1999 Fiscal Year Final Research Report Summary
Subcellular localization and regulatory mechanism of purine alkaloid
| Project/Area Number |
10640627
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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 |
植物生理
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| Research Institution | Ochanomizu University |
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Principal Investigator |
ASHIHARA Hiroshi Ochanomizu University, Department of Biology, Faculty of Science, Professor, 理学部, 教授 (00017211)
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| Co-Investigator(Kenkyū-buntansha) |
芦原 坦 お茶の水女子大学, 理学部, 教授 (00017211)
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| Project Period (FY) |
1998 – 1999
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| Keywords | Purine alkaloid / Caffeine / Metabolism / Metabolic Regulation / N-methyltransferase / S-Adenosylmethionine / Secondary metabolism / Chloroplast |
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| Research Abstract |
Subcellular localization and regulatory mechanism of purine alkaloid metabolism were investigated using several purine alkaloid producing plants. In preparation from young tea leaves, caffeine synthase (CS), a key enzyme in caffeine biosynthesis was associated with a purified chloroplast preparation obtained by using differential centrifugation and a discontinuous Percoll density gradient. CS was extracted from young tea leaves and purified 520-fold to apparent homogeneity and a final specific activity of 5.7 nkat mg-1 protein by ammonium sulfate fractionation and hydroxyapatite, anion-exchange, adenosine-agarose, and gel-filtration chromatography. The native enzyme was monomeric with an apparent molecular mass of 61 KD as estimated by gel-filtration chromatography and 41 KD as analyzed by SDS gel electrophoresis. The enzyme displayed a sharp pH optimum of 8.5. The final preparation exhibited 3-and 1-N-methyltransferase activity with a broad substrate specificity, showing high activity toward paraxanthine, 7-methylxanthine, and theobromine and low activity with 3-methylxanthine and 1-methylxanthine. The possible role and fine control mechanism of CS in purine alkaloid biosynthesis in tea leaves are discussed. The 20-amino acid N-terminal sequence for CS was obtained. Catabolism of caffeine was examined using various species of Coffea and Camellia plants. Degradation of [8-ィイD114ィエD1C] caffeine is negligible in leaves of almost all plants, but In it was catabolized rapidly by leaves of C. eugenioides primarily by a caffeine → theophylline → 3-methylxantine → uric acid → allantoin → allantoic acid → urea → COィイD22ィエD2 + NHィイD23ィエD2 pathway. Based on seven N-terminal residues of CS, a 1.31 kb sequence from cDNAs obtained from young tea leaves. High expression of the CS gene (TCS) was observed in young leaves. Thus, regulation of caffeine synthesis in tea leaves seems to be performed at the transcription level.
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[Publications] Kato, M. Mizuno, K., Fujimura, T., Iwama., Irie, M., Crozier, A. and Ashihara, H.: "Purification and characterization of caffeine synthease from tea leaves"Plant Physiol.. 120. 579-586 (1999)
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