Functional reconstitution of rat liver cytochrome P-450 with mesohemin

BIOCHEMICAL

Vol. 121, No. 1, 1984 May 31, 1984

FUNCTIONAL RECONSTITUTION

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 95-101

OF RAT LIVER

CYTOCHROME P-450

WITH MESOHEMIN

Lester M. Bornheim and Maria Almira Correia Department of Pharmacology and The Liver Center University of California, San Francisco, California Kevin Department Received

March

of Chemistry,

M. Smith

University

of California,

Davis,

California

5, 1984

SUMMARY: After allylisopropylacetamide-mediated "suicide" destruction of their prosthetic heme moieties, certain rat liver cytochrome P-450 isozymes can be effectively reconstituted by addition of exogenous hemin in vitro. We now report that two of these isozymes will equally accept mesohemin, a 2,4-diethyl heme-analog and result in a "meso-hemoprotein" with altered spectral but not functional characteristics.

In the 450 exist olism

of

liver,

microsomal

as isozymes

which

a variety

them as well. isolation

of

peptide

mapping

to contribute On the examined

apparently

porphyrin

IX

coordinated Although

(7).

the

cytochrome

rat

the

are

identical the

thiolate

region and rabbit P-450~~

not

that

the

P-450

homologous

and have been the

a cysteine

P-450 containing

residue

moieties

are

a cysteine

If,

monomers

con-

sequencing

and

indicated

feature

is

been

that believed

isozymes.

P-450

as iron has

on

the

residue

(3-5,12),

whole is

found as well this

by their

isozymes

(III)-protoshown

of the apocytochrome

isozymes,

as proposed

have

cytochrome

identified

metab-

following

of the various

the

P-

inducible

are

This

heme iron

phenobarbital-inducible ( 3-5,12).

isozymes

specificity of

are

Aminoacid

(2-6).

heme moieties

of

(1).

cytochrome

oxidative

isozymes

hemoproteins

of enzyme

hemoproteins,

the but

individual

substrate

hand,

apocytochrome

tetradecapeptide in both

are

for

chemicals

cytochrome

differential

In

to the

mole

various

moieties

other

shown

termed

responsible

of the

have

the

to the

only

collectively

different

heme per

of

apoprotein

not

analyses

and purification one prosthetic

their

are

structurally

Structural

taining

hemoproteins

to

be

(8-11).

heterologous,

a

to be conserved as in bacterial region

is

indeed

0006-291X/84 $1.50 95

Copyright 0 1984 by Acudemic Press, Inc. All rights of reproduction in any form reserved.

Vol. 121, No. 1, 1984 the heme binding to be quite

BIOCHEMICAL

site,

rigidly

very

the heme moiety of

the

constitute

heme moiety

In recent

through mide

years,

15).

Such

tional

P-450

is,

moiety

of

been

we are

has of

the

to

obtain

now

capable

lost This

site

appear

the

of

to

hemoprotein

of restoring

2,4-diethyl

in

result

both

P-

moieties. approach:

first

destroyed

the

now

with

50% of

heme (13and

func-

improved

this

reconstituted

us to examine of hemin,

fresh

structural

We have

as compared

analog

is

supplied

approximately

has permitted

re-

such as allylisopropylaceta-

exogenously

of

to

cytochrome

by an indirect

(13-15).

yields

inability

bacterial

P-450

of

relation-

heme and apocytochrome

substrate

shown

greater

to

cytochrome

with

by inactivation,

improvement

its

specifications

due to the

circumvented

a suicide

replacement

ness of mesohemin, the

been

replaced

as

this

Structure-function

contrast

of

and subsequently

so

for

structural

unexplored (in

has

the

P-450s.

remained

heme

turnover

fraction

20% (15).

cytochrome

problem

reconstitution

procedure That

this

catalytic

about

dissociation

prosthetic

(AIA)

specifications

known

holoenzyme

deliberate

the existing

is

have

mammalian

after

structural

little

of mammalian

the

450~~)

the

defined.

