BIOCHEMICAL
Vol. 86, No. 2, 1979
AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 350-357
January 30, 1979
SELECTIVE RADIOCHEMICAL LABELING OF TYPES A AND B ACTIVE SITES OF RAT LIVER MONOAMINE OXIDASE D.J.
November
18,
and K.Y.
1978
SUMMARY: By using clorgyline and deprenyl sitesof monoamine oxidase, respectively, labeled radiochemically with [3H]pargyline. sites in rat liver mitochondria indicated 1:3.3. Subunits containing the labeled A separated by SDS-gel electrophoresis , and 60,000 daltons. Pargyline monoamine flavin
moiety
towards
(MAO; E.C.
of the
MAO,since
flavoproteins (3).
enzyme
sequence
forms
to the
recently
(5).
tinguished
by the
form.
A basic
MAO activity
proteins
on differences Two forms effects
complete
per mole
of flavin.
contained
question
yet
components).
This
question
of protein
by the
is
not easily
purification,
since
0006-291X/79/020350-08$01.00/0 @ 1979
by Academic Press, Inc. in any form reserved.
350
from bovine for
specificity
this
when is
having liver
the MAO (4).
of multiple
and inhibitor A and B) can be dis-
selectively
inhibits
the different
of lipids
answered
--in vivo
moiety
the existence
or to different binding
specific
occurring
in a pentapeptide
is whether
are due todifferentapoenzymes
the
purified
The flavin
as types
which
to be answered
example,
inhibition
evidence
of MAO (designated
with
to be highly other
of
was administered
has been isolated
in substrate
inhibitor adduct
to either
when the drug
of clorgyline,
for
of reproduction
appears
with
which
(as,
Copyright All rights
reaction to bind
enzyme and is
modifications
techniques
a covalent
has been considerable
of MAO, based
sensitivity
forming
failed
Ps bound
Ser-Gly-Gly-Cys-Tyr
There
This
is stoichiometric
one mole of inhibitor bound
(1).
or to other
The reaction
is an irreversible
1.4.3.4),
[14CJpargyline
(2)
covalently
to block the types A and B active the remaining site could be selectively Titration of the A and B active that they were present in a ratio of and B catalytic sites could not be each had a molecular weight of
(N-benzyl-N-2-propynylamine)
oxidase
Pak
Department of Psychiatry of Pittsburgh School of Medicine Psychiatric Institute and Clinic Pittsburgh, PA. 15261
University Western Received
Edwards
types
of
post-translational or other
by using
enzyme is
the type
the
tightly
membrane standard bound
to the
A
BIOCHEMICAL
Vol. 86, No. 2, 1979
mitochondria
1 membrane
such as the
use of sonication
of the different environment.
and/or
binding
by removing
the possibility
selectively
lost,
preparation
contained
In order active
observed
but he could
sites
and deprenyl
be excluded.
Moreover,
out
problems,
selectively
pargyline.
before
the
labeled
respectively,
upon purifiA MAO was
the purified
the labeled
pargyline
enzyme
moieties
by using
clorgyline
are
to labeling
approach
subunits
to label
protein
prior
We have used this
and to characterize
of lipids
of type
that
we have used [3H]
were
[3H]
of the
form of the enzyme.
The A and 6 sites
the A and B sites,
activity
the possibility
membrane
with
enzymatic
inactive
these
properties
cannot
intact
site
A and B sites
not rule
observed
micro-
removal
the
properties
lipophilic
by incomplete
that
enzyme,
the natural
their
the
the
caused
of MAO in the
to block
may alter
that
a catalytically
to circumvent
solubilized.
remaining
(7)
to solubilize
them from
of the detergent
of MAO, McCauley
needed
detergents,
enzyme are due to artifacts
or to residual
the
The techniques
enzyme forms In addition,
solubilized
cation
(6).
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
the
to titrate
by SDS-gel
the
electrophoresis.
MATERIALS AND METHODS. Rat liver mitochondria were isolated according to the procedure of Autilio et al. (8) and resuspended in 0.2M potassium ohosohate pH 7. 4. MAO activity was assayed by a micro-radiochemical procedure as described by Edwards et al. (Q), using [14C]serotonin or[l4C]phenylethylamine as substrates. Protein concentrations were determined by the method of Hartree (lo), using ;Jovine serum albumin as standard. Electrophoresis was carried out in 7.5% polyacrylamide slab gels in the presence of sodium dodecyl sulfate (SDS), according to the procedure of Weber and Osborn (11). For determination of radioactivity, 2.5 mm gel slices were incubated overnight at 25" in 5 ml of a mixture containing PPO/POPOP/toluene cocktail and 4% Protosol (New England Nuclear). [3H] Pargyline (6.75 Ci/mmole) was prepared by New England Nuclear. RESULTS:
Fig.
selectively
1 shows the
label
either
results
obtained
from
the A or B active
sites
experiments
carried
out
to
of MAO with
C3H] pargyline.
. In both
cases,
a concomitant
the
incorporation
decrease
in enzymatic
was increased.
In addition,
reach
when the
a plateau
inhibition. decreased From these
of the
these
linearly
with
data,
the molar
activity
the amount amount
By replotting
["HI
the amount quantities
as the
of ["HI
of pargyline data,
label
amount
pargyline
added
we observed
of r3H]
increased
pargyline
bound
appeared
complete
(see sites
with
pargyline to
enzymatic
the per cent
of the A and B active
351
of added bound
produced that
nonlinearly
activity
insets, in the
Fig.1). homog-
Vol. 86, No. 2, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
IO