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Biosynthetic Studies on Isoprenoid-modified Proteins

类异戊二烯修饰蛋白的生物合成研究

基本信息

批准号：
07680671
负责人：
SAGAMI Hiroshi
金额：
$ 0.13万
依托单位：
TOHOKU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

An improved method was established to purify farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) synthases from bovine brain which are responsible for the synthesis of lipid precursors in the biosynthesis of isoprenoid modified proteins. First, the two enzymes were separated from each other by Butyl Toyopearl hydrophobic chromatography. FPP synthase eluted earlier than GGPP synthase. The former and the latter enzymes were further purified by affinity chromatography using geranylmethyl phosphonophosphate as a ligand followed by Mono Q ion exchange chromatography and by affinity chromatography using farnesylmethyl phosphonophosphate as a ligand followed by Mono Q ion exchange chromatography, respectively. The specific activities of the pure FPP and GGPP synthases were estimated to be 1,070 nmol/min/mg and 294 nmol/min/mg, respectively. Guinea pig polyclonal antibodies were raised against these two pure enzymes. The anti-FPP synthase polyclonal antibodies did not recognize GGPP synthase and the anti-GGPP synthase polyclonal antibodies did not recognize FPP synthase. When crude extracts of bovine brain and liver were examined by western blot analysis with the anti-FPP synthase antibodies, a polypeptide with ca. 40 kDa was commonly detected in the both crude extracts and a polypeptide with ca. 140 kDa was observed only for the extract of brain. On the other hand, when the anti-GGPP synthase antibodies were used for the similar experiments, a polypeptide with ca. 170 kDa was detected in the crude extracts of liver and brain. These results suggest novel occurrence of FPP synthase and GGPP synthase in liver and brain.

建立了一种从牛脑中分离纯化法呢基二磷酸（FPP）和香叶基香叶基二磷酸（GGPP）脱氢酶的方法。首先，通过丁基Toyoprotein疏水层析将两种酶彼此分离。FPP合酶比GGPP合酶更早洗脱。前者和后者分别通过使用香叶基甲基磷酸盐作为配体的亲和层析，然后通过Mono Q离子交换层析和使用法呢基甲基磷酸盐作为配体的亲和层析，然后通过Mono Q离子交换层析进一步纯化。纯FPP和GGPP酶的比活性估计分别为1，070 nmol/min/mg和294 nmol/min/mg。豚鼠多克隆抗体对这两种纯酶。抗FPP合酶多克隆抗体不识别GGPP合酶，抗GGPP合酶多克隆抗体不识别FPP合酶。当用抗FPP合酶抗体对牛脑和肝的粗提物进行蛋白质印迹分析时，发现一种具有约在两种粗提物中通常检测到40 kDa的多肽，并且在两种粗提物中均检测到约40 kDa的多肽。仅在脑提取物中观察到140 kDa。另一方面，当抗GGPP合酶抗体用于类似实验时，具有ca.在肝和脑的粗提物中检测到170 kDa。这些结果表明FPP合酶和GGPP合酶在肝和脑中的新出现。