Abstract
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 585 nm with excitation at 571 nm and has a p K a 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 K m of 200 μM and V max of 11 nmol/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.
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