Synthesis of microsomal membranes and their enzymic constituents in developing rat liver

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 20, No. 2, 1965

Gustav

Dallner,

Philip

Siekevitz

The Rockefeller

and George E. Palade

Institute,

New York,

N.Y.

Received May 27, 1965 The limiting of enzymatic proteins

membrane of the endoplasmic

activities

which

(Siekevitz,

is related

1963).

brane structure

(Ernster

and relations

et al.,

1962).

The question

of the ER membrane, the synthesis

is closely

connected

and perhaps

other membrane components, protein.

The developing

answering

this

cell

rat

to be a suitable

data indicate

ER (Rowatson

the differential

whether

and probably

rates

during

that

of

non-enzymic system

the fetal

and Ham, 1954; Peters

in scme enzymes (Nemeth,

This paper describes

arises

the mem-

dependent on, the synthesis

appears

Available

developed

1963) and is deficient

liver

enzyme activity

of the membrane-bound enzymes

such as phospholipids

question.

has a poorly

moreover,

of the enzyme within

biogenesis

with,

constitutive

of the enzymes in the membrane

of the ER system;

by the position

(ER) has a variety

to some of its

The location

to the function

is influenced

are ascribed

reticulum

hepatic et&.,

1954; Strittmatter,

of synthesis

for

1963).

of membrane-bound

enzymes in the ER of the newborn rat. Experimental. N.Y.)

were used.

(Ralston

livers

The standard

Purina Co.,

and non-starved Ernster

Rats of CFN strain

et al.

adult (1962).

some microsomes

Separation

St. Louis, rats

diet

(Carworth (supplied

MO.).

(go-day

old),

Liver

Farms, ad libitum)

were prepared

in the preliminary

of smooth and rough (having

135

attached

New City,

was Lab Chow

microsomes

(Because of the high glycogen are lost

Inc.,

from fetus, as described contents

newborn by

of these

low speed centrifugation.) ribosomes)

microsmes

was

Vol. 20, No. 2, 1965

performed 1963). were

with

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

a two-layered

TO separate treated

suspended

0.26% deoxycholate

that

The pellets,

only

membranes. binding

1963; Orrenius

DL-leucine-1-C (1.5 w/10 points.

particles,

TCA and extracted were dissolved

was excised

of the various

gas flow

Hepatocytes

on the cytoplasmic

surface

contains

The groups

of its

the appearance

developed

et al.,

1962;

intraperitoneally at selected

(1952).

ribosomes

(- 500 i). deposits

birth, (Figs.

birth

time with

The final

12%

pellets

and counted in a Geiger-

limiting

very

if

1 and 2).

N 80 1)

large

aggregates deposits

of

appear At 3 The ER

of both smooth and rough elements. are separated, relationship

however,

by

between smooth

established.

transport After

spacing

that seen in the adult.

The characteristic

both electron

of rough surfaced

any smooth ER elements.

of the latter

is already

number

The cytoplasmic

the glycogen

approaches

a large

(ribosome

membrane.

and relatively

few,

contain

ER, primarily

of ribosomes

and is comprised

of attached

ER and glycogen

low activity

b5 and CO-

were precipitated

developed

packing

and contain

birth

distances

Before

was injected

and were plated

the

experiments,

and fractionated

At the time of birth,

increased,

is highly

acid,

many free ribosomes

particles.

days after

Corp.)

of Siekevitz

and a moderately by tight

greatly

contain

(Ernster

In incorporation

at 3 days before

type characterized

glycogen

earlier

The pellets

counter.

of mitochondria;

matrix

2 hrs.

the supernates

fractions

by the procedure in 88% formic

Results.

large

while

1964a).

to 4 hrs.

0.5% concentration

and the amounts of cytochrome

et al.,

The liver

The proteins

Muller

at 40,000 rpm for

l4 (New England Nuclear 9).

et al --•

and the membranes, were re-

pigment were measured as described

Dallner,

to Ernster

DOi: was added to a final

Enzyme activities

(Dallner,

at 40,000 rpm was prolonged

then centrifuged

the ribosomal

cscl

both rough and smooth microsomes

both the riboscxnes

in 0.25 M sucrose;

containing

(DOC) according

centrifugation

containing

and the suspension contain

gradient,

membrane components,

with

(1962) , except

sucrose

birth 136

enzymes and phosphatases the enzymes can be divided

exhibit into

Vol. 20, No. 2, 1965

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TPNH

diaoh.

DPNH

diaph. /

TPNH-NT

/

/* /

red. /

DPNH-NT

red.

in days

Fig. 1. Electron trawport enzymes in hepatic microsames as a function enzyme activities (on protein basis), expressed as per of age* Specific cent of adult level, are plotted on the ordinata,

pa

66 Pore

200

-

n f 2! = i cn

\

i

AlPare -----------.

-.----W-S-

Age

Fig.

2.

Phosphatases

in hepatic

in days

microsames

137

as a function

of age.

Vol. 20, No. 2, 1965

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

several groups depending on the rates of increase in their Nucleoside triphosphatase cific

activity

at all

(using ATP as substrate)

activity.

1)

displays the samespe-

stages of embryonic and neonatal development.

Nucleoside diphosphatase (using TDPas substrate),

2)

TPNH-neotetrazolium

(NT) reductase, DPNH-cytochrome c reductase, DPNH-KCreductase and oxidative demethylation

activity

at 8 days after

(using aminopyrine as substrate)

birth

are still

3) DPNHdiaphorase activity

considerably

increase slowly and

lower than the adult value.

(assayed with ferricyanide

as electron

acceptor),

the amounts of CO-binding pigment and cytochrome b5, increase more rapidly than the enzymes in group 2.

