Inhibition of bovine plasma amine oxidase by superoxide dismutase active Cu(II) complexes

Vol.

96, No.

September

2, 1980 30,

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages 8234330

1980

INHIBITION

OF BOVINE PLASMA AMINE OXIDASE

BY SUPEROXIDE DISMUTASE ACTIVE CU(I1)

David

M. Dooley*

and Thomas

COMPLEXES

S. Coolbauqh

Department of Chemistry Amherst College Amherst, Massachusetts 01002 Received

August

14,

1980

Summary: Aqueous Cu*+ and Cu(I1) complexes of salicylate, lysine, and tyrosine decrease the rate of benzylamine oxidation by bovine plasma amine oxidase. Bissalicylato Cu( II) and Cu* inhibit non-competitively with respect to benzylamine. Lysine, tyrosine, Cu(EDTA)*-, Zn*+, and Co*+ do not inhibit, and erythrocyte Cu, Zn superoxide dismutase shows only sliaht inhibition of the amine oxidase. The data are most consistent with an inhibitory mechanism involving dismutation of 0: by the Cu(I1) complexes within a site relatively inaccessible to the enzyme superoxide dismutase. Excess lysine significantly decreases inhibition by the bis-lysine complex of Cu( II). For the ing

past

several

the catalytic

years

mechanism(s)

zymes catalyze

the

oxidative

intensive

effort

of copper-containing deamination

purified

amine

seedlings,

and the

physical

and kinetics

display

distinct

are

funqus

methods

(l-3).

consistent

used:

bovine

with

should

must most

have

pig

plasma,

pig

kidney, studied

in many respects,

be considered

the minimum

en-

.

been extensively

similar

steady-state

These

amines:

plasma,

niger

to elucidat-

oxidases.

RCHO + Hz02 + NH3

Althouoh

which

Nevertheless,

*To whom correspondence Abbreviations

from

Aspergillus

differences

of the mechanism. experiments

oxidases

amine

of primary

RCH2NH2 + 02 + H20 Highly

has been devoted

in any general and transient

mechanism

pea by

the enzymes formulation

phase

kinetics

in Scheme 1 (l-5).

be directed

Sal, salicylate; acid1

PIPES,

piperazine-N,N'-bis[2-ethane

sulfonic

0006-291X/80/180823-08$01.00/0 823

All

Copyright 0 I980 rights of reproduction

by Academic Press, Inc. in any form reserved.

Vol.

96, No.

2, 1980

BIOCHEMICAL

(1)

F: + RCH2NH2 -

(2)

Ered + D2 -

AND

BIOPHYSICAL

E.RCH2NH2 -

RESEARCH

COMMUNICATIONS

Ered + RCHO

E + HZ02 + NH3 Scheme 1

Apparently

step

enzyme and the

(1)

or step

substrate

pertinent one-electron superoxide remaining

amine

radical within

in this

have investigated dase by superoxide

anion the

on the

oxidases

depending

on the

steps. 02,

active

which

inhibition

dismutase

the

might

In fact, crude

of highly active

of copper

specific

Cu(I1)

of O2 by the

in the

reduction

One-electron then

site).

and utilization

role

is whether

been shown to inhibit the

limiting,

activation

and the

regard

or two-electron

have previously

ported

center

question

can be rate

(l-5).

Our own interests copper-containing

(2)

Cuzt,

kidney

purified complexes

Cu(lys),

amine

bovine

in

of O2 generates

as an intermediate

aqueous pig

A

of O2 proceeds

reduction exist

reaction.

and the

(probably and Cu(tyr)P

oxidase plasma

(6).

amine

results

the

are

We oxire-

herein.

