| Project/Area Number |
08680662
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (C)
|
| Allocation Type | Single-year Grants |
|---|
| Section | 一般 |
|---|
| Research Field |
Structural biochemistry
|
|---|
| Research Institution | Osaka City University |
|---|
Principal Investigator |
ITO Kazuo Osaka City University, Faculty of Science, Lecture, 理学部, 講師 (20183171)
|
|---|
| Project Period (FY) |
1996 – 1997
|
| Project Status |
Completed (Fiscal Year 1997)
|
| Budget Amount *help |
¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 1997: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1996: ¥1,900,000 (Direct Cost: ¥1,900,000)
|
|---|
| Keywords | Endo-beta-N-acetylglucosaminidase HS / Asparagine-linked Oligosaccharide / Glycoprotein / Epithelial Cell / Human Oral Cavity Epithelial Cell / Human Salive / Glycobiology / エンド-β-N-アセチルグルコザミニダーゼHS |
|---|
| Research Abstract |
An improved purification method was established for an effective purification of endo-beta-N-acetylglucosaminidase (Endo) HS,resulting that Endo HS was purified about 100-fold with activity recovery of about 100% using a chitin column. The purified Endo HS was separated into three multiple forms (Endo HS-I,II and III) with different isoelectric point by HPLC with Mono Q5/5 column. Purity of Endo HS-I and Endo HS-II was 100% and Endo HS-III,about 15%. Total amount of Endo HS-I and Endo Hs-II, about 67 pmole and 240 pmole, respectively was not sufficient for determination of the partial amino acid sequence for cloning of Endo gene. The result indicates that sufficient amount of Endo HS for amino acid sequencing must be obtained from human oral cavity epithelial cell obtained from 5-10 liter of human saliva for ourification. The multiple forms of Endo HS showed a quite similar substrate specificity. Endo HS was specifically released asparagine-linked oligosaccharides of bi, tri and tetrantennary complex types from native glycoproteins and asparagine-oligosaccharides regardless of the existence of the fucose residue at the proximal N-acetylglucosamine and side chains and the sialic acid at the side chains. On the other hand, Endo HS did not act on high-mannose and hybrid type oligosaccharides on glycoproteins. The comparison of the specificity of Endo HS with other enzymes means that Endo HS evolved after appearing the synthetic pathway of complex type oligosaccharides for control of the amount of asparagine-linked oligosaccharides of complex type on animal cell glycoproteins.
|
