| Project/Area Number |
63470116
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
製造化学・食品
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| Research Institution | The University of Tokyo |
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Principal Investigator |
TAKAHASHI Nobutaka The Univ. of Tokyo, Dept. Agric. Chem. Professor, 農学部, 教授 (10011826)
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| Co-Investigator(Kenkyū-buntansha) |
YAMANE Hisakazu The Univ. of Tokyo, Dept. Agric. Chem, Instructor, 農学部, 助手 (80090520)
YAMAGUCHI Isomaro The Univ. of Tokyo, Dept. Agric. Chem, Instructor, 農学部, 助手 (00012013)
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| Project Period (FY) |
1988 – 1989
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| Project Status |
Completed (Fiscal Year 1989)
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| Budget Amount *help |
¥5,900,000 (Direct Cost: ¥5,900,000)
Fiscal Year 1989: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1988: ¥4,400,000 (Direct Cost: ¥4,400,000)
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| Keywords | Gibberellin / Receptor / Binding-Protein / affinity chromatography / 結合蛋白 / アフィニティークロマトグラフィー / イムノアッセ イ |
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| Research Abstract |
TO examine assay systems for the detection gibberellin (GA) binding protein(S), antibodies specific to active GAs were prepared from rabbits and mice. The ELISA system to measure the competitive binding of GA_4 between the immobilized rabbit anti-GA-antibody and the soluble mouse anti-GA-antibody which was regarded as an imitation of GA binding proteins was developed. By this system, 100 fmol of GA binding proteins were detectable. This system was used together with the equilibration dialysis assay to detect GA binding protein from plant extracts, also examined.
An affinity column was prepared by coupling GA_4-17-norketone to Sepharose 4B or Amino-cellulofine at C-16 position using carboxymethoxylamine as a spacer. The adduct of the GA_4-nor-ketone is biologically active and expected to bind with GA binding proteins. A precolumn was also prepared by coupling gibberic acid to Amino-cellulofine via C-7 carboxyl group.
Using the assay systems and the per- and the affinity columns, purificat
… More
ion of GA binding proteins in the etiolated seedlings of Phaseolus vulgaris cv. Mantle was attempted. The epicotyls (ca. 3 kg) of the etiolated seedlings were homogenized in Tris beffer and filtered through cheese cloth. The filtrate was centrifuged (9,000 x g) to remove insoluble materials. Crude protein fraction was precipitated from the supernatant saturated with ammonium sulfate. The precipitates was analyzed and centrifuged (18,000 x g), and the supernatant was assayed by the ELISA and the equilibration dialysis. The supernatant contained proteins binding radioactive GA_4. Most of the bound GA_4 was replaced by both biologically active GA_3 and inactive GA_<34>. However, the replacement of the bound radioactive GA_4 by GA_3 were larger a little than that by GA_<34>4. The crude proteins in the supernatant were subjected to the chromatographic purification using the precolumn and the affinity column, and ca 150 mg of proteins having GA_4 binding activity, 40 % of which was still unspecific binding. Further purification using Sephacryl S-400 was unsuccessful to show a week binding activity in the eluate at ca, 200 KD. Most of the activity was disappeared after the gel filtration chromatography, Further purification procedures are being examined. Less
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