Pharrnaco~g~alResea~h Commun~ation~VoL 2~ Supplementl~ 1988

p-NITRO-PHENYLETHYLAMINE : A MAO A INHIBITOR ~Nb A MAO B SUBSTRATE

109

Vicente L., G6mez N., Heredero J., Martin M. and Unzeta M.

Biochemistry Department , Faculty of Medicine, Universidad Autonoma de Barcelona. Bellaterra. Barcelona. Spain.

Keywords: MAO, p-nltro-phenylethylamine, irreversible inhibition.

In order to elucidate which are the molecular mechanisms involved in the

selectivity for MAO A and MAO B , the activity of both forms of the enzyme

towards p-nitro-phenylethylamlne (p-NPEA) has been studied. The aim of

this work was to find out how the presence of a nitro group in the "pare"

position of the phenyl ring would modify the behavlour of phenylethylamine

(FEA) in front of the two forms of MAD. The nitro group increases the

dipole moment and consequently enhances the electrostatic interactions.

METHODS: MAO activity was measured in liver mitochondria prepared by a

standard centrifuga£ion method (Gomez et el., 1986) from Sprague-Dawley

rats, either fluorimetrlcally (Snyder and Handley, 1968) or by a radlome-

tt~c assay (Fowler and Tipton, 1981), the latter one using labelled

5-hydroxytryptamlne and PEA as MAO A and MAO B substrates respectively.

Separated MAO A and MAO B forms were obtained preincubating mitochondria

at 37°C for 30 min. with 3"IO-7M deprenyl or clorgyline respectively.

RESULTS

pNFEA as substrate. MAO activity towards 25 pM pNPEA was determined fluo-

rimetrically at different concentrations (I0 -I0 - 10 -3 M) of clorgyline

and deprenyl. The unimodal inhibition curves thus obtained showed that

pNPEA is metabolized preferently by MAO B.

9NPEA as inhibitor. Inhibition studies were carried out with the radiome-

tric method, pNPEA was shown to have a time dependent inhibitory effect

on MAO A, the IC50 values at O and 30 min. being 540 pM and 200 pM res-

pectlvely. The initial and progressive inhibition of MAO A was of the same

magnitude when the enzyme was added after prelncubatlng the tissue with

the amine for one hour, what discards the possibility that the inhibition

could be mediated by a product of pNPEA resulting from its metabolism by

mitochondria.

The analysis of the reversible step by measuring MAO A activity at 0 time

O03"t.--6989/88/201VOlO9-2/S03.00/O © 1988 The Italian Pharmacological Society

Pharmacologica/ Research Communications, Vo/. 20, Supplement IV. 1988 110

at different concentrations of pNPEA (102-103 pM) as inhibitor clearly

demonstrated a competitive mechanism with a Ki=215 +_2.

The time inhibition of MAO A in front of 250 ~M pNPEA suggested an irre-

versible mechanism; this was corroborated by dilution and centrlfugatlon

experiments. Therefore we measured the rate of inhibition of MAO activity

in front of different concentrations of pNPEA (40-300 ~M). When plotting

the reciprocals of those rates versus the reciprocals of pNPEA concentra-

=ions, the values of K 2 and K i obtained from the intercepts were .024 4-

.003 min -I and 205 Z 12 pM respectively.

With respect to MAO B, the time course of its activity in the presence of

10 and 50 ~M pNPEA indicated a reactivation after an initial inhibition;

this is consistent with the suhstrate behaviour of this compound we had

previously observed. Therefore it was a K m rather than a K i what had to

be determined. However, quenching problems disturbing the fluorimetric

readings when using relatively high concentrations of pNPEA forced us to

use the radlometrlc method to establish if pNPEA acted indeed by displa-

cing labelled PEA in a competitive way and, if so, consider the resulting

K i as the Km; the validity of this assumption is supported by McEwen et

el. (1969), who showed that amines acting as competitive inhlbitors of

MAO have K i values which are virtually the same as their Km when acting

as substrates. The K i obtained in this way was 4.5 ~ 0.5 ~M, about half

the K m corresponding to PEA (Gomez et el., 1986).

reversible inhibition _ irreversible .inhibition

K i (~M) _ Ki (~M) k 2 (min-l)

MAO A 2[5 ~ 2 2O5 ± t2 .024 i .O03

MAO B ~ . s ± .5 - /--

These results allow us to conclude that the localization of a nitro group

in the "para" position of the phenyl ring of the PEA molecule increases

the affinity of MAO B towards this compound and enables the molecule to

act as an inhibitor of the A form. The kinetic parameters of this inhibi-

tion suggest that it is mediated by an "active site directed" mechanism.

• -Fowler C.J. and Tipton K.F. Biochem. Pharmac. 30, 3329 (19gl). -G6mez N., Unzeta M., Tipton K.F., Anderson M.C. and O'Carroll A-M. Biochem. Pharmac. 35, 24, 4467 (1986).

-McEwen C.M., Sasaki G. and Jones D.C. Biochemistry 8, 3952 (1969). -Snyder S.H. and Handley E.D.J. Pharmacol. Exp. Ther. 163, 386 (1968).