Cell-free synthesis of epoxide hydrase by liver poly (A+)-RNA isolated from phenobarbital treated rats

Vol.

94, No.

May

30,

2, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

Pages 542-548

1980

CELL-FREE

SYNTHESIS OF EPOXIDE HYDRASE BY LIVER POLY ISOLATED FROM PHENOBARBITAL TREATED RATS *Cecil

Departments chemistry,

B. Pickett, Joseph

*Nina R. Rosenstein, *Remus Morin and Anthony Y. H. Lu

of Biochemical Regulation * and Animal Merck Sharp & Dohme Research Laboratories,

Received

March

(A+)-RNA

L. Jeter

Drug Metabolism Rahway, New

and RadioJersey 07065

31,198O SUMMARY

Total liver RNA has been isolated from normal and 8 day phenobarbital treated rats by guanidine thiocyanate &mercaptoethanol extraction and fractionated by oligo (dT)-cellulose chromatography to yield poly (A+)-RNA. Poly (A+)-RNA from normal and phenobarbital treated rats have similar translational activity in the rabbit reticulocyte cell-free system. However major alterations occurred in the polypeptide products directed by these two classes of RNA. The translation products directed by 8 day phenobarbital poly(A+)-RNA were immunoprecipitated with rabbit IgG prepared against purified rat liver epoxide hydrase. The immunoprecipitate was subjected to SDS-polyacrylamide gel electrophoresis and the radioactive products detected by fluorography. Analysis of the fluorogram revealed that the major immunoprecipitable product co-electrophoresed with purified epoxide hydrase. These data suggest that the primary translation product of epoxide hydrase messenger RNA has the same molecular weight as the mature form of the enzyme.

INTRODUCTION Recent

work

from

2,3,4) have demonstrated synthesized protein P-450, the

as high

while

mature

chloanthrene

(5,6,7,8,9).

P-450 part

to the

and P-448 of the

membrane

and proteins

precursors

protein

mature

However

species In contrast that

form

to these

into

system,

of the ER proceeds

Inc. reserved.

P-448

as a high

it

by a common

542

on rat

organelles

are

liver

weight

report

both

and form

the insertion

that as the

by Kumar

by 3-methyl-

precursor

Since

mechanism.

cytochrome

systems

is induced

memhrane whether

and

have indicated

is a recent which

review

to the mature

in cell-free

membranes.

is unclear

recent

of certain

reticulum,

molecular

the microsomal

0006-291X/80/100542-07$01.00/0 Cop.vrighr ‘1-11980 bv Academic Pres, AN rights of reproducrion in an-v form

studies

findings

cytochrome

I for

are processed

is synthesized

by microsomal

are incorporated

Ref.

which

of the endoplasmic

to be synthesized

monooxygenase

proteins

proteins

weight

(9) who found

appears

(cf.

the membrane.

membrane

and Padmanaban

processed

molecular

phenobarbital-inducible

enzyme

of laboratories

that secretory

transversing

an integral

major

a number

which

cytochromes an integral

of integral

is

Vol.

94, No.

In order

to examine

are assembled of epoxide plays

into

mutagens

from

hydrase

purpose

in the

of this

with

activation Since

treated

anti-rat

hydrase

proceeds

enzyme

is synthesized

liver

by which

activity

of epoxide

hydrase

by cell-free

hydrase

translation

weight

the

compound

immunoprecipitation

molecular

system

of carcinogens

messenger

IgG, whether

P-450

increases

we have used this

reticulum,

of a high

as the mature

of a variety

reticulum

on the biosynthesis

the cytochrome

administration

rats and subsequent

via processing

with

COMMUNICATIONS

of the endoplasmic

our attention

and inactivation

is to determine

epoxide

RESEARCH

proteins

in concert

phenobarbital

endoplasmic

study

BIOPHYSICAL

we have focused

hydrase

the template

phenobarbital

products

in the

(10,11,12,13,14).

