2003年11月
A nonradioactive 96-well plate assay for screening of trans-sialidase activity
ANALYTICAL BIOCHEMISTRY
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- 巻
- 322
- 号
- 2
- 開始ページ
- 139
- 終了ページ
- 147
- 記述言語
- 英語
- 掲載種別
- DOI
- 10.1016/j.ab.2003.07.016
- 出版者・発行元
- ACADEMIC PRESS INC ELSEVIER SCIENCE
Trans-sialidase (E.C. 3.2.1.18) catalyzes the transfer of preferably alpha2,3-linked sialic acid to another glycan or glycoconjugate, forming a new alpha2.3 linkage to galactose or N-acetylgalactosamine. Here, we describe a nonradioactive 96-well plate fluorescence test for monitoring trans-sialidase activity with high sensitivity, specificity, and reproducibility using sialyllactose and 4-methylumbel-liferyl-beta-D-galactoside as donor and acceptor substrates, respectively. The assay conditions were optimized using the trans-sialidase from Trypanosoma congolense and its general applicability was confirmed with recombinant trans-sialidase from Trypanosoma cruzi. Using this procedure, a large number of samples can be tested quickly and reliably, for instance in monitoring trans-sialidase during enzyme purification and the production of monoclonal antibodies, for enzyme characterization, and for identifying potential substrates and inhibitors. The trans-sialidase assay reported here was capable of detecting trans-sialidase activity in the low-mU range and may be a valuable tool in the search for further trans-sialidases in various biological systems. (C) 2003 Elsevier Inc. All rights reserved.
- リンク情報
- ID情報
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- DOI : 10.1016/j.ab.2003.07.016
- ISSN : 0003-2697
- PubMed ID : 14596820
- Web of Science ID : WOS:000186610400001