Studies of substrate recognition mechanism on brain glutamate decarboxylase

分子量为65，000的大鼠脑谷氨酸脱羧酶（GAD）同工酶在比活性为50 nmol/min/mg的酿酒酵母中表达。表达产物与GAD 65单克隆抗体反应，经SDS-PAGE和Western blot分析，均显示一条65 kDa的条带。人组氨酸脱羧酶（human histidine decarboxylase，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了HDC。表达系统显示出200 pmol/min/mg组胺产生活性，这远远优于任何组织样品的上级活性。SDS-PAGE分析表明，HDC的前体蛋白为74 kDa，为进一步研究脱羧酶的性质奠定了基础。

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.