MATERIALS AND METHODS: Bovine plasma amine oxidase was purified by a method developed by B. Mondovi and coworkers (7). After plasma collection, anonium sulfate fractionation, and dialysis (8), two affinity columns are employed: first, an AH-sepharose-4B column and, second, a Concanavalin A-sepharose-4B column. Preparations of specific activity -2000 units/mg were routinely obtained. SDS polyacrylamide gel electrophoresis indicated the enzyme to be 2 90% pure. Bovine ceruloplasmin was also purified to homogeneity (9); based on comparisons of visible absorption and CD spectra and SDS gel electrophoretic patterns, no ceruloplasmin could be detected in the amine oxidase preparations. Enzyme activity was measured at 22? 2OC using benzylamine as substrate in O.lM phosphate buffer, pH 7.2. Some experiments were carried out in 50mM PIPES, pH 7.2. One activity unit is defined as an O.OOl/min. absorbance increase at 250 nm (10). Protein was estimated from the absorbance at 280 nm using E]Em = 20.8 (1). Inhibition studies were also carried out by monitoring the benzylamine to benzaldehyde at 250 nm using the assay conditons where noted, the benzylamine concentration was 3.3mM in a final

oxidation of above. Except volume of 3 mls.

Cu(lys)p and Cu(tyr)n were synthesized by a published method (11). was prepared by the method of deAlare and coworkers (12) and recrystallized water. Cu(EDTA)'was prepared by mixing a 10% molar excess (to insure complexation) of Na2EDTA with CuSO, in water.

Cu(sal)* from complete

Bovine superoxide dismutase, Concanavalin A-sepharose-4B, and AH-sepharose-46 were obtained from Sigma. Benzylamine was purchased from Eastman. All chemicals were reagent grade and were used without further purification. Kinetics measurements were made on a Perkin-Elmer 576 UV-VIS spectrometer.

824

2

a5

a R B + 40@M R

Cu(sal)p

CU(lYS)*

CU(lYS),

Co(NO,),

phosphate

28

R

cuso,

O.lM

20

B

None

A:

24

A+400uMlys

CU(lYS)

assays.

al

A

CU(EDTA)~-

three

-

A

Cu(sal),

of

115

A

Co(N'J,),

averaqe

67

A

ZnSO,

the

70

A

cuso,

is

75

A

cuso,

entry

117

A

W

Each

117

A

1YS

lys

96

A

Cu(tyr12

buffer,

78

113

100

A

Cu(tyr)

-

-

-

-

-

-

-

-

-

-

-

-

-

99

-

58

99

pH 7.2.

-

-

-

-

-

-

-

121

78

120

75

a2

123

125

101

109

79

CU(lYS),

100

93

A

99 -

2

-

105

121

Activity -2

92

115

-1

PLASMA

97

-Exp.:

OF BOVINE

A

A

Conditions

DN THE ACTIVITY

A

Dismutase

Superoxide

Concentration (PM)

OF INHIBITORS

I

CU(lYS)P

Cu,Zn

Erthyrocyte

None

Inhibitor

EFFECTS

TABLE

4

R:

-

-

-

-

-

-

90

-

-

-

-

-

-

-

-

-

-

79

-

90

96 -

AMINE

1

3.7

-4.9

65.4

75.3

70.4

0.0

41.7

1.7

39.1

34.8

- 1.7

- 1.7

16.5

13.0

19.1

15.7

50mM PIPES

Exp.:

OXIOASE

-2

buffer,

-

-

-

-

-

0.0

35.5

0.8

38.0

32.2

- 1.7

- 3.3

16.5

9.9

17.4

13.2

Percent

pH 7.2.

-

-

-

-

-

0.0

-

41.4

0.0

-

-

-

-

-

-

-

20.2

-

7.3

Inhibition 3

-

-

-

-

-

6.0

-

-

-

-

-

-

-

-

-

-

17.7

-

5.8

-4

Vol.

96, No.