In contrast,

ships

then

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

hemoprotein. the

the

previously

the

relative

as the prosthetic

cytochrome reported effectivemoiety

of

cytochrome. MATERIALS

AND METHODS

Materials: AIA was a gift from Dr. W. Scott, Hoffman-La Roche, Inc. (Nutley,NJ). Reduced glutathione and hemin were obtained from Sigma Chemical Co. (St. Louis, MO). Mesohemin was prepared by the catalytic hydrogenation of protoporphyrinIX dimethyl ester (16) followed by iron insertion and hydrolysis (17). 7-Ethoxyresorufin (ER) and p-chloro-N-methylaniline (PCNMA) were obtained from Pierce Chemical Co., Rockville, IL, and Calbiochem, La Jolla, CA, respectively. Ethylmorphine (EM) was obtained from Merck Chemical Company. Benzphetamine (BZP) was a gift from Dr. J. F. Stiver, Upjohn Company, Kalamazoo, MI. Hemin refers to iron (III)-protoporphyrin IX chloride, and mesohemin refers to 2,4-diethyldeuterohemin, i.e. the vinyl groups at the 2 and 4 positions of hemin are substituted with ethyl groups in mesohemin. Experimental Protocol: Male Sprague-Dawley rats (250-300 g) were pretreated daily with sodium phenobarbital (PB, 80 rug/kg, i.e.) for five days. Twenty-four hours after the last injection of phenobarbital, rats were injected with AIA (200 mg/ kg, i.p. in distilled water) 1 hour before killing. Livers were then removed, weighed, perfused with ice-cold saline, and homogenized (50% w/v) in 0.25 M sucrose containing 10 mM glutathione, pH 7.4 at 4' C. Aliquots of liver homogenates were adjusted to 0.1 M with respect to potassium phosphate buffer, pH 7.4 and incubated in the presence or absence of hemin or mesohemin [5O@l, 96

Vol. 121, No. 1, 1984

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

dissolved in a small volume of 0.1 N NaOH and neutralized with phosphate buffer, pH 7.41 at 37" C for 15 min in a Gyrotory Water Bath Shaker (New Brunswick Scientific). Homogenates were diluted with 3 volumes of ice-cold 1.15% KC1 and centrifuged at 9000 x g for 10 min. Supernatants were decanted and sedimented at 105,000 x g for 60 min. Microsomal pellets were resuspended in 1.15% KC1 and resedimented at 105,000 x g for 30 min. The washed microsomes were resuspended in 0.1 M phosphate buffer, pH 7.4. Protein was determined by the method of Lowry, et al. (18) and cytochrome P-450 content was determined by the method of Estabrook, et al. (19) using an Aminco DW-2a spectrophotometer. Microsomes were assayed for N-demethylase activity with EM and BZP (final concentration, 2 mM) or PCNMA (final concentration, 1 mM), as substrates, as described previously The amount of formaldehyde generated was quantitated by the method of Nash (14). was measured by the direct fluorometric (20). The rate of ER 0-deethylation assay described by Burke and Mayer (21).

RESULTS AND DISCUSSION Treatment

of PB-pretreated

struction

of

dependent

mixed

tion

over

60% of hepatic function

29% of

original

0

values,

Cytochrome P-450

with

cytochrome

oxidase

of ER and N-demethylation

13 and

rats

activity,

AIA,

as expected,

resulted

content.

In parallel,

P-450 assessed

by monitoring

of PCNMA, EM and BZP was also respectively

ER

PCNMA

(Fig.

1, Table

reduced 1).

in

to 45, In addition,

EM

1.

Activity

Values are presented as a P-450 content and dealkylase activity. of the original microsomal cytochrome P-450 content (1.75 + 0.15 nmol/mg) and original N-dealkylase activities (123 + 5 pmol/mg/min, 87 + 11 nmol/mg/l5 min, 381 + 36 nmol/mg/15 min, and 292 + 17 nmol/mg/l5 min for 7-ER, PCNMA, EM, and BZP, respectively) in corresponding non-AIA-treated (PB-pretreatOn the bases of similar molar extinction coefficients (EM) for ed) controls. hemin and mesohemin solutions, their corresponding pyridine-hemochromogens, and heminand mesohemin-reconstituted cytochrome b5. EM for mesohemin-reconstituted cytochroma P-450s was also assumed to be the same as that of the native cytochrome P-450. cytochroma percentage

