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