RESULTS:

Effects

oxidation

are

(T.

amine

oxidase inhibit

than

Cu(sal),

in Table

method

activity the

(8).

enzyme but are CIA'+.

in the

BIOPHYSICAL

complexes

Essentially

I.

unpublished Cu(sal)n

to approximately

or aqueous

concentration

AND

dismutase

and D. M. Dooley,

by another

also

amine

of superoxide

presented

S. Coolbaugh

purified

BIOCHEMICAL

2, 1980

about

on the

identical data)

the a factor

were

amine

obtained

oxidase

CIA'+ (as CuSOs) inhibit Cu(l~s)~

of two to three

of CIA'+ and Cu(sal)2

of benzylamine

results

same extent.

plots

COMMUNICATIONS

rate

on plasma

and aqueous

Double-reciprocal

presence

RESEARCH

less

of rate are

and Cu(tyr), effective

versus

shown

benzyl-

in Figure

9r

Figure

1:

Inhibition of O.lM phosphate Cu(sal)*; -o-,

bovine plasma amine oxidase by Cu2+ buffer pH 7.2. -O-, no inhibitor; 200 pM CuSO,.

826

and Cu(sal)* -o-, 200

in UM

1.

Vol.

96, No.

pretreatment cell

of

RNase

protease

2).

where

precipitation

residual

BNase lane

is

BIOPHYSICAL

and

shown

that

to

included

RESEARCH

which

be

in

P does

prior

to

free

all

Koski of

our

COMMUNICATIONS

used

to

proteases.

inactivate

In

pretreatment

of

BNase (NB4)2SO4 Methods.

assays

of

some

the

KB

addition

mixtures

to

BNase

then

further

Even

after

relevant 2A,

fractions 2-4).

soluble

the

the

(see

To

digested

by we

A pretreated

apparent

with

exclude

BNase

of

gradient

where

were

BNase

A in

reconstruction

to

reaction

RNase

834

BNase mixtures containing

P activity.

the

fractionated

by

described

evident

in in

there

gradient,

presumably

presence control is

A pretreated with

from

particular,

activity

polyacrylamide that

mixtures, for

out

Tl

2.

enzyme

the

RNase

Fig.

the is

(N84)2S04

the

such

carried

A bound possibility

no

the the

BNase to

lane

in

sucrose

of

A was

this

containing

the

by

as

In

of

products

were

RNase

in

whereas

lC,

2B).

fractionated amount

sedimentation

residual

gradient

were

respect

mixtures

Nevertheless,

the

samples,

Figure

precipitated the

under

residue with

fraction

containing

experiments

in

(Fig.

size. the

last

velocity

bottom

of

small

is

residual

assayed

top

from

A and

result.

RNase

its

the

(NB4)2S04

mixtures

observed

some

activity

breakdown

the

shown

loses

a slight

pretreatment

fractions

gradient

These

by

so,

accumulated

A pretreated

A which

the

2-4)

BNase

samples,

at

as

steps

in

fractions

from

same

in

Even

result

Tl

mixtures

removed

end

these

precipitate

P.

the

Methods)

gradient

of

lanes

by

purified

BNase

because

evident

the

activity

and

RNase

RNase

unresuspended

sediment

same

P is

sucrose

fractionation,

for

The

BNase

pretreatment

of

shown).

with

not

the

purification (see

degradative

does

(8),

is

A oretreatment: and

P pretreated

Tl

assay

activity

(not

inactivation

lanes

RNaSe

RNase

Further

mixtures

(Fig.

RNase

centrifugation

these

the

been

Since

Tl

2).

gradient

due

has

PMSF

lane

conditions

and

and

P (1)

uretreatment:

lC,

the

BNase

AND

.

(Fig.

1C.

coli

P (7).

RNasel

be

IL

inhibitor

Methods)

by

BIOCHEMICAL

2, 1980

is

expected

of

BNase

treated seen

P in sample

in

the

sample

various

to

same

(Fig.

2B,

concentrations beads

and

P cleavage

both

yielded

products

containing

the

the might

pretreated

control

We detected

of

treated both

BNase

Vol.

96, No.

our

data.

