Ohmori E, Fukui T, Imanishi N, Yatsunami K, Ichikawa A
Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, Kyoto University.
J Biochem. 1990 Jun;107(6):834-9. doi: 10.1093/oxfordjournals.jbchem.a123134.
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.
通过硫酸铵分级沉淀、在pH 7.5和6.0条件下透析、在DEAE-琼脂糖CL-6B、苯基琼脂糖CL-4B和羟基磷灰石上进行层析、苯基超聚糖HPLC、单Q HPLC以及二醇-200凝胶过滤HPLC，从小鼠肥大细胞瘤P-815细胞中纯化组氨酸脱羧酶至电泳纯。在所使用的测定条件下，纯酶的比活性为800 nmol/分钟/毫克，与粗提物相比纯化了12500倍，产率为7%。事实证明，两步透析对于去除干扰酶纯化的因子至关重要。酶反应的最适pH为6.6，酶的等电点为pH 5.4。在十二烷基硫酸钠存在下的聚丙烯酰胺凝胶电泳中，酶的分子量约为53 kDa，在凝胶过滤中为110 kDa，在无十二烷基硫酸钠的聚丙烯酰胺梯度凝胶电泳中为115 kDa。在pH 6.8时，组氨酸的Km值估计为0.26 mM。
