1996 Fiscal Year Final Research Report Summary
Highly sensitive method of glycosidase determination
| Project/Area Number |
07558092
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Section | 試験 |
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| Research Field |
Structural biochemistry
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| Research Institution | Tokyo Institute of Technology |
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Principal Investigator |
YAMAGATA Tatsuya TIT,Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology Professor, 生命理工学部生体分子工学科, 客員教授 (10251654)
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| Co-Investigator(Kenkyū-buntansha) |
HYUGA Masashi TIT,Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology Resea, 生命理工学部・生体分子工学科, 教員 (50251658)
YAMAMOTO Hisao TIT,Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology Profe, 東京研究所探索1研究室, 室長
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| Project Period (FY) |
1995 – 1996
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| Keywords | glycosidase / zymography / p-nitrophenyl glycoside / b-galatosidase / hyaluronidase / endoglycoceramidase / artificial enzyme substrates |
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| Research Abstract |
p-Nitrophenyl glycosides have been widely used for the determination of glysodisases, but they cannot be applicable to substrate gel zymography which offers a very sensitive detection of glycosidases even in the presence of inhibitors, since p-nitrophenol resulting from the enzymatic cleavage of the substrates cannot stay in the gel. We have synthesized 4-nitro-2-alkylphenyl-beta-D-galactosides and-mannosides where alkyl chains were C4, C6, C8, C10, and C12. The glycosides thus synthesized having alkyl chains longer than C6 were successfully shown to serve as a good substrate for glycosidases in zymography.
Endoglycoceramidase cleaves the linkage between the oligosaccharide and ceramide of glysosphingolipids and has been shown to be a versatile tool for the structural study and function of glycosphingolipids. The smallest native substrate for the enzyme is lactosylceramide, but p-nitrophenyl-lactoside is not attacked by the enzyme. In order to use in gel substrate zymography, we have sy
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nthesized 2-N-alkylamino-4-p-nitrophenyl-beta-lactoside and shown this to be a good substrate for the enzymens obtained from Rhodococcus sp., Corynebacterium sp, and leech.Modification of the above mentioned substrated by sulfate ester at the position of 3'O- or6'O-has not impaired the availability of the substrate to endoglycoceramidase but has made the substrate exempted from the attack by exoglycosidases.
Atopic dermatitis with Staphylococcus aureus is frequently encountered disease, but to date no research has been directed to the roles of hyaluronan lyase secreted by this bacteria in the skin with this disease. Examination of microbial culture supernatants from the skin surface by substrate gel electrophoresis indicated 90 kDa hyaluronan degrading activity in 70% of specimens obtained from atopic dermatitis patients, while none in healthy volunteer specimens. Molecular weight, optimal pH and mode of action of hyaluronidase were the same as those of S.aureus and the bacterium was confirmed present in specimens from atopic dermatitis skin. The pronase extract of desquamation of atopic dermatitis skin contained unsaturated tetra-and disaccharides, specific degradation products of hyaluronan produced by bacterial hyaluronan lyase. Degradation of skin hyaluronan by Staphylococcal hyaluronan lyase may aggravate atopic dermatitis. From these findings, inhibition of bacterial lyase would appear to bring about remission of atopic dermatitis. Less
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[Publications] Nagatsuka,Y., Yamaki,M., Watarai,S., Yasuda,T., Higashi,H., Yamagata,T.,and Ono,Y.: "The Epstein-Barr virus (EBV) alters the B cell glycolipid which is recognized by the human monoclonal antibody to i-blood antigen." Virus Research. 43. 57-68 (1996)
Description
「研究成果報告書概要(和文)」より
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[Publications] Nagatsuka, Y., Yamaki, M., Watarai, S., Yasuda, T., Higashi, H., Yamagata, T., and Ono, Y.: "The Epstein-Barr virus (EBV) alters the B cell glycolipid which is recognized by the human monoclonal antibody to i-blood antigen." Virus Research. vol.43. 57-68 (1996)
Description
「研究成果報告書概要(欧文)」より
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