Project/Area Number |
06558094
|
Research Category |
Grant-in-Aid for Developmental Scientific Research (B)
|
Allocation Type | Single-year Grants |
Research Field |
Structural biochemistry
|
Research Institution | Tokyo University of Pharmacy and Life Science (1995) The University of Tokyo (1994) |
Principal Investigator |
TAKAHASHI Kenji Tokyo University of Pharmacy and Life Science, School of Life Science, Professor, 生命科学部, 教授 (70011533)
|
Co-Investigator(Kenkyū-buntansha) |
MURAMATSU Tomonari National Institute for Cancer Research, Department of Biophysical Chemistry, Lab, 生物物理部, 室 長 (70212256)
|
Project Period (FY) |
1994 – 1995
|
Project Status |
Completed (Fiscal Year 1995)
|
Budget Amount *help |
¥6,200,000 (Direct Cost: ¥6,200,000)
Fiscal Year 1995: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1994: ¥4,800,000 (Direct Cost: ¥4,800,000)
|
Keywords | Peptide libraty / Resin-bound peptide / substrate specificity / Systematic specificity analysis / Protease / Endopeptidase / Chymotrypsin / Subsite specificity / 化学合成 |
Research Abstract |
This project aimed to develop a new systematic method for analyzing the specificity of endopeptidases. For this purpose, we prepared resin-linked peptide libraries, in which only one specific position was occupied with various amino acid residues, and used these libraries as substrates for a protease. The hydrolysis products were analyzed quantitatively by automated peptide sequencing by Edman degradation. The results demonstrated the usefulness of this method. 1) We checked various resins and found that an aminomethyl-resin was most suitable as the solid support for synthesis of resin-bound peptide libraries. With these as substrates, we investigated the effect of spacers on the cleavage efficiency and found that pentaglycine was sufficient as a spacer, which increased the cleavage efficiency by chymotrypsin about 30-fold. 2) The peptide synthesis by the Fmoc-method using an equimolar mixture of Fmoc-amino acids at one specific position did not give an equimolar mixture of the desired peptides, a 10-times difference being obtained in the yields of respective peptides. This was overcome by changing the relative molar ratio of the Fmoc-amino acids in the mixture ; thus the difference in the yield could be reduced to within 3-fold for most amino acids. 3) We examined the usefulness of this method by applying it to study on the subsite specificities of chymotrypsin. As model peptide libraries, we prepared Arg-Pro-Xxx-Phe-Ser-Pro-Arg (Gly)_5-Resin, N-acetyl-Arg-Pro-Gly-Phe-Xxx-Pro-Arg- (Gly)_5-Resin and N-acetyl-Arg-Pro-Gly-Phe-Ser-Xxx-Arg- (Gly)_5-Resin, where Xxx was a mixture of various amino acids, submitted them to chymotryptic hydrolysis and analyzed the hydrolysis products. The results provided us with novel and interesting information on the P_2-, P_1'-, and P_2'-site specificities of chymotrypsin. 4) We purified various novel proteases to be examined by the present methods.
|