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

在胃里，组胺被认为是胃酸分泌的一种生理刺激剂。为了研究组胺合成的调节机制，用硫酸铵分级沉淀和DEAE-SepharoseCL-6B、Phenyl-SepharoseCL-4B、羟磷灰石、苯基超糖柱层析，从小鼠胃中纯化了组胺形成酶L-组氨酸脱羧酶。Mono Q和活性绿19-琼脂糖凝胶。纯化后的酶比活力为每毫克蛋白每分钟形成750nmoL组胺，比粗提物纯化37500倍，产率为1.6%。在十二烷基硫酸钠和100 kDa凝胶过滤条件下，用聚丙烯酰胺凝胶电泳法测得该酶的相对分子质量为54 kDa。测得该酶的等电点为pH 5.4。结果表明，L-组氨酸的Km值为0.29mM，编码小鼠胃酶氨基酸序列的单链基因大小为2.7kb。小鼠胃L组氨酸脱羧酶的这些分子和催化性质与肥大细胞瘤P-815细胞的酶非常相似。接下来，克隆了小鼠HDC基因5‘侧翼区的基因组DNA。该克隆在启动子区域含有一个类似TATA的框和一个GC-框，在5‘-侧翼区有几个可能的调节蛋白结合位点。将hDC-CAT融合基因的5‘端缺失构建体瞬时导入小鼠肥大细胞瘤细胞，发现-267~-43的序列是糖皮质激素和TPA促进hDC基因转录的调控元件(S)所必需的。

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.