The biosynthesis of cytochrome P450 by heavy and light rough endoplasmic reticulum of rat liver

Vol. 91, No. 1, 1979 November

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

14, 1979

Pages 95-l 01

THE BIOSYNTHESIS

OF CYTOCHROME P450 BY HEAVY

AND LIGHT ROUGH ENDOPLASMIC RETICULUM OF RAT LIVER Michael

B. Cooper,

Department

John A. Craft*,

of Biochemistry,

University

London Received

September

Margaret

R. Estall

College

WClE 6BT,

Great

and Brian

London,

R. Rabin

Gower Street

Britain

4, 1979

SUMMARY

The synthesis of cytochrome P450 by heavy rough endoplasmic reticulum and light rough endoplasmic reticulum has been examined in vitro, using immunochemical techniques. Contrary to previous indications the results show no evidence for preferential segregation of the cytochrome P450 m-RNA and that the presence of mitochondrial protein synthesis accounts for the differences that have previously been reported. Two morphologically be isolated

from rat

centrifugation studded the

membranes

closely

rough

endoplasmic

supernatant

destined

for

export,

Attempts heavy

rough

suggests

are might *

Current Glasgow,

of lamina

fractions

apossible

site

vesicles in vitro

isolated

Both

[Z].

and intrinsic mitochondria

functional the

to exist

address: Glasgow Great Britain.

types

membrane

of ribosome-

[l].

In contrast, mitochondrial

fraction

consists

of membrane

relationship site

are

College

between

[4,7,8],

the biosynthesis of Technology,

This

active

[4]. found

close

in

association

the two organelles. [6],

whilst

membranes

such a relationship of haemo-proteins. Cowcaddens

0006-291

95

of

proteins

membranes

[1,5].

of haem synthesis synthesis

for

proteins

from reticular

have been unsuccessful

are

This

by

stacks

from a post

g-forces.

can

can be prepared

mitochondria

is

reticulum

and have been shown to synthesize

of apo-cytochrome

be expected

with

at high

to separate

mitochondria the

and consists

reticulum,

[3,4],

endoplasmic

fraction

associated

ribosome-studded synthesis

of rough

The heavy rough

by centrifugation

in protein

Since

liver.

at low g-forces

light

discrete

distincttypes

Copyright All rights

Under

Road,

X/79/210095-07$01.00/0

@I 1979 by Academic Press. Inc. of reprodgctinn in any formreserved.

BIOCHEMICAL

Vol. 91, No. 1, 1979

conditions

of cytochrome

production

of haem and the

Various

attempts

of cytochrome cytochrome

of the

endoplasmic synthesis study

is

reticulum

which

in the biosynthesis

polysomes

rough

that

whilst

endoplasmic explanation both

of cytochrcme

types

[9,10].

of the presence

of nascent

[ll]

report

workers

have been contradicted restricted

has presented by the have

reticulum, for

the

site

synthesized other

in

the intracellular

to exclusively

A recent

preferentially

a possible suggests

reports

appears

[8,12].

[13]

by the heavy

we present

evidence

cytochrome

polysomes

the cytochrome

Earlier free

increase

has been reported

have been made to locate

with

RESEARCH COMMUNICATIONS

a co-ordinated

apo-cytochrome

P450 associated

membrane-bound that

P450 induction,

P450 synthesis.

and synthesis

AND BIOPHYSICAL

these

of membrane

light

inferred [14,15].

differences are

equally

to evidence rough preferential In this and provide active

P450 in vitro,

METHODS Male Sprague-Dawley were used in this study. phenobarbital (40 mg/kg sacrifice on day 5.

rats, 200-250 g, permitted food and water adlibitum Animals received a single i.p;injection of in 0.9% w/v NaCl) on each of 4 days prior to

Fractions of heavy and light rough endoplasmic reticulum were isolated from the same homogenate of liver. The heavy rough endoplasmic reticulum was prepared as previously described [4]. Light rough endoplasmic reticulum was prepared from the resultant post-mitochondrial supernatant by fractionation on a discontinuous sucrose gradient system as described by Blyth et al [16]. Protein synthesis in vitro was carried out as in 141, but using [14C] protein hydrolysate (50 m Ci/m atom carbon, Radiochemical Centre, Amersham) at 41.1Ci/ml and supplemented with those amino acids not present in the hydrolysate. Incorporation of radioactivity into total protein was estimated by precipitation with trichloroacetic acid [17]. Following protein synthesis in vitro, the membrane fractions were harvested by centrifugation at 105,000 g (max) for one hour at 40c. The membrane pellet was resuspended to 20 mg/ml protein in a solution containing sodium phosphate (O.lM) pH 7.7, glycerol (20% v/v), EDTA (ImM), and sucrose (0.25M). The suspension of membrane was solubilized by the addition of sodium cholate (3mg/mg membrane protein) and non-solubilized material removed by centrifugation as above. The resultant supernatant, which contained greater that 90% of the incorporated radioactivity was analysed by quantitative immunoprecipitation with a specific antibody to cytochrome P450 [4]. Protein was determined by the method of Lowry [18], RNA by the method of Fleck and Begg f19]. Phospholipid was extracted by the method of Folch 1201 and lipid phosphorus subsequently estimated by the method of Chen [21]. Succinate dehydrogenase was assayed by the method of Pennington [22], cytochrome ~450 by the method of Cmura and Sato [23] and glucose-6phosphatase by the method of Lsskes and Siekivitz 1241.

