| Project/Area Number |
01870005
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| Research Category |
Grant-in-Aid for Developmental Scientific Research (B).
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| Allocation Type | Single-year Grants |
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| Research Field |
General physiology
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| Research Institution | The University of Shizuoka |
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Principal Investigator |
HOSHI Takeshi Professor, School of Food and Nutritional Sciences, University of Shizuoka, 食品栄養科学部, 教授 (60004537)
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| Co-Investigator(Kenkyū-buntansha) |
ESAKI Sachiko Professor, School of Food and Nutritional Sciences, University of Shizuoka, 食品栄養科学部, 教授 (50046190)
KAMIYA Shintaro Professor, School of Food and Nutritional Sciences, University of Shizuoka, 食品栄養科学部, 教授 (60046183)
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| Project Period (FY) |
1989 – 1990
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| Project Status |
Completed (Fiscal Year 1990)
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| Budget Amount *help |
¥5,100,000 (Direct Cost: ¥5,100,000)
Fiscal Year 1990: ¥1,200,000 (Direct Cost: ¥1,200,000)
Fiscal Year 1989: ¥3,900,000 (Direct Cost: ¥3,900,000)
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| Keywords | H^+ / peptide cotransporter / Intestinal brush border membrane / Specific inhibitor / Molecular design / Dipeptidase activity / Non-hydrolyzable peptide structure |
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| Research Abstract |
In order for molecular design of specific inhibtors of the H^+/peptide cotransporter in intestinal brush border membrane, studies were made on substrate specificity of the carrier, side chain structures which strengthen the binding to the carrier site and hardly hydrolyzable structure of the peptide moiety. Dipeptidase activity of the outer surface of the brush border membrane was studied by two different ways. One was to measure the time-dependent decrease in concentration of a test dipeptide added to a high-K^+ high-pH medium in which an everted rat ileum was incubated. The increase in concentration of released amino acids was also measured simultaneously by means of a HPLC. The other was to analyze the transport potentials recorded in both Na^+-free and Na^+ containing (normal) media. In the absence of Na^+, only PD changes releated to H^+ current across the membrane is recorded, while in the presence of Na^+, PD changes due to both H^+ current associated with peptide transport and
… More
Na^+ current due to tranport of released amino acids are generated. It was found that Gly-Pro and Gly-Sar were not hydrolyzed at all, and Ala-Ala was hydrolyzed most efficiently. Other dipeptides were hydrolyzed at an intermediate rate. The order of sequence of rate of hydrolysis was as follows ; Ala-Ala>Gly-Leu>Ser-Gly>Ala-Leu>Leu-Gly>Ala-Gly>Leu-Ala>Gly-Gly. The order of hydrolyzability was quite similar to affinity scale of the dipeptidase isolated from soluble fraction of pig enterocytes by Noren et al. (1973). Prolinase activity found in pig enterocytes was not seen on the outer surface of the brush border membrane. In our experiments, Ala-Ala-Ala, which is not a substrate of pig enterocyte dipeptidase, was found to be raPidly hydrolyzed. This hydrolysis may be due to aminopeptidase N activity which is known to be present on the outer surface of the brush border membrane. From the results of this study, it is concluded that the structure similar to Gry-Pro or Gly-Sar is needed for the peptide moiety of the specific inhibitor of the H^+/peptide cotransporter. As for side chain structure necessary for increasing the binding affinity, phloretin-like structure proved to be effective. Accordingly, phloretin-Gly-Sar or phloretin-Gly-Pro is considered to be the first candidate of the effective specific inhibit or of the peptide carrier. Less
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