97

its

O-deethyla-

Restoration of Mlcrosomal Cytochrome P-450 Content and Dealkylase after AIA-mediated Destruction. PB-pretreated rats were given AIA (200 mg/kg, 1.p.) for 1 hour. Liver homogenates were prepared and incubated in the presence or absence of hemin or mesohemin (50+) at 37OC for 15 min. Microsomes were prepared and assayed for Fisure

de-

47,

BIOCHEMICAL

Vol. 121, No. 1, 1984 TABLE I

Restoration of Rat Liver and Dealkylase Activity

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Microsomal Cytochrome P-450 Content after AIA-Mediated Destruction DEALRYLASE ACTIVITY

Treatment

Cytochrome P-450 (nmol/mg protein)

Incubation

AIA

----

0.66 + 0.02

AIA

Mesohemin

0.83 -+ 0.04

AIA

Hemin

None

----

ER (pmol/ mg/min) 55+

homogenates

the spectrally

values

AIA treatment, trum was

the

shifted

was reverted

109 -+ 12

72+_

5

111 -+

9

146 -+

4

1.12 + 0.04

100+7

70+_

6

139+ -

4

232 -+

6

1.75 -+ 0.15

123 +

381 + 36

292 +

17

liver

homogenates

maximum

of the

450 to

detectable

ues.

such

with

microsomes spectrum

from

treated

2A,B).

PB-pretreated

On the rats

P-450

nm (Fig. not given

from

its

cytochrome

results

were

obtained

when mesohemin

was added

somes.

This

suggests

that

to 446 nm observed

treated

rat

of mesohemin binding

to

P-450

subtracting

the

fraction

the

microsomal

cytochrome

the

homogenates

into the

the

fraction spectral

which

is

shift incubated

apocytochrome, membrane.

characteristic

rather

than

Moreover,

contribution resistant

of to

the

the

spec-

reconstitution

when

incubated

with

aliquots

residual

of

mesohemin,

to 47% of original altered

to shift

val-

absorption

of mesohemin

to liver

the absorption

rat

Similar

liver

in microsomes

microfrom

AIA-

was due to incorporation

merely

to

its

absorption

mesohemin

AIA-mediated 98

absorption

to AIA-treated

mesohemin

with

P-450

450 nm maximum.

with

reconstituted

1).

Addition failed

to 64% Following

1, Table

a distinctly

AIA,

hemin,

was restored

hand,

was restored

2C).

rats

other were

in

with

on hemin

of

liver

P-450

(Fig.

however,

resulted

446

content

84 + 10

animals

incubated

cytochrome

452 nm, which,

reconstitution

were

P-450

CO-reduced

cytochrome

PB-pretreated

rats

same AIA-treated

a maximum at

83 _+ 11

AIA treatment

450 nm (Fig. the

5

3 individual

cytochrome

before

the spectrally

spectrum

AIA

hepatic

from

However,

such

7

min)

9

observed

towards

(nmol/mg protein/l5 57+-

from

from

BZP

3

detectable

of original

EM

40+

Values determined as described in Figure 1 Values represent the mean + S.E. from at least

when liver

PCNMA

could

spectrum

of

the

be visualized

cytochrome destruction).

nonspecific

P-450,

by (i.e.

Accordingly,

Vol. 121, No. 1, 1984

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNKATIONS

Hemm

None 440 450

440 450 I I ’ I ’ ’ I ’

I I I I I I

;

/

I

’

A!!