aim of increasing The

Epoxide

role

AND

the mechanism

the membrane,

hydrase.

a major

epoxide

BIOCHEMICAL

2, 1980

level with

of the

RNA in rat liver. of poly

(A+)-RNA

of the translation

the biosynthesis precursor

and

of epoxide

or whether

the

form. METHODS

RNA Isolations - Male Sprague-Dawley rats (120-170 g) were used in all experiments. Phenobarbital treated rats were given 1 mg/ml of sodium phenobarbital in their drinking water for a period of 8 days before sacrifice. Control animals were given water ad libitum. Total liver RNA was isolated by the guanidine thiocyanate-cesium chloriz mure described by Chirgwin, et al. (15). Total Liver RNA was resuspended in sterile water at 20-40 ml A units, heated 2 min. at 6S°C, rapidly cooled to room temperature and adjusted to eg M lrthium chloride, 0.5% SDS, 1 mM EDTA and 10 mM Tris-HCl pH 7.4. The RNA was passed through an oligo (dT)-cellulose column (Type T-3, Collaborative Research, Waltham, MA) which had been equilibrated with the same buffer. Bound poly (A’)-RNA was eluted from the column with 10 mM Tris-HCl pH The poly (A+)-RNA was adjusted to 0.2M potassium 7.4, 0.1% SDS and I mM EDTA. acetate, pH 5.0, and precipitated overnight with two volumes of absolute ethanol. Cell-Free Protein Synthesis - Poly (A+)-RNA was translated for 60 minutes in a mlcrococcal nuclease treated rabbit reticulocyte lysate (16) purchased from Bethesda Research Laboratories. The reaction mixture (90 ul) contained 30 ~1 of lysate, 25 mM Hepes pH 7.4, 10 mM creatine phosphate, 48 mM KC)5 87 mM K acetate, 1.2 mM 50 mM 19 amino acids (-methionine), 203yCi [ S]-methionine and l-2 ug of FZ$‘?..+)-RNA. To assess the incorporation of [ S]-methionine into protein, 2 uf of the reaction mixture was spotted on Whatman 3 MM paper and placed in cold 10% trichloroacetic acid. The filter papers were boiled for 10 min. in 5% trichloroacetic acid, rinsed thoroughly with cold 5% trichloroacetic acid, followed by 10096 ethanol, ethanol-ether (1:l) and finally ether. The filter papers were allowed to dry before being placed in Aquasol 2 and counted. Aliquots of the translation mixtures were mixed with an equal volume of 5.0 mM Tris-HCl pH 6.8, 2% SDS, 2% B-mercaptoethanol, and 20% glycerol, boiled for 2 min., then subjected to SDS-polyacrylamide gel electrophoresis (17). The radioactive products were visualized by autoradiography. Autoradiograms were scanned with a Helena Quik Scan Densitometer. Preparation of Antibodies Against Purified Epoxide Hydrase - Phenobarbital-induced epoxide hydrase was purified by the method of Knowles and Burchell (18) as modified by Guengerich, et al. (19). The purified protein gave a single band when subjected to SDS-polyacrylamide gel electrophoresis. Antibody against rat liver epoxide hydrase

543

Vol.

