| Project/Area Number |
02670984
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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 |
Physical pharmacy
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| Research Institution | Nagoya City University |
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Principal Investigator |
KURONO Yukihisa Nagoya City University, Faculty of Pharmaceutical Sciences, Research Associate Professor, 薬学部, 講師 (50080205)
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| Co-Investigator(Kenkyū-buntansha) |
IKEDA Ken Nagoya City University, Faculty of Pharmaceutical Sciences, Emeritus Professor, 名誉教授 (50080164)
OHTA Naoko Nagoya City University, Faculty of Pharmaceutical Sciences, Research Associate, 薬学部, 助手 (50117818)
YOTSUYANAGI Toshihisa Nagoya City University, Faculty of Pharmaceutical Sciences, Professor, 薬学部, 教授 (40080189)
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| Project Period (FY) |
1990 – 1992
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| Project Status |
Completed (Fiscal Year 1992)
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| Budget Amount *help |
¥2,400,000 (Direct Cost: ¥2,400,000)
Fiscal Year 1992: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1991: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1990: ¥1,500,000 (Direct Cost: ¥1,500,000)
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| Keywords | Human serum albumin / Drug binding / X-Ray crystal structure / Crystallization / Enzymatic activity / Enzyme kinetics / Characterization / アシル化ヒト血清アルブミン / X線結晶構造解析 / X線構造解析 / エステラ-ゼ様活性 / キャラクタリゼ-ション |
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| Research Abstract |
1. Crystallization of Human Serum Albumin (HSA) : Defatted HSA (Fraction V) was crystallized by the standard hanging drop technique. Plate or needle crystals of 0.1 to 0.2 mm in size were obtained repeatedly. The crystals of 0.5 to 2.0 mm are required for the x-ray structural analysis. Examination of the HSA purity by SDS-PAGE suggested the existence of several percents of the dimer and trimer. The monomer was isolated by gel-chromatography, end was crystallized. The crystals of 0.4-0.5 mm in size were obtained.
2. Crystallization of Acyl-HSA : The R-site of HSA was acylated with rho-nitrophenyl acetate and cinnamoylimidazole. By-products were removed by the dialysis and the acyl-HSA solutions were lyophilized. The acyl-HSA was crystallized, and the crystals of 0.2-0.3 mm in size were obtained.
3. Characterization of Drug Binding Sites of HSA : We have characterized the drug binding sites of HSA by applying the enzymatic activities of the protein. The results from this kinetic methodology will be compared with those from the x-ray crystallography. HSA has multiple reactive sites towards rho-nitrophenyl 4-guanidinobenzoate. The pH-profile for the catalytic rate constant suggested the involvement of groups with pKa's of 6.0 and 10.0 for the reactions (Published in Chem Pharm. Bull., 39, 1292 (1991)). Among amino acid rho-nitrophenyl esters, N-CBZ-alanine rho-nitrophenyl ester was most quickly reacted with HSA. The reactive site towards this substrate was R-site alone (Published in Chem. Pharm. Bull., 40,2169(1992)). Furthermore, the reactions of nitrophenyl phosphinates with HSA were studied kinetically and the reactive sites towards these substrates were explored.
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