Biosynthetic Studies on Isoprenoid-modified Proteins

建立了一种从牛脑中纯化法尼酯二磷酸合成酶（FPP）和香叶基香叶基二磷酸合成酶（GGPP）的改进方法，这些合成酶在类异戊二烯修饰蛋白的生物合成中负责脂质前体的合成。首先，用丁基Toyopearl疏水色谱法将两种酶分离。FPP合成酶比GGPP合成酶洗脱时间早。分别以香叶基甲基磷酸磷为配体、Mono Q离子交换层析和以法尼基甲基磷酸磷为配体、Mono Q离子交换层析亲和层析纯化了前者和后者酶。估计纯FPP和GGPP合成酶的比活性分别为1,070 nmol/min/mg和294 nmol/min/mg。制备了针对这两种纯酶的豚鼠多克隆抗体。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。用抗fpp合成酶抗体对牛脑和牛肝粗提物进行western blot检测，发现牛脑和牛肝粗提物中普遍存在约40 kDa的多肽，而牛脑粗提物中仅存在约140 kDa的多肽。另一方面，当使用抗ggpp合酶抗体进行类似实验时，在肝和脑粗提物中检测到约170 kDa的多肽。这些结果提示FPP合成酶和GGPP合成酶在肝脏和大脑中有新的出现。

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.