96

Vol. 91, No. 1, 1979

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE 1 Chemical

and enzymic content

of heavy end light

rough endoplasmic

Heavy rough endoplasmic reticulum

(mg

RNA RNA/mg protein)

reticulum

light rough endoplasmic reticulum

0.05 + 0.02

0.13 + 0.01

Phospholipid (ug Pi/mg protein)

3.2

Succinate dehydrogenase (umol x h-1 x mg protein -11

0.08 + 0.005 (19 _+ 5)

0.04 + 0.002 (5 + 2)

0.11 + 0.01 (28 + 5)

0.15 $ 0.02 (24 - 4)

0.45 + 0.05

1.40 + 0.05

+ 1.2

14.2

+ 1.5

Cytochrome ~450 (n mole x mg Protein-l)

The chemical end enzymic activities of the two membrane types were determined as outlined in the Methods. 2 The specific activity of succinate dehydrogenase is expressed as umol 2-(p-icdophenylj-3-(p-nitrophenyll-5tetrasolium reduced x h-1 x mg protein-l, and of glucose-6-phosphatase as umoles Pi released x h-1 x mg protein -1. The figures in parenthesis are the percentages of the total homogenate activity recovered in each fraction. RESULTS AND DISCDSSION The fractions characterized

by their

The presence reticulum

of heavy and light

of mitochondria is

clearly

dehydrogenase

present.

dehydrogenase

activity

The presence protein

chemical

and enzymic in the

indicated

incorporate trichloroacetic

fraction

A considerable

in this

were

shown in Table

specific

activity of the

is recovered

fraction

of succinate succinate

in this

results

of incubation,

radio-labelled

amino in a linear

acids fashion

97

both

fraction.

in decreased

into

membrane

material (figure

fractions

insoluble 1).

1.

endoplasmic

ratios.

the conditions

acid,

reticulum

of heavy rough

proportion

of the homogenate

of mitochondria

endoplasmic content

by the high

and phospholipid/protein Under

rough

in

In the data

RNA/

BIOCHEMICAL

vol. 91, No. 1, 1979

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

25-

zo* b -ii 2 a 15is 0% 10 -

I

6

12

INCUBATION

16

20

TIME

(MINS

24

20

1

FIGURE 1 Kinetics of acid insoluble

incorporation material

in vitro of [14c] by heavy and light

amino

acids

into

trichloroacetic

rough endoplasmic reticulum. Protein synthesis in vitro was carried out as described in the Methods. Incorporation by heavy rough endoplasmic reticulum is shown in the presence (A) and absence (0 I of 5 x 10s6M chloramphenicol. Incorporation by light rough endoplasmic reticulum is shown similarly in the presence !A) and absence (0 ) of the antibiotic.

presented is

1, incorporation

in figure

much greater

than

considered

that

activities

of the mitochondrial

heavy

rough

rough

discrepancy

rough

endoplasmlc

arises protein

because

endoplsamic

reticulum. of

We have

the varying

synthesizing

reticulum

system

levels present

and in the

fraction.

The antibiotic protein

this

by light

by heavy

synthesis

chloramphenicol, [25].

selectively

The inclusion

inhibits

of chloramphenicol

98

mitochondrial in

the protein

BIOCHEMICAL

Vol. 91, No. 1, 1979

synthesizing

incubation

containing

resulted

in a decreased

Further,

in the presence

same as that effect

on

found

membranes by both The is

types. types

result not

of

in

to be preferentially

rough

the

antibiotic

light

rough

(see figure fraction

using

analyses the

into

examined

incubation

located of reticulum

of radioactivity

the

in the chloramphenicol

in in

into

radioactivity reticulum.

Heavy endoplasmic

of

incubations

P450

chloramphenicol

P450 appears reticulum.

containing

protein

P450 is

can

cytochrome.

cytochrome

endoplasmic

diminished

cytochrome

[14C] precipitated precipitated by in fraction.

Where

heavy

synthesis,

the

unaltered.