2, 1980

O;,

produced

has previously kidney

at a site

been proposed

amine Both

BIOCHEMICAL

oxidase

the

and the

plexes

are non-competitive

inhibitors

with

tyrosine

inhibit

substituted not catalyze

inhibitory

cycle

Cuzt

or aqueous with

amine

in metal

dismutase,

of crude

that

pig

involving

simply

active

Co'+

sites

on proteins

are

in-

these

to benzylamine.

of the

also

plots

indicating

oxidase.

binding

dismutation,

Cu'+,

respect

blocking

plasma

superoxide

of the double-reciprocal

mechanisms

and subsequent bovine

for

COMMUNICATIONS

Cu,Zn superoxide

in the catalytic

intercepts

of Cu(sal)p

formation

to the

to participate

in the presence

complex

RESEARCH

(6).

slopes

inconsistent

BIOPHYSICAL

inaccessible

creased

are

AND

com-

Other

results

enzyme-inhibitor

site.

Neither

and Znzt,

which

(and

vice

show no inhibition

lysine

nor

can often versa),

be

but

of benzylamine

do

oxi-

dation. A reasonable tion

interpretation

of substrate-reduced

tested.

copper

and (2)

complex.

could

also

amine

oxidation.

superoxide

agents

than

oxidation

of the

manner,

reduced the

copper

enzyme,

fully

Efed'

reduced

have to be released

after

above

does not seem too

restrictive,

the

Cu, Zn superoxide

bovine

plasma

amine

EFed Scheme 2:

dismutase oxidase.

less

does not our

react

the

E;ed*02

(1)

The mechanism with

since

indicates

that

possibility

is

Et,d and E+ed represent 2 electron duced enzyme, respectively.

828

inhibition

requires

Eied, the

the

lack

the

redox form)

requirement

of inhibition

0; may be tightly that

by

that

a protonated

Cu complex E;,,*O;u El red

__t

of benzyl-

O2 or other

(or

to the

which

rate for

of an 0;

transfer

CU(EDTA)~-,

the overall

of O2 with

complexes

electron

since

0;

of reoxida-

dismutation

mechanism

Since

rate

copper

enzyme via

more slowly

especially

Another

are:

suggested

reaction

the

by the

decrease

EFed.

enzyme

that

likely

complexes.

enzyme,

would

decreased

reduced is

Scheme 2 summarizes active

is

possibilities

mechanism

in this

dismutase

one-electron

mechanistic

The latter

inhibit

results

enzyme by O2 is

Two outstanding

intermediate;

of these

0; dissociation

+ 02

and 1 electron

re-

by bound is

by

Vol.

96, No.

2, 1980

sufficiently second

BIOCHEMICAL

slow

molecule

such that of the

We have not experiments cause

results

plasma

oxidase

inconsistent

with

lie

to the

0; could

be the

Finally, oxidase oxide

left

would

dismutation

in solutions

ria

towards

mation would it

constants be less

(19) than

seems clear

to Cu(lys)~ tion

part

may arise

dismutase

active

tris-

is

not could

Cu(I1) -

decrease

as only

and that

negligible species

super-

possibility

have is that

significantly

to 0;

drives

the

Consideration

equilib-

of the

concentration

of Cu(lys)

containing

200 uM Cu(ly~)~,

superoxide

Cu(lys)

the

amine

bis-complexes

lysine

active.

total

plasma

or tetrakis-complexes

Another

formally

from

(l-3),

E:ed -

could

unlikely

less

of the

not

an observable

of bovine

lysine

(19).

the

in pig

Eked ---h(I)

addition

inhibition

Cu(lys)*

that

since

and Cu2+ contribute

is

site

formed.

by forming

which

and be-

of O2 are

of evidence

its

Excess

10 uM in a solution at least

a

in agreement.

reduction

Cu(I1) -i

after

upon the

containing

Cu2+ is completely

superoxide

of 02;

of Cuflysf

in solution

of aqueous

dominant

that

with

reported,

binding

process

by a variety

seems rather

indicates

in the

in this

titrations

Cu(ly~)~,

reaction

not altogether

the dioxygen

Eied -

noting.

in potentiometric concentrations

is

steps

out

of lysine

This

equilibrium

further

ruled

of Cu(ly~)~

be inactive.

