Evidence for a new P-450 hemoprotein in hepatic microsomes from methylcholanthrene treated rats

Vol. 24, No. 5, 1966

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

EVIDENCE FOR A NEW P-450 HEMOPROTEIN IN HEPATIC MICROSOMES FROM METHYLCHOLANTHRENE TREATED RATS N. E. Sladek Department

and G. J. Mannering

of Pharmacology, Minneapolis,

Received

suggested

work

tha.t

N-demethylase

from

that

N-demethylase barbital

this

results from

has been This

(PB).

normal,

that

in normal

rats

the metabolism

from MC treated supported

1965).

tance

of two forms

of P-450.

oxide

as the

for

Recently,

that

Using

reduced

The relative

concluded

not

Two-substrate

this

size

hemoprotein,

from

forms

P-450, which

normal

(EM) from of 3-CH3-MAB kinetic

for

the

studies

microsomal

participation systems

of the responsible

1965;

Ernster

and Orrenius,

and Sato

(1966a)

suggested

isocyanide they

rather observed

than Soret

of the two peaks was pH dependent

"microsomal

in two interconvertible

2-diethylamino-

conclusion.

--et al.,

ethyl

pheno-

(4 x 10s5 M)

the N-demethylation

Imai

sodium

that

of ethylmorphine

rats.

to

whose

by the enzyme system

in the NADPH dependent (Cooper

finding

with

in low concentration

has been presented

of many drugs

ligand

but

(3-CH3-MAB)

and in rats

rats

was b a sed on the

rats,

Remmer and Merker,

and 455 mu.

1966)

(MC) is administered

of treatment

and the N-demethylation

evidence

P-450,

as a result

of 3-CH3-MAB

EM and 3-CH3-MAB

Considerable

found

(SKF 525-A)

by the enzyme system

hemoprotein,

and Mannering,

cholanthrene

conclusion

PB and MC treated

employing

(Sladek

when methy

the N-demethylation

and PB treated

exists

laboratory

increased

2,2-diphenylvalerate

inhibited

authors

55455

the --de novo 3-methyl-4-monomethylaminoazobenzene

may be different

ethyl

Minnesota

of Minnesota

July 20, 1966

Previous

rats

University

at least are

in the

reduced

in a pH-dependent

for 1965;

the exis-

carbon

mon-

peaks

at 430

and the state, equilibrium."

Vol. 24, No. 5, 1966

If this

indeed

is

result

BIOCHEMICAL

the case,

in a change

forms

of P-450.

relative

of the

studies

treatment

causes

Pl-450,

if

are

view

that

P-450

plays

METHODS.

Male,

Holtzman

in a pH-dependent

groups

of four

of the

following

3) MC, 20;

appear equilibrium.

an important

animals

rats

role

which for

because

it

causes

days

Takemori,

1958).

to remove

hemoglobin

and processed

to obtain

microsomal

fractions

microsomal

Livers

plus

N-demethylase

were

soluble

times

has been described

reaction

rates.

2 x 10-3

M and 2 x 10m4 M, respectively.

reaction

rates

the

chromotropic

1958).

EM and 3-CH3-MAB

were acid

determined procedure

When EM was the

of Nash (Anders of the various Beckman Model

microsomal

preparations

varied

to preserve

amount

previously

measurements

and

of EM of the

1964).

Incubof

concentrations

of

was the substrate, of HCHO formed

(Takemori

using

Each cuvette

using

and Mannering,

by a modified

monoxide

was determined

669

1964)

linearity

in substrate

The carbon

perfused et al.,

et al.,

When 3-CH3-MAB

beam spectrophotometer.

injection, (Rubin

the and

The composition

HCHO was measured 1966).

for

the determination

(Rubin

the

was included

Mannering

the spectral

employed

described

2) PB, 40;

last

activities.

by measuring

substrate,

and Mannering,

DB dual

were

five

injections

1956;

the

and

the

responsible

previously

x g) for

were

into

Morphine

(Axelrod,

previously

and enzyme concentrations

divided

in the enzymes

x g) for

N-demethylase

P-450

of drugs.

