1996 Fiscal Year Final Research Report Summary
Biosynthetic Studies on Isoprenoid-modified Proteins
| Project/Area Number |
07680671
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Functional biochemistry
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| Research Institution | TOHOKU UNIVERSITY |
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Principal Investigator |
SAGAMI Hiroshi Tohoku Univ.Institute for Chemical Reaction Science Associate Professor, 反応化学研究所, 助教授 (10134058)
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| Project Period (FY) |
1995 – 1996
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| Keywords | Affinity Chromatography / Brain / Isoprenoid / Geranylgeranyl Diphosphate Synthase / Farnesyl Diphosphate Synthase / Prenyltransferase |
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| Research Abstract |
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.
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