The catalytic demethylation of N,N-dimethylaniline-N-oxide by liver microsomes

BI~~~MICAL

Vol. 13, No. 3, 1963

Flora Clayton

Foundation

Received

the oxidative first

described

LaDu,

et al.

present

by Mueller

and Miller

demonstrated fraction

supplemented

with

appears

to be of the

oxidase.

first

part

a&y1

N-oxide

cated

by the

MPH

of Chemistry

and oxygen

(Gillette

type

classified

the U-methyl

and the intermedf.ate last

i)

two steps

in the

"W%)2

to the

1957).

The enzyme

Drodie

1958)

then

micro-

CQmpQUndS

(1957)

is

by

the Qxidative

by Mason

compound

following

NAIIPR3

catalyzed

fcf.

N-oxide

studies

The isolated

et al. cm

was

compounds

system was localized

N-alkyl

suggested

catalyzing

Subsequent

the enzyme

amine

It has been

of the reaction

(1953).

soluble

tlssua

N-methyl

homogenates.

of lipid

and corresponding

soluble

that

of liver

of a variety

function

Department Texas

in mammalianliver

of lipid

(1955)

dealkylation

system

M. Ziegler’

Institute and the of Texas, Austin,

demethylation

the microsome

aldehyde

and Daniel

RESEARCH CO~UNICATIONS

23, 1963

August

somes when

Ii, Pettit

Biochemical The University

The enzyme system

in

AND BIOPHYSICAL

as a mixed that

in the

is oxidized

to the

degraded

as indi-

sequence

of

reactions:

0 R-B(ai3)2

02

Supported fn part G&09044-02. +Work American

by a grant

out during

carried Heart.

ASSOciatiQn,

from National

tenure

Institutes

as an Established

Inc.

193

of ffealth,

Investigator,

BIOC~~ICAL

Vol. 13, No. 3, 1963

AND BIOP~SI~L

RESEARCH CO~UN{CATiONS

While there was someevidence to support this reaction mechanism (Fish et al.

1955,1956), an earlier

N,N-dimethylaniline-N-oxide intermediate livar

this

microsomes catalyze

of N,N-dimethylaniline-N-oxide

reaction

methylation

gation from liver

by the liver

The oxtdative

de-

microsomes.

were isolated by differential

centrifu-

(1948).

demethylation assays were

erlenmeyer flasks at 38' in a Dubnoff

carried

out

shaker.

metabolic

in

10 ml

open

The complete

mediumcontained per ml: potassium phosphate, pff 7.5, 200

nmoles; magnesiumchloride,

citric

oxidative

homogenatesprepared in 0.25 g sucrose according to

the method of HogebooPl-et al.

moles;

and form-

The maximumrate of

is several fold greater than the overall

The microsoma fractions

reaction

the rapid de-

to methylaniline

of both WPH and oxygen.

of dimethylaniltne

by

The data presented in this report

Drodie 1938).

aldehyde in the absence

to be an

damethylation of N,N-dimethylaniline

damonstrate that rat and pig liver

qethylation

that the rate of

demethylatioa was too slow for it

in the oxfdative

mierosomss (cf.

report indicated

MDP+, 0.1 vole;

5 wales;

isocitrate,

5 poles;

semicarbazide, 1 mole;

h&D+, 5

and sufficient

iso-

dehydrogenase to reduce 0.5 Crmoleof NsDP+per minute per ml,

Tha concentration

of N,~dimethylaniline,

and of microsomal protein

N,N-di~thyla~line-~oxide

are given in the Table and Figure.

After

preincubating

with shaking for 4 minutes the react&on was started by

adding either

the microsomas or the substrate.

action mixture were withdrawn at intervals containing

sufficient

concentration

6.0 p! trichloroacetit

of 0.6 &

The deproteinized

Aliquots

and pipetted

of the re-

into tubes

acid to give a final supernatant solutions were

assayed for formaldehyde by the method of Nash (1953).

The rates of

formsldehyde formation reported in this paper are corrected small

amount

for the

of formaldehyde produced in the absence of substrate.

