Relative turnover rates of subunits of rat liver fatty acid synthetase

Vol. 48, No. 6, 1972

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

RELATIVETURNOVER RATESOF SUBUNITS OF RAT LIVER FATTY ACID SYNTMETASE John Tweto*, Peter Dehlinger, and Allan R. Larrabeet Department of Chemistry University of Oregon Eugene, Oregon 97403 Received

June 15,1972;

Revised

August

1,1972

Summary--The subunits of the multienzyme complex rat liver fatty acid synthetase are degraded with half lives which are related to their molecular weight in a manner similar to that found in the laboratory of Schimke for soluble, membraneand ribosomal proteins from rat liver. The rates of turnover of all the subunits of fatty acid synthetase appear to be slower than the rate of exchange of the enzyme prosthetic group, 4'-phosphopantetheine. Rat liver

fatty

tains all the activities fatty

acids.

acid synthetase (FAS) is a multienzyme complex which connecessary for the --de novo synthesis of long chain

Although the enzyme complex readily

mately equal sized subunits, attempts to isolate complex have been unsuccessful. guanidino-labeled half life

dissociates into two approxithe individual

enzymes of the

Previous turnover studies on FAS employing

arginine and tritiated

pantothenate showed that the average

of the subunits of FAS is 3 to 4 days and that the prosthetic

of FAS is exchanged at a rate which is more than 10 times the protein rate (1). FAS tryptic

In the present study we have examined the relative peptides and FAS subunits solubilized

dodecyl sulfate

group turnover

turnover rates of

by the ionic detergent sodium

(SDS). Materials

and Methods

The care of the rats and the preparation of the enzyme were as previously described (1).

For the 3H/14C studies FAS was further

gradient centrifugation Isolation

purified

by sucrose

(2).

of FAS Peptides - Trypsin digests were prepared essentially

as de-

scribed by Hayes and Larrabee (3) with the exception that the amount of trypsin was lowered to 3% of the weight of sample. At the end of the trypsin * Present Address: Department of Anatomy and Cell Biology, University burgh Medical School, Pittsburgh, Pa. 15213 + Present Address: Department of Chemistry, Memphis State University, Tennessee 38111, and to whomcorrespondence should be addressed.

Copyright @ I9 72 by Academic Press, Inc. AN rights of reproduction in any form reserved.

1371

treat-

of PittsMemphis,

Vol. 48, No. 6, 1972

BIOCHEMICAL

ment an undigested precipitate digested with crystalline fractionated

(15% of original

pepsin.

on Aminex A-5

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

material)

was subsequently

The peptides from these digests were

resin&

a

modification

(3) of the procedure de-

scribed by Jones (4). Hydrolysis

of Peptides and Quantitation

graphy of the trypsin

HCl (about 3 ml).

and the peptides hydrolyzed

The tubes were sealed in a vacuum

for 40 hours at llO°C, followed by evaporation of

The sample from each tube was applied to a sheet of Whatman3 MM

chromatography paper and electrophoresis with 0.1 M potassium carbonate buffer, 150 ma for 2 hours.

was performed on a water cooled plate pH 10.2, at 200 volts

per inch, 100 to

Arginine was located by comparison with a standard.

isolated arginine was eluted from the paper with water, acidified, quot taken for counting and for arginine quantitation reaction

from chromato-

and pepsin digests were dried in glass tubes and dis-

solved in constant boiling

the Xl.

of Arginine - Fractions

(5).

When a sample of arginine from intact

of protein was hydrolyzed

and the hydrolysate

The

and an ali-

by meansof the Sakaguchi FAS was required,

a sample

treated as described for peptide

hydrolysis. Sodium Dodecyl Sulfate

(SDS) Disc Gel Electrophoresis

concentrated by ammoniumsulfate

precipitation

- Samplesof FAS were

and dialyzed

overnight

at 30°C

against 0.2% mercaptoethanol and 0.2% SDS in 0.05 M sodium phosphate, pH 7. Acrylamide gels (5%) were made according to the procedure of Weber and Osborn (6) with SDS incorporated

into the gels.

Five mg of protein were applied

large gel column (19 x 60 mm)and electrophoresis the tracking

dye reached the bottom of the gel.

to

a

was performed at 30 ma until The buffer

in the reservoirs

was 0.1 M sodium phosphate, pH 7, with 0.2% SDS. At the end of the run the gel was frozen on a block of dry ice and stored at -2O'C. was performed by extruding blade fixed at right

the frozen gel from a tube into the path of a razor

angles to the gel axis.

advancing a calibrated placed in a scintillation

Sectioning of the gel

One mmsections were obtained by

screw against the bottom of the gel. vial

and dissolved by addition

1372

Each slice was

of 0.5 ml of 30%H202

Vol.

48,

No.

