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物。为了研究组胺合成的调控机制，采用硫酸铵分离和DEAE-Sepharose CL-6B、Phenyl-Sepharose CL-4B、羟基磷灰石、Phenyl-Superose的柱层析，从小鼠胃中纯化组胺形成酶l-组氨酸脱羧酶（HDC）至电泳均匀性。单Q和活性绿19琼脂糖。纯化酶的组胺比活性为750 nmol / min / mg蛋白质，比粗提物纯化37500倍，产率为1.6%。在十二烷基硫酸钠存在下，聚丙烯酰胺凝胶电泳估计酶的分子量为54 kDa，凝胶过滤估计酶的分子量为lOO kDa。测定酶的等电点pH为5.4。l -组氨酸的Km值估计为0.29 mm。检测了编码小鼠胃酶氨基酸序列的单个mRNA，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能值与肥大细胞瘤p -815细胞酶非常相似。然后，分离出包含小鼠HDC基因5′侧区的基因组DNA克隆。该克隆在启动子区含有一个tata - like box和一个GC-box，在5'侧区含有几个可能的调节蛋白结合位点。用HDC- cat融合基因的5'缺失构建体短暂转染小鼠肥大细胞瘤细胞，发现-267到-43的序列是参与HDC基因与糖皮质激素和TPA转录增加的调控元件所必需的。

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.