TY - JOUR
T1 - A long-wavelength fluorescent substrate for continuous fluorometric determination of α-mannosidase activity
T2 - Resorufin α-d-mannopyranoside
AU - Coleman, Daniel J.
AU - Kuntz, Douglas A.
AU - Venkatesan, Meenakshi
AU - Cook, Gabriele M.
AU - Williamson, Staci P.
AU - Rose, David R.
AU - Naleway, John J.
N1 - Funding Information:
The authors thank Heather Strachan and Kelley Moremen at the Complex Carbohydrate Research Center and Department of Biochemistry and Molecular Biology, University of Georgia, for data regarding the enzyme activities toward various mannosidase substrates. In addition, we thank Anthony P. Guzikowski and Michael Strain for help with NMR analyses. This work was funded in part by Grant 1R43MH079542 from the National Institutes of Health–National Institute of Mental Health (to J.J.N., D.J.C., G.M.C., and S.P.W.). D.R.R. received funding from the Canadian Institutes for Health Research ( MOP79312 ) and the Mizutani Foundation ( 080032 ).
PY - 2010/4
Y1 - 2010/4
N2 - A simple and reliable continuous assay for measurement of α-mannosidase activity is described and demonstrated for analysis with two recombinant human enzymes using the new substrate resorufin α-d-mannopyranoside (Res-Man). The product of enzyme reaction, resorufin, exhibits fluorescence emission at 585nm with excitation at 571nm and has a pKa of 5.8, allowing continuous measurement of fluorescence turnover at or near physiological pH values for human lysosomal and Drosophila Golgi α-mannosidases. The assay performed using recombinant Drosophila Golgi α-mannosidase (dGMII) has been shown to give the kinetic parameters Km of 200μM and Vmax of 11nmol/min per nmol dGMII. Methods for performing the assay using several concentrations of the known α-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.
AB - A simple and reliable continuous assay for measurement of α-mannosidase activity is described and demonstrated for analysis with two recombinant human enzymes using the new substrate resorufin α-d-mannopyranoside (Res-Man). The product of enzyme reaction, resorufin, exhibits fluorescence emission at 585nm with excitation at 571nm and has a pKa of 5.8, allowing continuous measurement of fluorescence turnover at or near physiological pH values for human lysosomal and Drosophila Golgi α-mannosidases. The assay performed using recombinant Drosophila Golgi α-mannosidase (dGMII) has been shown to give the kinetic parameters Km of 200μM and Vmax of 11nmol/min per nmol dGMII. Methods for performing the assay using several concentrations of the known α-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.
KW - Continuous fluorescent enzyme assay
KW - DGMII
KW - Drosophila melanogaster mannosidase II
KW - Fluorogenic substrate
KW - GMII
KW - Golgi mannosidase II
KW - Kinetic assay
KW - Res-Man
KW - Resorufin α-d-mannopyranoside
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U2 - 10.1016/j.ab.2009.11.039
DO - 10.1016/j.ab.2009.11.039
M3 - Article
C2 - 20026005
AN - SCOPUS:77649178895
SN - 0003-2697
VL - 399
SP - 7
EP - 12
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 1
ER -