via

in Scheme 2 as no definitive

are

copper

enzyme species

are worth

activity

been observed

(15-18)

the equilibrium

effects

COMMUNICATIONS

enzyme have been

of copper

in the absence

by Cu(ly~)~ dismutase

that

is

predominant

the

plasma

that

involvement

For example,

far

bovine

One-electron

which

RESEARCH

regenerated

enzyme copper

oxidases

(15).

intermediate,

required.

in the amine

the

enzyme is

included

role

BIOPHYSICAL

complex.

has been presented

amine

Cu(1)

copper

on other

Good evidence

oxidized

explicitly

on its

AND

dismutase

activity

and Cu*+.

At pH 7.2 the

in solutions

of Cu(sal)2.

are

probably

Cu(sa1)

for-

+ Cu2+ but

attributed concentraThe pre-

and Cu(sal)!$-

(20). ACKNOWLEDGEMENTS:

This

work

We thank

Patricia

Jenkins

sor Jim

Fee for

much helpful

for

was supported assistance

by an NIH BRSG Grant with

advice.

829

the

gel

electrophoresis

(5 SO7 RR07110-11). and Profes-

Vol.

96, No.

2, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

REFERENCES: 1.

2. 3. 4. 5. 6. L: 9. 10. 11. 12. 13. 14. 15. 16. 1;: :::

Yasunobu, K. T., Ishizaki, H., and Minamiura, N. (1976), Molec. Cell. Bio*. , 13, 3-29. MalmstrUm, B. G., Andreasson, L. E. and Reinhammar, B. (1975) The Enzymes, 3rd ed., V. XIIB, (P. Boyer, ed.), p. 507-579, Academic Press, New York. Kapeller-Adler, R. (1970), Amine Oxidases and Methods for Their Study, Wiley, New York. Mondovi, B. (1972), Adv. Biochem. Psychopharm., V. 5 (E. Costa and M. Sandler, ed.), pp. 181-183, Raven Press, New York. Ogura, Y., Suzuki, H., and Yamada, H. (1972), Molecular Mechanisms of Enzyme Action, pp. 15-35, Univ. of Tokyo Press, Tokyo. Younes, M. and Weser, U. (1978), Biochem. Biophys. Acta 526, 644-647. Sabatini, S., Turini, P., and Mondovi, B., personal communication. Yamada, H. and Yasunobu, K. T. (1962), J. Biol. Chem. 237, 1511-1516. T. S., and Jenkins, P., in preparation. Dooley, D. M., Coolbaugh, Tabor, C. W., Tabor, H. and Rosenthal, S. M. (1954), J. Biol. Chem. 208, 645-661. Stevens, C. M. and Bush, J. A. (1950), J. Biol. Chem. 183, 139deAlvare, L. R., Goda, K., Kimura, T. (1976), Biochem. Biophys. Res. Comnun. 69, 687-694. Younes, M., Lengfelder, E., and Weser, U. (1978), Biochem. Biophys. Res. Commun. 81, 576-580 and references cited therein. Joester, K-E., J-ng, G., and Weser, U. (1972), FEBS Lett. 25, 25-28. Barker, R., Boden, N., Cayley, G., Charlton, S. C., Henson, R., Holmes, M. C., Kelly, I. D., and Knowles, P. F. (1979), Biochem. J. 177, 289-302. Grant, J., Kelly, I., Knowles, P., Olsson, J., and Pettersson, G. (1978), Biochem. Biophys. Res. Commun. 83, 1216-1224. Kleutz, M. D. and Schmidt, P. G. (1977), Biochemistr 16, 5191-5199. Kleutz, M. D., Adamsons, K., and Flynn, JdBiochemistry 19, 1617-1621. Brookes, G. and Pettit, L. D. (1976), J. Chem. Sac. Dalton Trans., 42-46. O'Young, C.-L. and Lippard, S. J. (1980), J. Amer. Chem. Sot. 102, 4920-4924 and references cited therein.

830