| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1993: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 1992: ¥1,400,000 (Direct Cost: ¥1,400,000)
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| Research Abstract |
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.
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