A long-wavelength fluorescent substrate for continuous fluorometric determination of alpha-mannosidase activity: resorufin alpha-D-mannopyranoside.
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.
Coleman
Daniel J
DJ
Marker Gene Technologies Inc., University of Oregon Riverfront Research Park, Eugene, OR 97403, USA.
Kuntz
Douglas A
DA
Venkatesan
Meenakshi
M
Cook
Gabriele M
GM
Williamson
Staci P
SP
Rose
David R
DR
Naleway
John J
JJ
D000818:Animals; D004330:Drosophila; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000737:chemistry*; D005470:Fluorometry / Q000379:methods*; D006056:Golgi Apparatus / Q000201:enzymology; D057166:High-Throughput Screening Assays; D006801:Humans; D007700:Kinetics; D008247:Lysosomes / Q000201:enzymology; D008362:Mannosides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D010078:Oxazines / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D017026:Swainsonine / Q000737:chemistry / Q000494:pharmacology; D043323:alpha-Mannosidase / Q000235:genetics / Q000378:metabolism*
D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D008362:Mannosides; D010078:Oxazines; D011994:Recombinant Proteins; C549458:resorufin alpha-D-mannopyranoside; D043323:alpha-Mannosidase; D017026:Swainsonine