N-methylaniline

was estimated by vapor phase chromatography using a

194

BIOCHEMICAL

Vol. 13, No. 3, 1963

AND BIOPHYSICAL

column packed with 5 per cent polyethylene

RESEARCH COMMUNICATIONS

glycol

succinate on chromo-

was distilled

under vacuum. Analy-

sorb P at 182". Commercial N,N-dimethylaniline

sis by vapor phase chromatography indicated

that it was free from con-

taminants other than traces of N-methylaniline. aniline-N-oxide ment.

was synthesized by Dr. Rowland Pettit

The melting point of the recrystallized

ported,

The N,N-dimethylof our Depart-

N-oxide was 150" [re-

152" (Belov and Savich 1947)]. The data summarized in Table I demonstrate that pig liver

somescatalyze

the demethylation of N,N-dimethylaniline-N-oxide

rate greater than the oxidative

proximately

that N-methylaniline

(at pR 9.0)

is produced at ap-

the samerate as formaldehyde during the catalytic

ation of the N-oxide. component catalyzing non-dializable

at a

demethylation of the corresponding di-

methyl amine. Vapor phase chromatography of ether extracts of the reaction mediumindicate

micro-

Recent experiments indicate

demethyl-

that the microsomal

the demethylation of the N-oxide is heat labile,

and appears to be an enzyme. The rate of formaldehyde

production is a linear over a relatively

function

of microsomal protein concentration

wide range (0.2 to 4.0 mg per ml) and the reaction

is also linear with time for at least the first

ten minutes.

A graphical determination of the Michaelis constant for N,N-dimethylaniline

in rat liver

q icrosomes gave a Kmof 1.42 x 10-3 g. The

Michaelis constant for the N-oxide was determined in both rat and pig liver

microsomes from the Lineweaver-Burke plot shown in Figure I.

The high Kmvalue (139 x 10e3 kl) observed with both rat and pig liver microsomesmay be due to the very limited polar N-oxide (cf.

lipid

solubility

of the

Brodie 1958).

The data presented in this report demonstrates that the rate of N,N-dimethylaniline-N-oxide ficiently

demethylation by liver

microsomes is suf-

high for the N-oxide to be considered as an intermediate

the oxidative

demethylation of N,N-dimethylaniline. 195

in

BIOCHEMICAL

Vol. 13, No. 3, 1963

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Table I Demethylation Activity

of Pig Liver Microsomes Substrates N,N-dimethylN,N-dimethylaniline aniline-Noxide mumolesformaldehyde per q in per

Additions

Complete system II

5.4

15

minus KADP+and NADPKgenerating system

0

14

minus NADPHgenerating system and oxygen

0

17

minus WDPB generating system, NbIIt and p&C12

0

17

1)

minus q icrosomes

0

0

II

minus microsomes plus boiled microsomes

0

0

II 0

II

II

Concentration of substrates, protein, 2.6-3.3 mg per ml.

3 pies

per ml; concentration

0.20-

0.16-

0.12I 7

/

0.08-

I

0.02

/

/ ?'

0.04

/I

//

/

//

0.06

/

//

/

/

//

0.08

of microsomal

P/'

0.10

[+ x lo-3 Figure I.

mg

Lineweaver-Burke plot of the effect of substrate concentration on the rate of demethylation of N,N-dimethylaniline-Noxide.

[Sl = Mcdarity v - qmoles formaldehyde produced per min. per mg q icrosomal protein per - Pig liver microsomes Km = 139 x 10-3M Vmax = 175.4 voles min per mg --- - Bat liver microsomes Km- 139 x 10-3K Vmex - 71.4 qusoles per mia per mg

Vol. 13, No. 3, 1963

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

References 1.

Belov, V. N., and Savich, 257

K.

K., J. Gen. them. (U.S.S.R.),

17,

(1947).

2.

Brodie, B. B., Ann. Kev. Biochem., 57, 427 (1958).

3.

Fish, M. S., Johnson, N. Pi., Lawrence, E. R., Horning, g. c., Biochim. et Biophys. acta, l8, 564 (1955).

4.

Fish, H. s., Sweeley, C. C., Johnson, N. M., Lawrence, E. P., and Kerning, E. c., Biochim. et Biophys. acta, 2l, 196 (1956).

5.

Gillette, J. B., Brodie, B. B., and LaDu, B. N., J. Pharmacol. Exptl. Therap., 119, 532 (1957).

6.

Eogeboom, G. Ii., Schneider, W. C., and Palade, G. P., J. Biol. Chem., 172, 619 (1948).

7.

LaDu, B. N., Gaudette, L., Trousof, N., and Brodie, B. B., J. Biol. Chem., 214, 741 (1955).

8.

Mason, Ii. S., Science, 125, 1185 (1957).

9.

Mueller,

10.

G. C., and Miller,

J. A.,

J. ~i01. Chem., 202, 579 (1953).

Nash, T., Biochem. J., 55, 416 (1953).

197