Irreversible binding of dopa and dopamine metabolites to protein by rat liver microsomes

Vol. 66,, No. 4, 1975

BIOCHEMICAL

IRREVERSIBLS

BINDING

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

OF DOPA AND DOPAMINE METABOLITES

TO PROTEIN

BY RAT LIVER

MICROSOMES

M. Scheulen, P. Wollenberg, H.M. H. Kappus and H. Remmer Institute of Toxicology, University D 74 Tiibingen, WilhelmstraBe Recieved

August

25,

Bolt,

of Tubingen, 56, Germany

1975

SUMMARY: Rat liver microsomes catalyze NADPH-dependent irreversible binding of metabolites of DOPA and DOPAmine to microsomal protein and to BSA. Binding is inhibited by cysteine and the singlet oxygen quencher 1,4-diaza-bicyclo(2.2.2)octane. Irreversible binding to BSA is also catalyzed by mushroom tyrosinase, xanthine oxidase, and NADPH-cytochrome c reductase. The results suggest that in the microsomal system the participation of the hemoprotein, cytochrome P-450, is not an absolute requirement for the irreversible binding of metabolites of DOPA and DOPAmine to proteins. Adverse

reactions

a-methyl-DOPA and hepatic the

include

is

DOPA in

the

The catechol

sinase these

enzymic

Abbreviations: pine; BSA, bovine

covalent

are action

(l),

and Wureel

(4)

to

of rat reported

to proteins liver

described

proteins also

discus-

of a-methyl-

and 2-hydroxyto a large

microsomes (5).

to DOPA and DOPAmine

1396

extent

and mushroom

!Che present and discusses

DOPA, L-3,&dihydroxyphenylalanine* DABCO, l,&diaza-bicyclo(2.2.2foctane; serum albumin.

Cop.vright o I9 75 by Academic Press, Inc. All rights of reproduction in my form resemed.

(2),

effects.

2-hydroxy-estradiol bound

have

they

binding

with

hepatitis

serum

catechol;

protein

of these

estrogens,

as previously findings

of

therapy

anemia

of this

pathogenesis

ethynylestradiol,

during

of a-methyl-DOPA

due to autoxidation implications

wing

Gottlieb

(3).

binding

sed the

observed

immunohemolytic

necrosis

irreversible

which

frequently

report

follotyro-

extends

a possible DOPAmine,

Vol. 66, No. 4,1975

mechanism

BIOCHEMICAL

of covalent

proteins

by liver

MATERIALS

binding

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of these

natural

compounds

to

microsomes.

AND METHODS

[3-14ClDOPA and Lthylamine-l4 C]DOPAmine were obtained from Radiochemical Centre, Amersham. Male Wistar rats (200 - 260 g) were used. Rat liver microsomes were prepared as described by Remmer et al. (6). Microsomal NADPH-cytochrome c reductase (EC 1.6.2.4.) was solubilised and purified according to Omura and Takesue (7). Microsomal incubations containing BSA, a NADPH regenerating system and 14C-DOPA or l4C-DOPAmine were carried out as described elsewhere (8). After incubation, the microsomes and supernatant were separated by ultracentrifugation (9). Microsomal pellets were washed twice with KCl-Tris buffer (4 vol. 1.15 % KC1 plus 1 vol. 0.25 M Tris-HCl buffer, pH 7.5) and dialyzed against distilled water for 24 hr. This treatment removed all free and reversibly bound radioactivity from the microsomal pellet. That removal was complete was proved by additional solvent extractions and measurement of radioactivity of the remaining supernatants and determination of radioactivity irreversibly bound to protein as previously described for imipramine (10). The supernatants of ultracentrifugation, which contained the BSA added to the microsomal incubation mixtures, were mixed with 1 vol. charcoal suspension (1 '$ norit A plus 0.01 $ dextran in 0.1 M Tris buffer, pH 7.5), shaken at room temperature for 30 min and centrifuged. This procedure was repeated twice. The supernatants thus obtained were dialyzed against distilled water for 24 hr. Gel filtration on Sephadex GlOO in 10 mM Tris-HCl buffer, pH 7.8, showed that the radioactivity remaining after these procedures was confined only to the albumin pee&. Incubation of albumin containing bound DOPA or DOPAmine metabolites which was isolated by gel filtration chromatography with cold excess DOPA or DOPAmine did not remove any radioactivity from the protein. Incubations of 14C-DOPA with tyrosinase (EC 1.10.3.1.) and BSA were performed as previously described using estrogens as substrates (5). For incubations with xanthine oxidase (EC 1.2.3.2.) or cytochrome c reductase, the tyrosinase was replaced by 8 mu/ml xanthine oxidase plus 0.8 mM hypoxanthine or 112 mu/ml NADPH-cytochrome c reductase plus a NADPH regenerating system (6), respectively. Radioactivity was determined according to the method of Bray (11) and protein was measured by the method of Lowry et al. (12). RESULTS Rat