4) TPNHdiaphorase (measured with ferricyan-

ide) and TPNH-cytochrome c reductase activity at the first activity

day after

5) Finally,

rises immediately following

of the adult liver, 1957).

birth.

indicating

reach the adult level

glucose-6-phosphatase (G6Pase) greatly

exceeding the activity

described (Nemeth, 1954; Weber and Cantero,

in G6Pase, TPNH-cyt. c and TPNH-NTreductase activities

Increases

were inhibited

as previously

birth,

quickly

by actinomycin D (2x0.8 mg/kg) and puromycin (2x10 mg/kg),

that the assays measured increase in enzyme amount.

In the developing liver and TPMI-cyt.c

the newly appearing enzymes, such as G6Pase

reductase initially

have higher specific

activities

rough microsomes, and are presumably synthesized there (Pig. 3).

in the On the

other hand, ATPase, which does not change during development, is found in both subfractions, the adult rat liver

but is concentrated mainly in smooth microsomes. In these enzymes are distributed

smooth microsomes (Dallner,

equally between rough and

1963).

Pig. 4 shows the incorporation

of 1eucine-C14 into the membrane

proteins of rough and smooth microsomes. The labeled amino acid was injected into P-hour old rats and the membraneswere isolated hours later.

As can be seen, the specific

of the rough microsomes

early time point,

activity

fraa 0.5 to 32

of the membraneprotein

is higher than that of the smooth microsomes at an

but is lower later

on.

138

Vol. 20, No. 2, 1965

BlOCHEMlCAl

1

I -1

TPNH-cyt.

I 0

c red.

I +,

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I +2

ATPose

I

I +3

-I

0

+I

+i?

+3

DOYS

Fig. 3. Distribution developing rat liver.

I

of sane enzymes in rough and smooth microsanes of

I 3

I 6

I, 9

12 Hours

Fig. 4. Incorporation Z-hour old rats.

1 IS after

, IS

, 21

, 24

, 27

, 30

injection

of 1eucine-C 14 into microsomal membranesof

139

Vol. 20, No. 2, 1965

BIOCHEMICAL

Discussion. indicates

AND BIOPHYSICAL RESEARCH COMMJNICATIONS

Electron microscopic evidence (Dallner et al.,

that at birth,

1965b)

rat hepatocytes already have an ER system, includ-

ing somesmooth membranes. However, as the data of this paper show, only after

birth

do many of the constitutive

membraneenzymes appear.

question arises whether these new enzymes are integrated

The

into already exist-

ing membranes, or whether a small amount of new membrane(containing and non-ensymic protein) specific

is synthesized concomitantly

enzyme protein.

To this intent

an inquiring

during the samedevelopmental period was carried ported in a subsequent paper (Dallner et al., The above data also throw somelight microsomal electron transport et al.,

chains.

1964a, 1964b; Sato et al.,

lipids

with the synthesis of into lipid

synthesis

through and will

be re-

1965a).

on the components of the two

The available

1964) indicates

evidence (Orrenius the following

electron

flow diagram for the microscmal TPNH oxidase: ___3 ;PT & ferricyanide+cyt.c

Xil -CO-binding 4 NT

TPNH+F

pigment+detoxicatjon

,$ demethylation

fn the microsomes of newborn rats the low detoxication be due to a deficiency ductase, for it

system

activity

appears to

of the component Xl, here measured as TPNH-NTre-

is this latter

activity

which seemsto be rate limiting

in

the neonatal microsomal TPNHchain. Cytochrome b5 (Strittmatter protein

(Strittmatter

reductase (Dallner,

and Ball,

and Velick,

1952), the DPNHoxidizing

1956) and the PCMBsensitive

1963) appear to be integrated

flavo-

DPNH-NT

in the following

sequence

for the microsanal DPNHoxidase: DP~JH-

ferrzyanide Again, the rate limiting

cytocyome b B? 5 J# cyt.c

y2 -

ypD-

%C factor

of the chain appears to be a component


140

Vol. 20, No. 2, 1965

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

This work has been supported by a grant from the National Institutes of Health (AM-01635); and a travel

grant to G. D. from the Swedish Cancer

Society.

References Dallner, Dallner,

G. (1963) Acta Path. Microbial. Stand., Suppl. 166. G., Siekevitz, P. and Palade, G. E. (1965a) Biochem. Biophys. Res. Cournuns. This issue. Dallner, G., Siekevitz, P. and Palade, G. E. (1965b). In manuscript. Ernster, L., Siekevitz, P. and Palade, G. E. (1962) J. cell Biol. 2, 541. Howatson, A. F. and Ham, A. W. (1955) Cancer Res. l5, 62. Nemeth, A. M. (1954) J. Biol. Chem. 208, 773. Orrenius, S., Dallner, G. and Ernster, L. (1964a) Biochem. Biophys. Res. Communs.l4, 329. Crrenius, S., Dallner, G., Nordenbrand, K. and Ernster, L. (1964b) Abstracts, 6th Intern. Congr. Biochem., NewYork, p. 326. Peters, V. B., Kelly, G. W. and Dembitzer, H. M. (1963) Ann. N.Y. Acad. Sci. In, 87. Sato, R., Cmura, I. and Nishibayashi, H. (1964) 9 Intern. Symp. on Cxidases and Related Cxid. Red. Systems, Amherst, Mass. Siekevitz, P. (1952) J. Biol. Chem. 195, 549. Siekevitz, P. (1963) Ann. Rev. Physiol. 25, 15. Strittmatter, C. F. (1963) Arch. Biochem. Biophys. 102, 293. Strittmatter, C. F. and Ball, E. G. (1952) Proc. Nat. Acad. Sci. 3S, 19. Strittmatter, P. and Velick, S. F. (1956) J. Biol. Chem. 221, 277. Weber, G. and Cantero, A. (1957) Cancer Res. l7, 995.

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