A long-wavelength fluorescent substrate for continuous fluorometric determination of α-mannosidase activity: Resorufin α-d-mannopyranoside

Daniel J. Coleman, Douglas A. Kuntz, Meenakshi Venkatesan, Gabriele M. Cook, Staci P. Williamson, David R. Rose, John J. Naleway

Research output: Contribution to journalArticlepeer-review

7 Scopus citations

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 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.

Original languageEnglish (US)
Pages (from-to)7-12
Number of pages6
JournalAnalytical Biochemistry
Volume399
Issue number1
DOIs
StatePublished - Apr 2010
Externally publishedYes

Keywords

  • Continuous fluorescent enzyme assay
  • DGMII
  • Drosophila melanogaster mannosidase II
  • Fluorogenic substrate
  • GMII
  • Golgi mannosidase II
  • Kinetic assay
  • Res-Man
  • Resorufin α-d-mannopyranoside

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint

Dive into the research topics of 'A long-wavelength fluorescent substrate for continuous fluorometric determination of α-mannosidase activity: Resorufin α-d-mannopyranoside'. Together they form a unique fingerprint.

Cite this