sulfate fractionation, dialyses at pH 7.5 and 6.0, chromatographies on DEAE-Sepharose CL-6B, Phenyl-Sepharose CL-4B and Hydroxylapatite, Phenyl-Superose HPLC, Mono Q HPLC, and Diol-200 gel filtration HPLC. Under the assay conditions used, the pure enzyme exhibited a specific activity of 800 nmol/min/mg, which constituted 12,500-fold purification compared to the crude extract, with a 7% yield. The two-step dialysis turned out to be essential for removing the factor(s) which interfered with the enzyme purification. The optimum pH for the enzyme reaction was 6.6 and the isoelectric point of the enzyme was pH 5.4. The molecular mass of the enzyme was found to be approximately 53 kDa on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, 110 kDa on gel filtration, and 115 kDa on polyacrylamide gradient gel electrophoresis in the absence of sodium dodecyl sulfate. The Km value for histidine was estimated to be 0.26 mM at pH 6.8.
Ohmori
E
E
Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Kyoto University.
Fukui
T
T
Imanishi
N
N
Yatsunami
K
K
Ichikawa
A
A
D000818:Animals; D002262:Carboxy-Lyases / Q000302:isolation & purification*; D004795:Enzyme Stability; D006640:Histidine Decarboxylase / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D012515:Mast-Cell Sarcoma / Q000201:enzymology*; D051379:Mice; D008970:Molecular Weight; D014407:Tumor Cells, Cultured / Q000201:enzymology
D002262:Carboxy-Lyases; D006640:Histidine Decarboxylase