A microtitre plate assay for measuring glycosidase activity.
Glycosidases perform a wide range of functions in physiology and pathology, and are potential targets for the treatment of diseases such as influenza, cancer, AIDS and diabetes. This paper reports a convenient discontinuous colourimetric assay for the measurement of glycosidase activity. The assay utilises 4-nitrophenyl- substrates and quantities of product are determined by measuring absorbance at 405 nm. This assay is performed in a 96 well microtitre plate and has been used to characterise the properties of seven different glycosidases from bacteria, yeast and higher eukaryotes and their kinetic parameters determined. Assays in the presence of known inhibitors showed that inhibition modes can be determined, and IC(50) and K(i) values calculated. This assay appears to be of widely applicable and of general utility for the measurement of glycosidase activity and the evaluation of inhibitors.
Ball
Andrea L
AL
Department of Pharmacy & Pharmacology, University of Bath, Claverton Down, Bath, United Kingdom.
Chambers
Kirsty A
KA
Hewinson
Meera
M
Navaratnarajah
Sambavi
S
Samrin
Lamia
L
Thomas
Nesta
N
Tyler
Abigail E H
AE
Wall
Amanda J
AJ
Lloyd
Matthew D
MD
D000818:Animals; D001236:Aspergillus oryzae / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology* / Q000627:therapeutic use; D004926:Escherichia coli / Q000201:enzymology; D006026:Glycoside Hydrolases / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D012680:Sensitivity and Specificity
D004791:Enzyme Inhibitors; D006026:Glycoside Hydrolases