1989 Fiscal Year Final Research Report Summary
Genetic Engineering Modification of Aequorin for Detection of Metal Ions
| Project/Area Number |
63880020
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| Research Category |
Grant-in-Aid for Developmental Scientific Research
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| Allocation Type | Single-year Grants |
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| Research Field |
物質生物化学
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| Research Institution | Osaka Bioscience Institute |
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Principal Investigator |
TSUJI Frederick I. Osaka Bioscience Inst. 2nd Dept. Head, 第2研究部, 部長 (80201755)
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| Co-Investigator(Kenkyū-buntansha) |
THOMPSON Eric M. Osaka Bioscience Inst. 2nd Dept. STA Fellow, STAフェロー (90203661)
NAGANO Kozo the University of Tokyo Pharmaceutical Sci. Assistant Prof., 薬学部, 助教授 (30012636)
HAYAISHI Osamu Osaka Bioscience Inst. Director, 所長 (40025507)
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| Project Period (FY) |
1988 – 1989
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| Keywords | Aequorin / Calcium-binding protein / Metal ions / Model building / Prediction of secondary structure / Prediction of three dimensional structure / グラフィックス・ソフトウェア |
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| Research Abstract |
Aequorin is a small (189 amino acid residues, M_r=21,400) monomeric calcium-binding protein from the jellyfish, Aeguorea victoria, which emits light in the presence of calcium. The protein consists of apoaequorin (apoprotein), coelenterazine (substrate), and molecular oxygen bound in a complex. On binding calcium, an intramolecular reaction takes place in which the apoprotein acting as an oxygenase catalyzes the oxidation of coelenterazine by oxygen, yielding light, carbon dioxide, and coelenteramide. The apoaequorin may be regenerated into aequorin by incubation with coelenterazine, dissolved oxygen, EDTA, and 2-mercaptoethanol.
In 1985, we cloned the complementary DNA for apoaequorin and showed that the amino acid sequences of the calcium-binding sites of aequorin are homologous to those of the corresponding calcium-binding sites of bovine calmodulin. In order re-design the aequaorin molecule so that it would emit light with other bivalent metal ions, both theoretical and laboratory studies were carried out. Model building studies were performed using a VAX computer with Insight (display) and Discover (energy minimization) programs. Site-directed mutagenesis was undertaken initially to determine the relationship between structure and function by replacing the three cysteine residues in all permutations. The seven modified aequorins were tested for activity and it was found that the aequorin with all three cysteine residues replaced by serine had the best possibility of producing light with cadmium ion.
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