6, 1972

BIOCHEMICAL

two days at

AND

and incubation

for

were

dissolved

in 10 ml of Aquasol

mine

(Packard

Inst.

Co.).

days to eliminate

long

It

temperature.

room

and clarified

was necessary

lived

turnover

pantetheine,

reported

of a rapidly

turning

terial

acyl

labeled

to test

activity

Each isolated

1 show that

all

peptides

usually

differing

reflect

experimental

units

of the complex.

cpm/pg which sample

compares

have

or slightly

radioactivity

demonstrated

that

the pantothenate

tography

of peptide

specific

radioactivity

did

the rapid

turnover

of prosthetic

compounds

in

Tl with

the cytosol,

and not

to a rapid

exchange

a with

approximately represents Rechroma-

two peptides

These

turnover

for

to ACP.

showed

due to its

was 53

injected

and thus

analogous

could

the sub-

observed

contained

differ. is

for

rats

Tl

gradient

significantly group

utilizing

protein

a more shallow not

rates

of the digest

containing

radioactivity,

of the peptides

peptide

T2 20% of the pantothenate

in Table

discrepancy

of 55 cpm/pg

experiment

FAS radio-

reported

This

turnover

the value

An earlier

from

results with

of a protein

whose

imply

that

pantothenate subunit

con-

4'-phosphopantetheine. To further

nents,

with

U-14C-arginine

specific

at most.

different

acids.

and the specific

arginine

be

of the purified

The results

similar

of two or three

amino

with

digests

to bac-

it would

exists,

injected

was hydrolyzed,

the existence

analogous

complex

of radioactive

and pepsin

analyzed

FAS.

several

4'-phospho-

by assuming

FAS

were

was measured.

peptides

taining

trypsin

favorably

pantothenate

80% and peptide

rats

The mean specific

of undigested

radioactive

by a pulse

arginine

error

for

group,

If such a protein

(7).

peptide

by a factor

prosthetic

in the

possibility, from

of the isolated

slices

of 0.1 ml of Hya-

the samples

be explained

species

activity

resulting

isolated.

gel

by addition

bound

could

(ACP)

this

The solubilized

to store

covalently

protein

specific for

COMMUNICATIONS

and Discussion

(1).

protein

carrier

and the peptides were

earlier

to a higher

In order

of the

over

RESEARCH

phosphorescence. Results

The rapid

BIOPHYSICAL

investigate

we performed

a double

the relative label

turnover

experiment

1373

similar

rates

of FAS protein to

that

described

compoby

Vol. 48, No. 6, 1972

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table 1. Specific Radioactivity of Arginine from Trypfic and Peptic Peptides: Five 200 g rats were each injected with 100 pci of [UCl-L-arginine (273 mci/mmole). Ten hours later the animals were killed and the arginine from the resulting peptides was isolated and analyzed. T and P refer to tryptic and peptic peptides, respectively. Peak No.

cpm/pg

Tl T2 T3 T4 T5 T6 T7 T8 T9

Arias

peritoneal

(8).

Rats

injection

and their

approximates decay

the

subjected count

livers

removed

initial

isotope

sented

experiment

geneous

turnover result

larger

rates

relationship

of protein between

gure

for 3 shows

a wide similar

variety data were

hours

figure

This

rate

of soluble for

indicates

The purified

ratios

obtained

proteins

--et al.

higher

are preobtained

gels

due to

by the which

is pre-

in a manner

by Weber and Osborn in which

both

and the animal of fluctuation

3H/14C

a systematic

FAS subunits,

simultaneously the extent

is

as indicated

experiment,

The

of FAS have hetero-

there

in SDS acylamide

the

of SDS.

in 14C radioactivity

of the

were

FAS was

of 3H/14C ratios

by Arias

rates

1374

animals

14C level,

the presence

the subunits

the control

administered

days.

In addition

weight

to migration

and the

The range

turnover

snd the molecular

level,

count

losses

the

in-

Thus 3H radioactivity

tritium-carbon

greater

an intra-

by a second

injection

in

that

given

days later

electrophoresis

degradation.

3H and l4C-leucine later.

of three

as discussed

the relative

were

FAS purification.

suggests

from relatively

sumed to be related scribed

and the

since,

three

incorporation

gel

strongly rates,

ratios,

for

state

the second

1 and 2 respectively.

in this

ratios

after

a period

disc

of the gel

in Figures

3H/14C

after

to acrylamide

profile

followed

Two hours

arginine

79 62 34 43 27 44 41 18

in the steady

of l4 C-leucine

in radioactivity

cpm/ug

TlO Tll T12 T13 Pl P2 P3 P4

maintained

of 3H-leucine.

killed

Peak No.

