Studies of substrate recognition mechanism on brain glutamate decarboxylase

脑谷氨酸脱羧酶底物识别机制的研究

• 批准号: 06670155
• 负责人: UENO Hiroshi
• 金额: $ 1.34万
• 依托单位: KYOTO UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06670155/
• 关键词: Neurotransmitter; GABA; GAD; HDC

Rat brain glutamate decarboxylase (GAD) isozyme having molecular weight of 65,000 was expressed in Saccharomyces cerevisiae having a specific activity of 50 nmol/min/mg. The expressed enzyme reacted with anti-GAD65 monoclonal antibody and showed a 65 kDa band on SDS-PAGE and Western blot analysis. Detailed culture conditions were examined.Human histidine decarboxylase (HDC) is a close relative of GAD.In order to compare the substrate specificity of two decarboxylases, we have expressed HDC in Saccharomyces cerevisiae. The expression system exhibited 200 pmol/min/mg histamine producing activity which is far superior than any tissue samples. SDS-PAGE analysis gave 74 kDa band corresponding to precursor form of HDC.The expression system we have developed will provide strong hand on further progress in characterizing decarboxylases.

分子量为65,000的大鼠脑谷氨酸脱羧酶(GAD)同工酶在酿酒酵母中表达，比活性为50nmol/min/mg。表达的酶与抗GAD65单克隆抗体反应，并在SDS-PAGE和Western blot分析中显示出65 kDa条带。检查了详细的培养条件。人组氨酸脱羧酶(HDC)是GAD的近亲。为了比较两种脱羧酶的底物特异性，我们在酿酒酵母中表达HDC。该表达系统表现出 200 pmol/min/mg 的组胺产生活性，远远优于任何组织样品。 SDS-PAGE 分析给出了对应于 HDC 前体形式的 74 kDa 条带。我们开发的表达系统将为表征脱羧酶的进一步进展提供强有力的支持。

项目成果

Kanai et al: "A novel heterologous gene expression system in Saccharomyces cerevisiae using the isocitrate lyase gene promoter from Candida tropicalis" Appl. Microbiol. Biotechnol.44. 759-765 (1996)

Kanai 等人：“使用来自热带假丝酵母的异柠檬酸裂解酶基因启动子的酿酒酵母中的新型异源基因表达系统”。

Kanai at al: "A Novel heterologous gene expression system in Saccharomyces cerevisiae using the isocitrate lyase gene promoter from candida tropicalis" Appl. micrubiol. Biotechnal.44. 759-765 (1996)

Kanai 等人：“使用来自热带假丝酵母的异柠檬酸裂解酶基因启动子的酿酒酵母中的新型异源基因表达系统”。

Kanai, T., Atomi, H., Umemura, K., Ueno, H., Teranishi, Y., Ueda, M., Tanaka, A.: "A novel heterologous gene expression system in Saccharomyces cerevisiae using the isocitrate lyase gene promoter from Candida tropicalis" Appl.Microbiol.Biotechnol. 44. 759

Kanai, T.、Atomi, H.、Umemura, K.、Ueno, H.、Teranishi, Y.、Ueda, M.、Tanaka, A.：“使用异柠檬酸裂解酶基因的酿酒酵母中的新型异源基因表达系统