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2, 1980

BIOCHEMICAL

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was raised in rabbits by intradermal injections (8 sites) of 200 ug of purified enzyme mixed with Freud’s complete adjwant. Booster injections consisting of 70 ug of epoxide hydrase mixed with Freud’s incomplete adjuvant were given at approximately weekly intervals. Rabbit IgC was partially purified from the antiserum by DEAE-Affi gel blue affinity chromatography. Antibodies against epoxide hydrase gave a single sharp immunoprecipition band in Ouchterlony immunodiffusion gels when reacted against either purified epoxide hydrase or sodium cholate solubilized microsomes isolated from phenobarbital treated rats. The antibody did not cross react against either purified cytochrome P-450 from phenobarbital treated rats or cytochrome P-448 from 3 methylcholanthrene treated rats. Immunoprecipitation of Epoxide Hydrase - After a 60 min. incubation period the translatron mixtures (270 ul) were adjusted to 5% SDS, boiled for 3 mins. and diluted I:10 with 2.5% Triton X-100, 190 mM NaCI, 50 mM Tris-HCI pH 7.4 and 6 mM EDTA. The mixture was preincubated for 5 min. with heat-inactivated formaldehyde fixed S. aureus cells (20 ul) to decrease non-specific binding during immunoprecipitation (207: Tnecefls were removed by centrifugation and twenty microliters of anti-rat liver epoxide hydrase IgG was added to the supernatant. The mixture was incubated at room temperature for 1 hr. followed by overnight incubation at 4OC. Antigen-antibody The cells complexes were precipitated by the addition of 100 ul of S. aureus cells. were washed three times with 0.1% SDS, 10 mM Tris-HCI -7Trc,2 mM EDTA then boiled for 3 min. in SDS sample buffer containing 5% SDS and 100 mM DTT. The S. aureus cells were removed by centrifugation and the supernatant containing the immunz -tated translation product was analyzed by SDS-polyacrylamide gel electrophoresis. The radioactive product was detected by fluorography (21) using EN HANCE purchased from New England Nuclear.

RESULTS In order

to assess the translational

phenobarbital

treated

both fractions

of poly (A+)-RNA

trichloroacetic

acid insoluble

at various control

times

(Fig. The

were

during

translation

[35S]

the

translated

radioactivity

60 minute

treated

of poiy

reticulocyte

rats

(A+)-RNA

system,

determined period.

stimulated

total

from

equal

in the cell-free

incubation

products

directed

by control

to SDS-polyacrylamide

visualized

densitometric

system

Both

and of

and the total of the mixture (A+)-RNA

p0ly

protein

normal

concentrations

on aliquots

synthesis

Analysis

scans of these autoradiograms

phenobarbital

treated

weights,

synthesized

by control

identified

rats

50- 52,000,

from

to a similar

poly (A+)-RNA

by cell-free

translation

the

and

synthesis

or synthesized

544

which

that

of at

in greatly

immunoprecipitation

poly-

(Fig. 2a) or the liver

poly (A+)-RNA

least

appeared

(A+)-RNA

radioactive

of the autoradiogram

27,000)

and

poly

and the

(Fig. 2b) revealed

directed 29,000

and phenobarbital

gel electrophoresis

by autoradiography.

(molecular

have

activity

in the rabbit were

DISCUSSION

1).

subjected

peptides

from

rats

and phenobarbital

extent

AND

3 polypeptides either

reduced that

to be not

amounts. one

We of

the

Vol.

94, No.

BIOCHEMICAL

2, 1980

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

3.2 t 0

2.8

E a 2

24

!? 00 &

2.0

X

.4

Time Figure

I

(mm)

Poly (A+)-RNA from control (0) and 8 day phenobarbital treated rats (a) were translated in vitro using the rabbit reticulocyte cell-free system. During the incubation, -2 ~1 aliquots were rev ved and counted as described in the Methods s tion to assess 73 the incorporation of [ SJ-methionine into protein. Incorporation of [ g-methionine into protein is based on the total translation mixture (90 ~1).

MW x 1O-3

a 94

-

68

-

45

-

30

=

21

-

94U

-0

68K

45K

30K

--’ A

B

c

Migration

-

Figure

2a

Autoradiogram of the [35S] labeled polypeptides directed by liver poly (A+)-RNA from phenobarbital treated rats (A) control rats (B) and by the rabbit reticulocyte lysate in the absence of exogenous poly (A+)-RNA (Cl. The arrows represent the major alterations which occur in the polypeptides directed by poly (A+)-RNA from phenobarbital treated animals. The molecular weight markers were phosphorylase B (94K), BSA (68K), ovalbumin (45K), carbonic anhydrase (3OK) and soybean trypsin inhibitor (21K).