52 34 57 62 44 58 52 45 55

--et al.

jection

arginine

(6).

deFi-

isotopic killed

two

in 3H/14C

ratios

BIOCHEMICAL

Vol. 48, No. 6, 1972

SOOk

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I

I

I

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8

I

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32

40

48

56

GEL SLICE NUMBER Figure

1

200 g rats were maintained on Purina lab chow in an artificial light At zero time each was cycle alternating twelve hours on twelve hours off. administered 100 uci [U-14C]-L-leucine (312 mci/mmole)(New England Nuclear) via intraperitoneal injection. Three days later each rat was injected with 750 uci 4,5-[3H]-L-leucine (50 ci/mmole)(Schwartz-Mann) and two hours later The purified FAS was dissociated in the rats were killed and FAS prepared. SDS, subjected to acrylamide gel electrophoresis and the gel sliced and counted.

Two

inherent

in

served

in the

of isotope followed

These

control

three that

results

the

membrane with

half

lives

and ribosomal

prompted which

from

proteins

presented

here

consistent

in equilibrium

with

by the observed

has been

found

for

that

are

a pool protein

soluble

not

rat

with

occurs.

rate

correlation

1375

where

weight in which

from

rat

This

it

degraded

FAS subunits,

degradation

gel.

plasma

are all

a model

first

of the

molecular

ob-

case it

proteins,

liver

is

the order

In this

of Schimke,

membrane

subunit

proteins

shown,

the length

laboratory

from

ratios

was administered

of dissociated

size-degradation the

over

microsomal

to their

form

%I-leucine

the

are related

in the dissociated

experiment,

steadily

proteins,

in the 3H/14C

of 14 C-leucine.

which

The data of FAS are

rose

to those

the soluble

proteins,

is,

by administration

3H/14C ratio

are similar

was shown that

that

was reversed;

days later

variation

In another

experiment.

injection

was found

No systematic

the technique.

model

(9,10,11). the subunits and it

is

is

of FAS subunits liver.

This

model

is

Vol. 48, No. 6, 1972

BIOCHEMICAL

1 0

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1 16

AND BIOPHYSKAL

1

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24

GEL

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RESEARCH COMMUNICATIONS

I

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58

SLICE NUMBER

Figure 2 The ratio DPM3H/DPM14C of the data shown in Figure 1 is plotted as a function of slice number. Slicing began at the top of the gel. The dotted lines represent the standard deviation of the ratio which was calculated by a computer taking into account statistical error in counts, pipetting error in preparing standards, and error incurred in interpolating the automatic external standards. A straight line fitted to the graph utilizing a least squares fit, weighted by the standard deviations shown, had a slope of 0.262 per gel slice and an intercept of 21.7.

similar

to that proposed for the turnover of rat liver

membranesand ribosomes

(9,lO). Acknowledgement This investigation was supported by Grant GB-18998 from the National Science Foundation. John '&et0 is the recipient of a training grant (5-TOl-GMO0444) from the Public Health Service, Department of Health, Education and Welfare. We also wish to thank Dr. Joel Ivey for the computer program. REFERENCES 1.

meto, J., (1971).

Liberati,

M. and Larrabee, A. R., 2. -Biol.

1376

Chem.246, 2468

Vol. 48, No. 6, 1972

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS

v t 25

1

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GEL SLICE NUMBER Figure

3

A control experiment was performed in a similar fashion to the experiment described in Figures 1 and 2, except a mixture of 50 uci [14C]-L-leucine and 750 pci [311]-L-leucine was injected into each of two 240 g rats. Two hours after injection the rats were killed. A straight line fitted to the graph utilizing a least squares fit, weighted by the standard deviations shown, had a slope of 0.008 and an intercept of 11.6.

2. 3. 4. 5. 6. 7. 8. 9. 10. 11.

Tweto, J., and Larrabee, A. R., J. Biol. Chem. in press, (1972). Hayes, L., and Larrabee, A. R., Biochem. Biophys. --Res. Commun. 45, 955 (1971). Quant. Biol. 29, 297 (1964). Jones, R. T., Cold Spring Harbor m. Satake, K., and Luck, J. M., Bull. Sot. --Chim. Biol. 40, 1743 (1958). Weber, K., and Osborn, M., J.Kl.Chem. 244, 4406 (1969). -Vagelos, P. R., Majerus, P. W., Alberts, A., Larrabee, A. R., and Ailhaud, G. P., Fed. Proc. 2, 1485 (1966). Arias, I.M., Doyle, D.,tnd Schimke, R. T., -J. --Biol. Chem. 244, 3303 (1969). Dehlinger, P. J., and Schimke, R. T., J. Biol. Chem. 246, 2574 (1971). Dice, J. F., and Schimke, R. T., J. BioL Chem. 24J, 98 (1972). Dehlinger, P. J., and Schimke, R.-T.,ochem. Biophys. &. Commun. UJ, 1473 (1970).

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