Mesohemm

A

hlesohem~n vs None CC) (A)

440

450

I I I

I I I

’

I

I I I

I I I

440 I

450 I

420 430 440 450 460

Wavelength (nm 1 Figure 2. CO-reduced Cytochrome P-450 Absorption Spectra of Hepatic Microsomes Before and After Reconstitution with Hemin or Mesohemin. Rat liver microsomes were prepared as described in Figure 1. Cytochrome P-450 was determined by the method of Estabrook, et al. (9) in which both sample and reference cuvettes were bubbled with CO, the baseline recorded, and then sodium dithionite added to the sample cuvette. Spectrum 2A was obtained with liver microsomes from AIA-treated rats. Spectra ZB, and C were determined after incubation of liver homogenates from such rats with hemin or mesohemin, respectively. Spectrum 2D represents the absorption difference between the sample cuvette in 2C and the sample cuvette in 2A. optical

subtraction

of the CO-reduced

somes of an AIA-treated after

mesohemin

spectrum

with

tion

spectrum

with

mesohemin

ation

rat

reconstitution

appears

to be unique

into

P-450

spectrum

of

results

in

It b5 also

is

microsomes

a CO-reduced

difference

This

latter

isozyme(s)

relevant shifts

the

that

Soret from

absorp-

reconstituted

to note

of the hemoprotein

micro-

corresponding

2D).

the microsomal

of liver

incorpor-

absorption

max-

413 to 406 nm and

(22).

reported content

for

forms

that

440 nm (Fig.

moiety.

and reduced

423 to 411 nm, respective.y As previously

2C),

apocytochrome

p-450

from

maximum at

as the prosthetic

ima of the oxidized

2A),

(Fig.

an absorption

of mesohemin

cytochrome

(Fig.

cytochrome

is

(14,15,23-25), associated

the with

99

hemin

mediated

a corresponding

increase

increase

in

in its

Vol. 121, No. 1, 1984 mixed

function

and PCNMA was whereas

oxidase

activity

restored

to

of

1, Table

chrome

P-450

ethylation

with

values

(Fig.

as effective noteworthy

1).

as hemin

in

that

by polycyclic with

absorption

of these

isozymes

contrast,

neither

methylase

activity.

tion)

of the

inflicted tion,

with

appears

Whether

compared

BZP N-demethylation,

its

prosthetic

indicate

that

only

that

P-450

is

just

appear

to significantly

refractoriness

of AIA,

BZP

loss

of mesohemin

into

may be more than

one of which

is

capable

"CO-re-

shift.

(rapid

degradaapoprotein In addi-

whereas

mesohemin

restoration the

In

EM N-de-

to be determined.

Such partial

is

reconstitution

restore

N-demethylase

restoration.

the

damage to the

remains

is

inducible

spectral

reflects structural

It

induction

the structural

mesohemin

only

O-de-

(PB-induced)

largely

such cytochrome

in a similar

there

PCNMA and

mesohemin

are

results

or

of cyto-

of the maximum to 448 nm in

incorporation

rats

may be

apocytochrome

one isozyme of accepting

as

responsible mesohemin

as

moiety.

Collectively, can very

of

of hemin,

for

that

reconstitutes

rate

reconstitution

also

partial

AIA-treated

FM, BZP, and

not

intriguing

metabolite

in only

to that

is

from

hemin-mediated

PCNMA and ER dealkylases.

or irreversible

successfully

a slower

but

this

apocytochrome

to result

due to

appear

nor

by a reactive hemin

It

mesohemin

hemin

thus

isozymes

shift

spectrum.

of

respectively,

81% after

similar

reconstituting

a characteristic

to

values

homogenates

after

findings

hydrocarbons

original

of EM, BZP,

50, 87, and 89% of original

two particular

aromatic

associated duced"

these

liver

to 29,

fully

N-demethylation

increased

N-demethylation

These

is,

84% of

hand,

increased

1, Table

and

in

other

mesohemin,

of ER were

That

likewise P-450

On the

1).

79,

was

cytochrome

1).

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

(Fig. 36,

ER 0-deethylation

reconstitution (Fig.

BIOCHEMICAL

our

findings

effectively

function

to be quite

promiscuous

indicate with in

that

mesoheme their

some cytochrome

as the

prosthetic

prosthetic

P-450 moiety

isozymes and thus

acceptance.

Acknowledgement: We thank Ms. Elizabeth Winckler for graciously typing this manuscript. These studies were supported by NIH grants AM-26506 (MAC), We acknowledge the use of the Liver Core GM-09163 (LMB), and HL-22252 (KMS). Facility in spectrophotometry (AM-26743). 100

Vol.

121,

No.

1, 1984

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

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