| Budget Amount *help |
¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 1997: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 1996: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Research Abstract |
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.
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