1990 Fiscal Year Final Research Report Summary
Studies on Anti-Amnesiac Effect of Specific Inhibitor for Prolyl Endopeptidase
| Project/Area Number |
01571217
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
Biological pharmacy
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| Research Institution | Nagasaki University |
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Principal Investigator |
YOSHIMOTO Tadashi Nagasaki University, School of Pharmaceutical Sciences, Assistant Professor, 薬学部, 助教授 (60088870)
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| Project Period (FY) |
1989 – 1990
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| Keywords | anti-amnesia / amnesia / inhibitor / prolyl endopeptidase / peptidase / cloning / nucleotide sequence / gene |
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| Research Abstract |
Prolyl endopeptidase (EC 3.4.21.26) is widely distributed in animal tissues and seems to play some role in the metabolism of biologically active peptides. We found the potent inhibitors specific for the enzyme. Z-Gly-Pro-CH2Cl. Z-Pro-prolinal and the related compounds show an antiamnesic effect on experimental amnesia in rat and mouse induced by scopolamine or electro-convulsive shock. This antiamnesic effect seems to be produced by the inhibition of the enzyme in brain. In order to clarify the mechanism. Function relationship of the inhibitors for the enzyme was studied and the enzyme gene was cloned.
1. Study on active site of prolyl endopeptidase : Structural requirements of N-blocked L-proline derivatives as specific inhibitors for prolyl endopeptidase were investigated using a series of substrate analogs. Replacement of L-proline by its D-isomer remarkably reduced the inhibition. Introduction of a sulfur atom in proline and/or in the penultimate pyrrolidine rings significantly increased the inhibition, but the introduction of Oxygen rather diminished the activity. A peptide linkage between the proline and the Pyrrolidine ring was also require to keep the inhibitory activity. A benzyloxycarbonyl group was most effective as an N-blocked component of the inhibitors.
2. Cloning of prolyl endopeptidase gene : Amino acid sequence of the proteolytic peptides of the enzyme were determined by Edman degradation after isolation by HPLC. Probes for Southern analysis were prepared following the amino acid sequences. The gene encoding prolyl endopeptidase from Flavobacterium meningosepticum was isolated and the entire nucleotide sequence of the coding sequence was determined. The transformant of E. Coli DH1 harboring pFPH5K50 with a 2.5 kbp fragment showed high prolyl endopeptidase activity. By this project. The structure at active site of prolyl endopeptidase and nucleotide sequence of the enzyme gene were clarified.
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[Publications] Tsuru, D., Naotsuka, A., Kobayashi, R., Oda, K., Murao, S. and Yoshimoto, T.: "Inhibition of Scytalidium lignicolum acid protease B by 1-Diazo-3-pheny1-2-propanone, ," Agric. Biol. Chem.53. 1305-1312 (1989)
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[Publications] Tsuru, D., Naotsuka, A., Kobayashi, R., Yoshimoto, T., Oda, K., and Murao, S.: "Inactivation of Scytalidium lignicolum acid protease B with 1, 2-epoxy-3-(4'-azido-2'-nitorophenoxy)propane," Agric. Biol. Chem.53. 2751-2756 (1989)
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[Publications] Yoshimoto, T., Oyama, H., Takeshita, T., Higashi, H., Xu, S-L and Tsuru, D.: "Nucleotide sequence of the neutral protease gene from Bacillus subtilis var. amylosacchariticus" J. Ferm. Bioeng.,. 70. 370-375 (1990)
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[Publications] Yoshimoto, T., Yamamoto, N., Ogawa, H., Furukawa, S., and Tsuru, D: "Specific inhibition of dipeptidylaminopeptidase IV by a new synthetic inhibitor, L-thioprolylthiazolidine" Agric. Biol. Chem.55. 1135-1136 (1991)
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