AND DISCUSSION

liver

microsomes

DOPA end l4 C-DOPAmine (Tab.1). a singlet

Binding oxygen

is

catalyze to both effectively

quencher

(13).

an irreversible microsomal inhibited This

1397

protein

binding

and albumin

by cysteine

may suggest

of l4&

that

and DABCO, in micro-

Vol. 66, No. 4, 1975

TABLF:

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1

binding of 14 C-DOPA and 14 C-DOPAmine metabolites to protein and BSA as catalyzed by rat liver microsomes

Irreversible microsomal

Incubations, performed at 37' for 90 min, contained DOPA or l4C-DOPAmine, 0.1 mM BSA and 1 mg microsomal ml. Mean values + SD (n = 4) are shown.

14Cs per

0.1 mM protein

nmol DOPA metabolites mg microsomal protein Bound

to 100 nmol

Complete system Boiled microsomes Without NADPH regenerating system With 1 mM cysteine With 10 mM lysine With 5 $ ethanol With 200 mM DABCO in 5 $ ethanol

BSA

Bound

5.85 0.65

+ 0.28 rf 0.06

18.0 1.28

+ 1.10 + 0.08

2.49

+ 0.03

1.17

1 0.13

1.91 3.78 5.12

+ 0.14 + 0.13 + 0.20

1.35 15.3 13.8

3 0.47 2 1.58 k 2.59

2.08

+ 0.17

4.03

2 0.96

nmol DOPAmine mg microsomal Complete Without nerating

system NADPH regesystem

somes the

transformation

activated

oxygen

sulfhydryl

using

is

catalyzed

also

5.28

2 0.44

of the

12.5

+ 0.21

6.20

catechol

to an intermediate

model

the

in the

resulted

observation,

systems

irreversible

comprised

microsomal

+ 0.38

known to transform

Incubation system

8.20

metabolites protein

substrate

capable

+ 0.34

may occur

of reacting

by

with

compounds.

Studies which

to 1 mg microsoma1 protein

confirm

this

view.

catechols

into

o-quinones

binding

presence

in along

the

oxidase

irreversible

with

NADPH-cytochrome

the

of DOPA to albumin

of a superoxide

of xanthine

findings c reductase

(5),

also

(Tab.2).

(OF) generating

/ hypoxarthine

binding

1398

Tyrosinase,

(14 - 16)

of DOPA to BSA. This

of Aust

et al.

may generate

(17)

that

superoxide,

BIOCHEMICAL

Vol. 66, No. 41975

TABLE 2 Irreversible by tyrosinase,

binding of xanthine

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

to BSA as catalyzed l4 C-DOPA metabolites oxidase and NADPH-cytochrome c reductase

I cubations (1 ml), performed at 37' for 60 min, contained 0.1 P4C-DOPA and 0.1 mM BSA. Mean values + SD (n = 4) are shown. = not determined. n.d.

