1997 Fiscal Year Final Research Report Summary
Analysis of the processing mechanism of endothelin-converting enzyme and the development of its inhibitors as novel therapeutics for vascular complications.
| Project/Area Number |
08671167
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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 |
内分泌・代謝学
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| Research Institution | KYUSHU UNIVERSITY |
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Principal Investigator |
TAKAYANAGI Ryoichi Kyushu University, Faculty of Medicine, Assistant Professor, 医学部, 講師 (30154917)
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| Project Period (FY) |
1996 – 1997
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| Keywords | Endothelin / Endothelin-converting enzyme / Endothelial cell / Big endothelin / ECE / ET / GFP |
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| Research Abstract |
1. Big ET-1 analogues with the inhibitory activity on endothelin-converting enzyme-1 (ECE-1). Two big endothelin-1(big ET-1) analogues with the inhibitory activity on ECE-1, [F^<21>]big ET-1(18-34) and [A^<31>]big ET-1(18-34), were found by various amino acid substitution of big ET-1. Kinetics analyses showed that [F^<21>]big ET-1(18-34) is a competitive inhibitor and [A^<31>]big ET-1 (18-34) is a non-competitive inhibitor, suggesting that ECE-1 recognizes the P1 position and the C-terminal portion in a different fashon.
2. Clarification of intracellular site for big ET-1-conversion. It is important for the drug design to know where in the cell the big ET-1 is converted, however, the intracellular site for the conversion of big ET-1 has been controversial. In the present study, using a chimera of ECE-1 and green fluorescent protein (GFP), visible signal in living cells, it was revealed that endogenous big ET-1 is converted intracellularly, not on the cell surface.
3. Analyses of the properties of ECE-1 by mutagenesis of ECE-1 cDNA.Mutagenesis of human cDNA revealed that glycosylation of the extracellular C-terminal domain of ECE-1 is essential to the enzymatic activity, and that the transmembrane domain plays an important role in the completion of normal glycosylation.
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