20.

20 hrs after

(9,000

MC

forms

intraperitoneal

sulfate,

drugs

(100,000

that

strengthen

1) saline;

as described

fractions

and 3-CH3-MA6 mixture

removed

daily (mg/kg):

narcotic

result,

Both

also

90 to 100 g were

a decrease

of EM and other

(Pl-450).

in the metabolism

received

four

pH might

suggesting

studies

could

in the

in two interconvertible

These

weighing

each,

materials

N-demethylation

ation

to exist

a shift

at a given

does occur,

of P-450

interconvertible

such a change, peaks

4) PB, 40 + MC, 20; 5) morphine

in the series

incubation

MC causes

moiety

of the

of a new hemoprotein

form,

which

equilibrium

such a shift

formation

when in reduced

in the protein

430 and 455 Soret

show that the

a change

pH dependent

Specifically,

heights

The current

in the

then

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

procedure

difference

spectrum

a recording (1 cm light

path)

Vol. 24, No.5,

contained

1966

microsomal

buffer,

pH 7.4,

(final

pH, 7.0).

30 sec.

preparation

Carbon

The ethyl

isocyanide

the ligand

that

ethyl

instead

to 250 mg of liver,

of dithionite was bubbled

absorption,

into

0.2

M phosphate

in a final

volume

of 3 ml

one of the

cuvettes

for

450 - 500 mu, was used as the estimate

difference

isocyanide

of CO.

500 mu and between 455 peaks,

equivalent

monoxide in

except

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and a few milligrams

The difference

of P-450. manner

BIOCHEMICAL

spectra

(final

was determined

concentration,

The differences

between

455 mu and 500 mu were

3.45

absorption

used as estimates

in a similar mM) was used as

at 430 mu and of the 430 and

respectively.

RESULTS.

In Table

centage

increases

equal.

Thus,

1 it in all

not only

N-demethylase

can be seen that

activities

six did

when rats

categories the

were

in EM N-demethylase

the increase

-ABLE 1. Changes in microsomal hemoprotein activities in res ponse to phenobarbital,

in P-450,

with

PB, per-

approximately and 3-CH3-MAE!

but the

increases

in

levels and N-demethylase methylcholanthrene and

x Yethylchol' anthrene

Phenobarbita

treated

of measurement

increases

parallel

were

Methylcholanthrene + Phenobarbital

I

Morphine

130 peak' % of control)

336 (*17)

140 (*14)

378 (*71)

65 (*14)

155 peak2 % of control)

386 (*29)

400 (*61)

723 (5135)

68 (*13)

130 peak t 455 peak3 % of control)

353 (*20)

226 (*28)

488 (*98)

66 (*14)

l-4504 % of control)

353 (*15)

251 (*42)

547 (*108)

72 (*14)

I-~~W;My~;y~-demethylas

309 (*21)

441 (+32)

784 (*38)

62 (*lo)

325 (*lo)

107 (*4)

377 (*41)

59 (*4)

% of control) :M N-demettjylase activity

% of control) Control: 0.075. Control: 0.037. 3 A00 as in ' 2 AOO455-500; AoD430-500; and 2; Control: 0.112. 4 AOD450-500; Control: 0.085. 5 umol HCHO formed/g of liver/hr; Figures

in

Control: parentheses

2.90.

6 umol HCHO formed/g

represent

S.E.

670

of liver/hr;

Control:

8.10

Vol. 24,No.5,

both

BIOCHEMICAL

1966

430 and 455 peaks were

tionalities

between

maintained

the M-demethylase

treatment.

the parallelisms

3-CH3-MAB

degree.

