the study of N-Glycosidase A

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the study of N-Glycosidase A

Post  jiangxiao on Sun Aug 07, 2011 7:48 am

There are many biological chemistry in our life, all these things involved in our food. Now introduce a substance--N-Glycosidase A
N-Glycosidase A is an amidase and cleaves N-glycans? between asparagine and the carbohydrate chain thereby converting asparagine to aspartic acid. The enzyme is an excellent tool to isolate both intact oligosaccharide and peptide moieties for the structural analysis and the functional examination of each moiety. The oligosaccharide is first released as a glycosylamine, which hydrolyzes spontaneously under the acidic conditions of the reaction to the reducing end containing glycan and to ammonia. The pH-optimum of the enzyme is very broad between 4.0 and 6.0. Over pH 7.0 the activity decreases rapidly. In contrast the enzyme retains a portion of activity even at pH 2.N-Glycosidase A is purified from sweet almond meal and shows one band corresponding to a molecular weight of 52.5 kD upon SDS-PAGE (see fig.). The enzyme was first described by Takahashi. The enzyme itself is a glycoprotein.
When using this preparation in combination with the DIG Glycan Detection Kit* it has to be taken into account, that method A (digoxigenin-labeling in solution) should not be used because of the interference of glycerol. Therefore, it is recommended to use method B (digoxigenin-labeling on blots) instead, or to remove glycerol by dialysis before method A is carried out. After dialysis the enzyme solution is stable for at least 1 week at 2-8° C.
N-Glycosidase A cleaves all types of asparagine bound N-glycans including high mannose-, hybrid-, biantennary-, triantennary- and tetraantennary complex types, provided that the amino-group as well as the carboxyl group are present in peptide linkage. N-Glycosidase A can also cleave a single N-acetylglucosamine residue from the peptide, albeit at a slower reaction rate. In contrast to N-glycosidase F* from Flavobacterium meningosepticum, N-glycosidase A from almonds can degrade N-linked glycans carrying a fucose linked _ (1-3) to Asn-GlcNAc. This structural motif is present in plant glycoproteins and is also found in insect glycoproteins.[url= ]N-Glycosidase A[/url][url= ]N-Glycosidase A [/url]


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