Biopharmacological study on the regulation of L-histidine decarboxylase activity in gastric acid secretion

在胃中，组胺被认为是胃酸分泌的生理刺激剂。为研究组胺合成的调控机制，采用硫酸铵分级分离和DEAE-SepharoseCL-6 B、Phenyl-SepharoseCL-4 B、羟基磷灰石、Phenyl-Superose柱层析，从小鼠胃中分离纯化了组胺形成酶--L-组氨酸脱羧酶（HDC）。Mono Q和活性绿色19-琼脂糖。纯化的酶表现出750 nmol组胺形成每分钟每毫克蛋白质的比活性，这构成了37,500倍的纯化相比，粗提物，产率为1.6%。在十二烷基硫酸钠存在下，通过聚丙烯酰胺凝胶电泳估计该酶的分子量为54 kDa，通过凝胶过滤估计该酶的分子量为100 kDa。测得该酶的等电点为pH5.4。L-组氨酸的Km值估计为0.29 mM。检测编码小鼠胃酶氨基酸序列的单个mRNA，发现其大小为2.7 kb。小鼠胃L-组氨酸脱羧酶的这些分子和催化性质值与肥大细胞瘤P-815细胞中的酶十分相似。接着，分离包含小鼠HDC基因的5 ′-侧翼区的基因组DNA克隆。该克隆在启动子区含有一个TATA样盒和一个GC盒，在5 '侧翼区含有几个调节蛋白的推定结合位点。用HDC-CAT融合基因的5 ′缺失构建体瞬时转染小鼠肥大细胞瘤细胞，发现从-267到-43的序列对于参与用糖皮质激素和TPA增加HDC基因转录的调节元件是必需的。

In the stomach, histamine is considered to be a physiological stimulant of gastric acid secretion. In order to study the regulatory mechanisms of histamine synthesis, the histamine-forming enzyme, L-histidine decarboxylase (HDC) was purified to electrophoretic homogeneity from mouse stomach using ammonium sulfate fractionation and column chromatographies of DEAE-Sepharose CL-6B, Phenyl-Sepharose CL-4B, hydroxylapatite, Phenyl-Superose. Mono Q and Reactive Green 19-agarose. The purified enzyme exhibited a specific activity of 750 nmol histamine formed per min per mg protein, which constituted a 37,500-fold purification compared to the crude extract, with a l.6% yield. The molecular mass of the enzyme was estimated to be 54 kDa by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate and lOO kDa by gel filtration. The isoelectric point of the enzyme was determinwd to be pH 5.4. The Km value for L-histidine was estimated to be 0.29 mM.The single mRNA encoding the amino acid sequence of the mouse stomach enzyme was examined and its size was found to be 2.7 kb. These molecular and catalytic property values of the L-histidine decarboxylase of mouse stomach are quite similar to those of the enzyme from mastocytoma P-815cells. Next, the genomic DNA clone including 5'-flanking region of the mouse HDC gene was isolated. This clone contained a TATA-Iike box and a GC-box in the promoter region, and several putative binding sites for regulatory proteins in the 5'-flanking region. With mouse mastocytoma cells transiently transfected with 5'deletion constructs of HDC-CAT fusion gene, it was found that the sequence from -267 to -43 is essential for the regulatory element(s) involved in the increased transcription of the HDC gene with glucocorticoid and TPA.