Thus the

PB treated whereas

peaks were

treated that

received

where

crease

in the 430 peak nor to the

the suggestion

of Imai

than

treated

obtained Imai

different

both

animals

and Sato

terconvertible

treated

be a function

curves

were

(1966a),

constructed

microsomes

from

normal,

microsomes

from

normal

and Sato,

from

MC treated

(1966a)

singly

the

intercepted

1) for

who used microsomes rats

gave an intercept

be predicted

concept

rats, were

from untreated at pH 6.9,

in the 430

represented

It also

supports

represent when rats

from results

of a pH dependent

in-

of the 430 and 455 peaks pH 7.4.

This

constant.

the 430 and 455 peaks

6'71

However,

or MC.

obtained

their

in-

a quantitative

430 and 455 peaks

compounds.

equilibrium

rats

the

and the pigments

these

at about

PB and MC treated and PB treated

would

the heights

of the pH dependent (Fig.

neither

be PB, morphine

The results

what

in developing

(1.37).

the same precursor.

that

with

plotted

respectively)

from MC treatment.

it

P-450

from

PB and MC were

The curves

should

Imai

that

in normal,

sums of the increases

whether

derived

and Sato

hemoprotein, pH's.

to the

observed, to a similar

very simi'lar and 0.51,

MC,

not seen.

increased

employed,

results

one form of the hemoprotein.

with

from

equal

the view are

were

with

was much greater

were

that

of treatment,

by the 430 and 455 peaks

two rather

about

rats

in the 455 peak bears

in P-450

strengthens

0.56

MC the ratio

the increase

are

regardless

(0.49,

PB and morphine

increase

in P-450

groups

treated

or the 430 peak were

455:430

were

as a result

were

treated

ratios

in the studies

observation

when rats

455 peak were

of the

changes

depressed

and the

Unlike

and 455 peaks,

hand,

were

N-demethylase

in the group

the increases

other

The same propor-

and spectral

in EM N-demethylase

and morphine

relationship

activities

in PB and morphine

no increases

whereas

proportions.

and hemoprotein

On the

observed

Essentially

were

of equivalent

when enzyme activity

of morphine

This

also

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

obtained

with

derived

the same as that

which

intercept When similar

the intercepts

rabbits.

at

from

observed

The microsomes reflects

a pH

by

Vol. 24, No. 5, 1966

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

MC

I 6.0

6.5

7.0

25

8.0

Treated

I

1

II

1

I

6.0

6.5

7.0

7.5

8.0

6.0

6.5

7.0

7.5

8.0

PH Fig.

1. Dependence of isocyanide difference from normal rats and rats treated with same as that described in the text for that 1.0 M rather than 0.2 M phosphate

dependent

equilibrium

microsomes.

This

The intercept a mixture

constant is

interpreted

observed

with

of the original

the

DISCUSSION.

These

pretation involved volving

in the this

could

with

which

not function involving

would

A second protein the

for

rats If

must

the

than

6.9.

a single

hemoprotein

in normal

and PB treated

hemoprotein

is

ethyl

to two forms

isocyanide,

it

is

MC treatment

contributes

poorly

interpretation

results

is

original

with

state,

is

inmay or

by peaks

of a new hemo-

activity, of EM.

but which

does

The reaction

by SKF 525-A.

not only

on the

but also

672

which

but after

in two forms

N-demethylase

a given

rats

The reaction

in the formation

not inhibited

of MC treatment,

One inter-

The hemoprotein

in the N-demethylation

insists

forms

in its

distinguishable

to 3-CH3-MAB

new hemoprotein

as the result

EM and 3-CH3-MAB.

by SKF 525-A.

inhibited

from

by the original

interpretations.

of both

result

interconvertibility

comtamination

at a pH lower

normal

a new hemoprotein.

to several

N-demethylation

two interconvertible

occur

without

seen with

are subject

or functions this

evidence

that

results

at 430 mu and 455 mu. protein

from

from MC treated

be measured

may not be interconvertible reacting

as strong

microsomes

intercept

assumes

different

and the new hemoprotein.

of the new hemoprotein hemoprotein,

quite

spectra on pH using microsomes MC and PB. Methodology was the the data given in Table 1 except buffer was employed.