2 into

cytochrome

by antibody/[14C] trichloroacetic rough reticulum

P450

by heavy

and

acid Light endoplasmic

rough reticulum

Control

0.05

+ 0.01

0.10

_+ 0.02

+chloramphenicol

0.10

+ 0.02

0.12

2 0.01

Protein synthesis in vitro was carried out for 15 minutes, at which time incorporation-] amino acids was still linear. Subsequent recovery and solubilization of the two types of endoplasmic reticulum were as described in the Methods. Immunoprecipitations using a specific antibody to cytochrcme P450 were carried out on aliquots of the solubilized membranes and contained between 2CO0 and 4000 cpm. The data shows the fraction of radioactivity precipitated by the antibody and gives the means and standard deviations from three separate experiments. Where indicated, chloramphenicol was included in the rotein synthesizing incubations in vitro at a concentration of -% M. 5 x 10

99

of

by the two

to the

2.

rough

rough

of cytochrome

biosynthesis

light

results

table

had no

reticulum

proteins

antibody

the

activity

endoplasmic

specific

in

TABLE The incorporation of light rough endoplaemic

by light

the biosynthesis

shown

was then

synthesizing

rough

a specific

are

acids

Theprotein

of heavy

amino-acids.

Chloramphenicol

amino

1).

reticulum

incorporation

membrane.

of incorporation

inclusion

endoplasmic

incorporation

the

of membrane

included

Although

of the

We have

these

endoplasmic

of radio-labelled

of radio-labelled

in the

comparisons

heavy rough

incorporation

the

reticulum

the mitochondria distort

for

incorporation

endoplasmic

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

BIOCHEMICAL

Vol. 91, No. 1, 1979

The presence rough

reticulum

P450.

synthesis is

of chloramphenicol

endoplasmic

cytochrome

is

identical

inhibited,

The results

of this

endoplasmic

reticulum;

of the cytochrome

light

where

of

mitochondrial

protein

of radiolabelled

amino

acids

types.

study

provide

RNA for

associated

containing

on the biosynthesis

incorporation

membrane

of messenger

mitochondrially

conditions

the

RESEARCH COMMUNKATlONS

in incubations

has no effect

Thus under

in both

segregation

,AND BIOPHYSICAL

cytochrome

membrane

and is

no evidence

in the

contrary

for

P450 with fraction

of heavy

to the proposal

is preferentially

located

the preferential

on the

that

light

rough the biosynthesis

rough

endoplasmic

reticulum. ACKNOWLEDGEMENTS We thank

the Cancer

financial laboratory

support,

Research

Campaign

and the Nuffield

and Medical Foundation

Research for

provision

Council

for

of

facilities.

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16.

Shore, G-C., and Tata, J.R. (1977). J. Cell. Biol. 72, 714-725. Dallner, G., and Ernster, L. (1968). J. Histochem. Cytol. 16, 611-632. Shore, G.C., and Tata, J.R. (1977). J. Cell Biol. 72, 726-743. Craft, J.A., Cooper, M.B., Estall, M-R., Rees, D.E. and Rabin, B.R. 96, 379 - 391. (1979). Eur. J. Biochem. Parry, G. (1975). Ph.D. thesis, University of London. 51-53. Granick, S., and Gumpei, U. (1963). J. Biol. Chem. 238, 821-827. Sargent, J.R., and Vadlamudi, B.P. (1968). Biochem. J. 107, 839-849. Negishi, M., Fuji-Kurijama, Y., Tashiro, Y., and Imai, Y. (1976). Biochem. Biophys. Res. Commun. 71, 1153-1160. Marver, H.S. (1969) in "Microsomes and Drug Oxidations, eds. Gillette J.R., Conney, A-H., Cosmidies, G.J., Estabrook, R.W., Fouts, J.R., Mannering G.J., (t pp. 495-515 Academic Press, New York and London. Baron, J., and Tephly, T.R. (1969). Biochem. Biophys. Res. Commun. 36, 526-532. Ichikawa, Y., and Mason, H.S., (1974). J. Mol. Biol. 86, 559-575. Craft, J.A., Cooper, M.B., Shephard, E.A., and Rabin, B.R. (1975) FEBS. Lett. 59, 225-229. Bhat, K.S., and Padmanaban, G., (1978). Biochem. Biophys. Res. Commun. 84, 1-6. Correia, M.A., and Meyer, U. (1975). Proc. Nat. Acad. Sci. USA, 72, 400-404. Meyer, U., and Meier, P.J. (1977). Experientia 33, 807. Blyth, C.A., Freedman, R.B., and Rabin, B.R. (1971). Eur. J. Biochem. 20, 580-586.

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Craft, J.A., Cooper, M.B., and Rabin, B.R. (1978). FEBS Lett. 88, 62-66. Lowry, O.M., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. Fleck, A., and Begg, D. (1965). Biochim. Biophys. Acta. 108, 333-339 Folch, J., Lee, M., and Sloane-Stanley, G.H. (1957). J. Biol. Chem. 226, 397-409. Chen, P.S., Toribara, T.Y., and Warner, H. (1956). Anal. Chem. 28, 1786-1791. Pennington, R.J. (1961). Biochem. J. 80, 649-654. Gmura, T., and Sato, R. (1964). J. Biol. Chem. 239, 2379-2385. Leskes, A., Siekivitz, P., and Palade, G.E. (1971). J. Cell Biol. 49, 264-287. Buchanan, J., Primack, M.P., and Tapley, D.F. (1970). Endocrinology, 87, 993-999.

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