Biopharmacological study on the regulation of histamine biosynthesis

纯化的小鼠肥大细胞瘤细胞的L-组氨酸脱羧酶（HDC）是由两个相同的53 kDa亚基组成的二聚体，而cDNA推导的HDC的大小为74 kDa。为了阐明组胺合成的调控机制，我们对HDC酶的翻译后修饰进行了研究，并对HDC基因的5 '侧翼区进行了分析。在Sf 9细胞中表达的重组74 kDa HDC具有低的酶活性并且存在于颗粒部分中，并且在体外被猪胰腺弹性蛋白酶转化为具有高催化活性的可溶性53 kDa HDC形式。对内源性活性蛋白酶进行了研究，结果表明，对苄脒敏感的蛋白酶负责加工。然后，使用大鼠嗜碱性细胞系RBL-2 H3研究了74 kDa和53 kDa HDC物质的代谢。在RBL-2 H3细胞中，74 kDa酶的周转速度非常快，而53 kDa HDC的周转速度很慢，这表明泛素蛋白酶体系统负责74 kDa HDC的降解。将具有HDC基因的5 ′-侧翼区和氯霉素乙酰转移酶（CAT）基因的融合DNA转染入人嗜碱性白血病KU-812-F细胞或人上皮癌HeLa细胞。CAT分析表明，HDC基因-1003 ~+99区域含有两个正调控元件和一个负调控元件。序列分析表明，在-520位点存在一个核因子c-Myb结合基序，KU-812-F细胞核提取物中含有与该基序结合的因子，而HeLa细胞核提取物中不含有与该基序结合的因子。这些结果表明，在HDC基因的5 '侧翼区中至少有一个元件，包括c-Myb结合基序，负责HDC的组织特异性表达。

Purified L-histidine decarboxylase (HDC) of mouse mastocytoma cells is a dimer consisting of two identical 53 kDa subunits, whereas the size of cDNA-deduced HDC is 74 kDa. In order to clarify the regulatory mechanism of histamine synthesis, we studied on the post-translational modification of HDC enzyme and also analyzed the 5'-flanking region of the HDC gene. The expressed recombinant 74 kDa HDC in Sf9 cells had low enzyme activity and was present in the particulate fraction, and 74 kDa HDC was converted into its soluble 53 kDa HDC form with a high catalytic activity by porcine pancreas elastase in vitro. A search was made for endogenously active proteinase, and it was shown that benzamidine-sensitive proteinase was responsible for the processing. Then, metabolism of both 74 kDa and 53 kDa HDC species was investigated using rat basophilic cell line RBL-2H3. In RBL-2H3 cells, the turn-over rate of 74 kDa enzyme was very fast, while that of 53 kDa HDC was slow, and it was suggested that ubiquitinproteasome system was responsible for the degradation of 74 kDa HDC.Next, to identify the regions that regulate the tissue-specific expression of HDC,a fusion DNA with the 5'-flanking region of the HDC gene and chloramphenicol acetyltransferase (CAT) gene was transfected into human basophilic leukemia KU-812-F cells or human epithelial carcinoma HeLa cells. CAT analysis revealed that the region from -1003 to +99 of the HDC gene contained two positive and one negative regulatory elements, Sequence analysis showed a nuclear factor c-Myb binding motif at position -520, and the nuclear extract of KU-812-F cells, but not that of HeLa cells contained a factor which can bind to this motif. These results suggest that at least one element in the 5'-flanking region of the HDC gene, including c-Myb binding motif, is responsible for the tissue-specific expression of HDC.