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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¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 1996: ¥3,500,000 (Direct Cost: ¥3,500,000)
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
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