Advanced Search

Journal Navigation

Journal Home

Subscriptions

Archive

Contact Us

Table of Contents

Sign In to gain access to subscriptions and/or personal tools.
Journal of Biomolecular Screening
This Article
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
1087057106291585v1
11/7/836    most recent
Right arrow References
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to Saved Citations
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Request Reprints
Right arrow Add to My Marked Citations
Citing Articles
Right arrow Citing Articles via Web of Science (1)
Right arrow Citing Articles via Google Scholar
Right arrow Citing Articles via Scopus
Google Scholar
Right arrow Articles by Wu, Y.-W.
Right arrow Articles by Tsai, Y.-H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wu, Y.-W.
Right arrow Articles by Tsai, Y.-H.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

A Rapid Transglutaminase Assay for High-Throughput Screening Applications

Yu-Wei Wu

Graduate Institute of Pharmacy, Taipei Medical University, Taipei, 110, Taiwan

Yu-Hui Tsai

Graduate Institute of Pharmacy, Taipei Medical University, Taipei, 110, Taiwan, Graduate Institute of Cell and Molecular Biology, Taipei Medical University, Taipei, 110, Taiwan

Transglutaminases (TGs) are widely distributed enzymes that catalyze posttranslational modification of proteins by Ca2+-dependent cross-linking reactions. The family members of TGs participate in many significant processes of biological functions such as tissue regeneration, cell differentiation, apoptosis, and certain pathologies. A novel technique for TG activity assay was developed in this study. It was based on the rapid capturing, fluorescence quenching, and fast separation of the unreacted fluorescent molecules from the macromolecular product with magnetic dextran-coated charcoal. As few as 3 ng of guinea pig liver transglutaminase (gpTG) could be detected by the method; activities of 96 TG samples could be measured within an hour. The Km of gpTG determined by this method for monodansylcadaverine (dansyl-CAD) and N, N-dimethylcasein was 14 and 5 µM, respectively. A typical competitive inhibition pattern of cystamine on dansyl-CAD for gpTG activity was also demonstrated. The application of this technique is not limited to the use of dansyl-CAD as the fluorescent substrate of TG; other small fluor-labeled TG substrates may substitute dansyl-CAD. Finally, this method is rapid, highly sensitive, and inexpensive. It is suitable not only for high-throughput screening of enzymes or enzyme inhibitors but also for enzyme kinetic analysis.

Key Words: transglutaminase (TG) • TG activity • TG kinetics • magnetic dextran-coated charcoal • fluorescence

This version was published on October 1, 2006

Journal of Biomolecular Screening, Vol. 11, No. 7, 836-843 (2006)
DOI: 10.1177/1087057106291585
Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?