mM

nmol

DOPA metabolites 100 run01 BSA Xanthine oxidase NADPH-cytochrome and hypoxanthine c reductase and NADPH reg. system

Tyrosinase

Complete system Boiled enzyme With 1 mM cysteine With 1 mM glutathione With 10 mM lysine

34.4 1.4 0.46

led

the

us to

binding

versibly

bound

microsomal

n. d.

f. 3.6

6.8

+ 0.73

n.d.

involvement

of this

when incubated

in

the

DOPA is presence

suggest

participation

of the

hemoprotein,

an absolute

requirement

the

These

Tab.2,

enzyme

results

obtained developed that

estrogens

(18),

oxidizes

catechols

radical

+ 0.12

13.7 + 1.2 3.2 + 0.88 n.d.

for

the

irreof

that

the

in

in

the

cytoirrever-

of DOPA to proteins.

The results originally

0.78

possible

+ 0.62 + 0.23 n.d.

of DOPA. As shown in

is not

binding

10.92

c reductase.

system P-450,

7.4 1.3

to albumin

NADPH-cytochrome

sible

35.2

examine

irreversible

chrome

0.46

i 2.3 + 0.05 + 0.05

anion,

which

with for

DOPA are

the

activated

metabolic oxygen

to an intermediate is

able

consistent

with

activation formed

in

, probably

to react

with

a concept,

of 2-hydroxyliver the

microsomes o-semiquinone

sulfhydryl

groups

of

proteins. Preliminary bind

experiments

DOPA irreversibly

and that

this

binding

show that to proteins of DOPA is

human , perhaps

inhibited

DOPA. 1399

liver

microsomes

by similar by the

analog

also

mechanisms cl-methyl-

Vol. 66, No. 4, 1975

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

REFERENCES 1. Murad, F. (1968) J. Am. Med. Ass. 203, 171-173. 2. Hoyumpa, M. and Conell, A.M. (1973) Am. J. Dig. Dis. 18, 213-222. T.A. and Gall, E.A. (1973) J. Am. Med. 3. Rehman, O.U., Keith, Ass. 224, 1390-1392. A.J. and Wurzel, H.A. (1974) Blood 43, 85-97. 4. Gottlieb, H.M. and Kappus, H. (1974) J. Steroid Biochem. 5, 5. Bolt, 179-184. 6. Remmer, H., Greim, H., Schenkman, J.B, and Estabrook, R.W. ,'"',~?kern 67 249-257 7. !I~%~~ ~e"~~s~eS~~yn~'o~~9~~~ 8. Kappus, H., Bolt, H.MI ad Remmer,'H. (1972; AcEa Endocr: 71, 374-384. H.M. and Remmer, H. (1973) Steroids 22, 9. Kappus, H., Bolt, 203-225. Pharmacol. 24, 10. Kappus, H. and Remmer, H. (1975) Biochem. 1079-1084. Bray, G.A. (1960) Anal. Biochem. 1, 279-285. Farr, A.L. and Randall, R.J. E Lowry O.K., Rosebrough, N.J., (195lj J. Biol. Chem. 193, 265-275. T. (1968) J. Am. Chem. Sot. 90, C. and Wilson, 13. Quannes, 6527-6528. I. (1969) J. Biol. Chem. 244, 14. McCord, J.M. and Fridovich, 6049-6055. I. (1969) J. Biol. Chem. 244, 15. McCord, J.M. and Fridovich, 6056-6063. D.M. end McCormack, J.J. (1971) Biochem. Pharmacol. 16. Valerino, 20, 47-55. T.C. (1972) Biochem. S.D., Roerig, D.L. and Pederson, 17. Bust, Biophys. Res. Commun. 47, 1133-1137. M. Bolt, H.M., Kappus, H. and P., Scheulen, 18. Wollenberg, Z. Physiol. Chem., in press. Remmer, H.; Hoppe-Seyler's

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