An improved approach to steady-state analysis of monoamine oxidases

Rona R. Ramsay, Aldo Olivieri, Andrew Holt

Research output: Contribution to journalArticlepeer-review

19 Citations (Scopus)


The search for new monoamine oxidase
inhibitors aims to identify potential lead compounds that
are more potent and selective than current drugs for use in
treating a variety of neuropsychiatric and neurodegenerative
conditions. An integral part of this process is a kinetic
examination of monoamine oxidases in the presence of the
inhibitor, to determine potency and selectivity and to
obtain information on mechanism. To date, kinetic data
obtained with a probe substrate have been analysed by
fitting to the Michaelis–Menten equation which describes a
unireactant process in which velocity is related to substrate
concentration in a rectangular hyperbolic manner. In this
study, we present evidence that monoamine oxidase
activity is often not adequately described by this approach.
We outline a novel equation strategy that takes account of
substrate and inhibitor binding to oxidised and reduced
enzyme forms, and quantifies differences between substrates
and inhibitors in this regard. When combined with
plate reader-based experimental techniques that allow large
numbers of substrate and inhibitor concentrations to be
used, and the global nonlinear regression facilities of
GraphPad Prism software, this straightforward approach
allows more appropriate analyses of monoamine oxidase
by non-experts than has previously been possible.
Keywords Monoamine oxidase Steady-state kinetics
Equations Inhibition GraphPad Prism
Monoamine oxidases (MAOs) are targets for therapeutics
designed to treat numerous neuropsychiatric and neurodegenerative
conditions (Youdim et al. 2006). To date,
drugs targeting MAOs have been developed as inhibitors
of enzymatic activity, designed to increase the availability
of biogenic amine transmitters and neuromodulators,
to decrease formation of reactive oxygen species
and also for in vitro use to probe mechanisms of MAO
catalysis. Preclinical development and experimental use
of MAO inhibitors require determination of inhibitor
potency and mechanism in steady-state assays of enzyme
activity. Such assays typically involve inclusion of the
inhibitor, at several concentrations, in assays of MAO
activity versus a range of substrate concentrations
extending either side of the KM value. Historically, initial
rate data have then been plotted and analysed by fitting
to a hyperbolic or linearised version of the Michaelis–
Menten equation. Analyses are rapid and easy to do with
regression software, and interpretation of results appears
Original languageEnglish
Pages (from-to)1003-1019
Number of pages17
JournalJournal of Neural Transmission
Issue number7
Early online date14 Jun 2011
Publication statusPublished - Jul 2011


  • Monoamine oxidase
  • Steady-state kinetics
  • Inhibition
  • Equations
  • GraphPad Prism


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