type

formation

of a new hemo-

on the association of drug

metabolism.

of each of Thus

Vol. 24, No. 5, 1966

the form

BIOCHEMICAL

associated

represented 455 peak

is involved

combine

A third

metabolism,

Because levels

(Sladek

the

hemoproteins,

each

one being

were

that

the

P1-450,

which

a basis

whereas

MC produced

with

for

the

drugs

that

of

can be

occur

when

of the hemo-

with

would

type

one type

of drug

of drug

in P-450

after

rate

other

limiting

with

of hemoprotein

seen after

can also

MC treatment

it

caused

Thus,

if

in the

the 455 peak is

This

The

In Table

430 peak.

involved

not,

that

1 it little

the

N-demethylation then

is

in

functional

be interpreted is

overall

microsomes.

in P-450,

by the

study

in the

interpretations.

an increase

the amount

MC treatment

in a previous

of EM in normal

the 430 peak is

increased

increased

to mean

due to an increase

in the

N-demethylation

of 3-CH3-MAB,

research

was supported

by USPHS grant

but not

in

in the

of EM.

ACKNOWLEDGMENTS:

This

The authors

gratefully

Mrs.

Ham, Miss

Sheila

that

in a different

is not

represented

associated

functions

N-demethylation

the

the observations

changes

was concluded

P-450

to EM N-demethylation.

increase

grateful

that

it

provide

MC has not respect

with

case,

the 430 and 455 peaks

more involved

the N-demethylation

associated

with

spectral

of EM was not

1966)

of EM and the hemoprotein effect,

with

who found

involved

increased,

in the hemoprotein

hemoprotein

in this

deny the interconvertability

Accordingly,

N-demethylation

findings

increase

associated

hemoprotein.

state.

of P-450

can be seen that

the form

metabolism,

(1966b)

of drug metabolism

the other.

involving

current

in a type

conforms

upon the

would

and Mannering,

reaction

and Sato

microsomal

or at least than

of drug concept

depending

natural

different

metabolism

This

interpretation

in its

represent

type

and Imai

with

involved

of EM, whereas

a second

two groups

drugs

while

in

(1966)

into

protein

430 peak is

of 3-CH3-MAB.

Remmer et al., divided

the

by the N-demethylation

N-demethylation

these

with

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

to Mr.

acknowledge Janice

Holen

Don Shoeman for

the able

technical

and Miss

Margaret

the

673

synthesis

of ethyl

No. GM-12543. assistance

Kuhfeld. isocyanide.

of We are

Vol. 24, No. 5, 1966

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

References Anders, M. W. and Mannering, G. J., Mol. Pharmacol., in press, July, 1966. Axelrod, J., Science, 124, 263, 1956. Cooper, D. Y., Levin, S., Narasimhulu, S. and Rosenthal, O., Sciences, 147, 400 (1965). Ernster, L. and Orrenius, S., Federation Proc., 24-, 1190 (1965). Imai, Y. and Sato, R., Biochem. Biophys. Res. Cornnun., 23, 5 (1966a). Imai, Y. and Sato, R., Biochem. Biophys. Res. Cornnun., z, 620 (1966b). Mannering, G. J. and Takemori, A. E., J. Pharmacol. Expm. Therap., 127, 187 (1959). Remmer, H. and Merker, H. J., Ann. N. Y. Acad. Sci., l& 79 (1965). Remmer, H., Schenkman, J., Estabrook, R. W., Sasame, H., Gillette, J., Narasimhulu, S., Cooper, D. Y. and Rosenthal, O., Mol. Pharmacol., 2, 187. (1966). Rubin, A:,‘ Tephly, T. R. and Mannering, G. J., Biochem. Pharmacol., 13, 1007 (1964). Sladek, N.‘E. and Mannering, G. J., Federation Proc., 25, 418 (1966). Takemori, A. E. and Mannering, G. J., J. Pharmacol. Exptl. Therap., 123, 171 (1958).

674