A bioluminescent assay for monoamine oxidase activity.
This article describes a novel two-step homogeneous bioluminescent assay for monoamine oxidase (MAO) that is simple, sensitive, and amenable to high-throughput screening. In the first step, MAO reacts with an aminopropylether analog of methyl ester luciferin. In the second step, a luciferin detection reagent inactivates MAO and converts the product of the first step into a luminescent signal. The amount of light produced is proportional to the amount of MAO and the time of incubation in the first step, but the luminescent signal is stable in the second step with a half-life greater than 5h. The assay has high precision, is more sensitive than current fluorescent methods, and can accurately measure the binding constants of known substrates and inhibitors. An automated screen of the Sigma-RBI Library of Pharmacologically Active Compounds (LOPAC(1280)) revealed a surprisingly high percentage of MAO inhibitors (16%) with a low false hit rate (0.9%). This implies that a significant number of compounds interact with the MAO enzymes and suggests that it is important to include MAO assays in drug metabolism studies. Other advantages of this bioluminescent assay over comparable fluorescent assays are discussed.
Valley
Michael P
MP
Promega Corporation, Madison, WI 53711, USA. mike.valley@promega.com
Zhou
Wenhui
W
Hawkins
Erika M
EM
Shultz
John
J
Cali
James J
JJ
Worzella
Tracy
T
Bernad
Laurent
L
Good
Troy
T
Good
Dave
D
Riss
Terry L
TL
Klaubert
Dieter H
DH
Wood
Keith V
KV
D000818:Animals; D007700:Kinetics; D008156:Luciferases / Q000378:metabolism / Q000493:pharmacokinetics; D008163:Luminescent Measurements / Q000379:methods*; D053858:Metabolic Networks and Pathways; D008738:Methyl Ethers / Q000737:chemistry / Q000493:pharmacokinetics; D051379:Mice; D008930:Mitochondria, Liver / Q000378:metabolism; D008995:Monoamine Oxidase / Q000032:analysis* / Q000378:metabolism*; D008996:Monoamine Oxidase Inhibitors / Q000378:metabolism*; D012680:Sensitivity and Specificity
D008738:Methyl Ethers; D008996:Monoamine Oxidase Inhibitors; D008156:Luciferases; D008995:Monoamine Oxidase