| Literature DB >> 20026005 |
Daniel J Coleman1, Douglas A Kuntz, Meenakshi Venkatesan, Gabriele M Cook, Staci P Williamson, David R Rose, John J Naleway.
Abstract
A simple and reliable continuous assay for measurement of alpha-mannosidase activity is described and demonstrated for analysis with two recombinant human enzymes using the new substrate resorufin alpha-d-mannopyranoside (Res-Man). The product of enzyme reaction, resorufin, exhibits fluorescence emission at 585 nm with excitation at 571 nm and has a pK(a) of 5.8, allowing continuous measurement of fluorescence turnover at or near physiological pH values for human lysosomal and Drosophila Golgi alpha-mannosidases. The assay performed using recombinant Drosophila Golgi alpha-mannosidase (dGMII) has been shown to give the kinetic parameters K(m) of 200 microM and V(max) of 11 nmol/min per nmol dGMII. Methods for performing the assay using several concentrations of the known alpha-mannosidase inhibitor swainsonine are also presented, demonstrating a potential for use of the assay as a simple method for high-throughput screening of inhibitors potentially useful in cancer treatment. Copyright 2009 Elsevier Inc. All rights reserved.Entities:
Keywords: Drosophila melanogaster mannosidase II; GMII; Golgi mannosidase II; Res-Man; continuous fluorescent enzyme assay; dGMII; fluorogenic substrate; kinetic assay; resorufin α-D-mannopyranoside
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Year: 2009 PMID： 20026005 PMCID： PMC2824011 DOI： 10.1016/j.ab.2009.11.039
Source DB: PubMed Journal: Anal Biochem ISSN： 0003-2697 Impact factor: 3.365