A long-wavelength fluorescent substrate for continuous fluorometric determination of alpha-mannosidase activity: resorufin alpha-D-mannopyranoside.

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.

• Authors: Daniel J Coleman, Douglas A Kuntz, Meenakshi Venkatesan, Gabriele M Cook, Staci P Williamson, David R Rose, John J Naleway
• Journal: Analytical biochemistry (Anal Biochem) Vol. 399 Issue 1 Pg. 7-12 (Apr 01 2010) ISSN: 1096-0309 [Electronic] United States
• PMID: 20026005 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)
• Copyright: Copyright 2009 Elsevier Inc. All rights reserved.

Chemical References

• Enzyme Inhibitors
• Fluorescent Dyes
• Mannosides
• Oxazines
• Recombinant Proteins
• resorufin alpha-D-mannopyranoside
• alpha-Mannosidase
• Swainsonine

Topics

• Animals
• Drosophila
• Enzyme Inhibitors (chemistry, pharmacology)
• Fluorescent Dyes (chemistry)
• Fluorometry (methods)
• Golgi Apparatus (enzymology)
• High-Throughput Screening Assays
• Humans
• Kinetics
• Lysosomes (enzymology)
• Mannosides (chemical synthesis, chemistry, metabolism)
• Oxazines (chemical synthesis, chemistry, metabolism)
• Recombinant Proteins (genetics, metabolism)
• Substrate Specificity
• Swainsonine (chemistry, pharmacology)
• alpha-Mannosidase (genetics, metabolism)