Figure

2b

Densitometric point to products

scans of lane A (lower the molecular weight range directed by poly (A+)-RNA

scan) where from

545

and lane B (upper scan) of figure the major changes occur in the phenobarbital treated animals.

2. Arrows translation

Vol.

94, No.

2, 1980

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

MW x 1O-3 b

68

-

45

-

A Figure

3

Fluorogram of [35S]-labeled epoxide hydrase immunoprecipitated from translation products directed by poly (A+)-RNA isolated from phenobarbital treated rats. Lane A represents the immunoprecipitation using pre-immune IgG. Lane B represents immunoprecipitation using anti-epoxide hydrase IgG. Co-electrophoresis of unlabeled authentic epoxide hydrase is indicated by the arrow.

polypeptides

in the 50-52,000

observations). unknown.

template

activity (6),

translatable

it

of

In order

total

immunoprecipitated

liver that

primary

by

poly

(AC&RNA

anti-rat

liver

hydrase Identical amide

radioactive (Fig.

gels.

RNA

which

molecular seem

were obtained

gels.

When pre-immune

was detected

(Fig. 3, lane A).

weight

polypeptides

to drastically

selectively

translation from

epoxide

alter with

increases

product

the

of epoxide

phenobarbital hydrase

treated

the other

level

of

hydrase, animals

the position

when the immunoprecipitates

product mobility of purified were

migrated

546

were

translation

as a

as purified

epoxide

epoxide

hydrase).

run on 7.5% polyacryl-

or IgG was used, no immunoprecipitable

Since the primary

the

IgG and the immunoprecipitate

The immunoprecipitable

represents

serum

(unpublished

is in agreement

band and had the same electrophoretic

3, lane B; the arrow

results

not

P-450

polypeptides.

the

with

does

compound

RNA for several

run on 10% SDS-polyacrylamide single

this

is cytochrome

and 27,000

messenger

detect

directed

range

phenobarbital

does appear

to

weight

of the 29,000

Although

messenger

polypeptides

molecular

The identity

remains

studies

I3

product

of epoxide

product hydrase

Vol.

94, No.

messenger mature

BIOCHEMICAL

2, 1980

RNA

form

proceed

appears

to have

of the enzyme,

via processing

biosynthesis

of the

cytochrome

P-448.

which

functional

equivalent

(I).

epoxide

hydrase

Dubois,

with

terminal

residue.

regard

proteins,

by which

investigation

experiments

it

by our

is inserted

sequence

into

into

of methionine

microsomal

is

of secretory rat liver

of many

secretory

as the

of epoxide

by the membrane

than

a sequence

of purified

ER but unlike

the

hydrase

contain

sequence

does not

rather

epoxide

the NH*-terminal

it is not removed

this protein

or insertion

presence

that

of the protein

that

as the

resembling

P-450

the NH2-terminal

and the

therefore,

thereby

it might

the signal

daltons,

of this enzyme

of cytochrome

we feel

protein,

COMMUNICATIONS

50,000

demonstrate

resembles

to hydrophobicity

for insertion

secretory

The mechanism under

that

weight,

precursor;

to the signal

It is probable,

serves as a signal

weight species

of the

RESEARCH

the biosynthesis

et al. (22) have sequenced

and found

proteins

that

phenobarbital-inducible

translation

BIOPHYSICAL

molecular

molecular

form

proteins

of most

we suggest

as the mature

is the

the same

of a high

Since our cell-free synthesized

AND

NH2-

hydrase

the signal

sequence

associated

protease.

membranes

is currently

laboratory. ACKNOWLEDGEMENTS

Petras

The authors would like to thank Joan Kifiyanski, and John Reminger for their help in preparing

Jack Kath, Bill Koptis, this manuscript.

Rosemary

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