An in vitro increase in aspartyltranscarbamylase activity

Vol. 22, No. 6, 1966

AN IN

BIOCHEMICAL

lrITR0

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

INCREASE IN ASPARTYLTRANSCARBAMYLASE ACTIVITY* Raizada

MSU/AEC Plant

Received

Research

February

A number

tion

observed

with

have demonstrated

(cf.

Nisman

an increase

in a cell-free

When grown

of this

mutant

vitro

in ATCase activity

partially

Pathology

inhibited

of specific

is

cell-free

greater

by RNase, but

if

mutant

is

activity

of E. coli. about

and Schachman,

here

dependent

amino

12 per

cent

The in

1965).

on the presence

enzyme preparation

exogenous

communica-

(ATCase)

requiring

(Gerhart

enzymes

The following

ATCase contitutes

reported

+t and a dialyzed

in activity

1964).

from a uracil of uracil,

protein

The increase

and Pelmont,

amounts

of the soluble

of ATP, GTP and Mg

synthesis

of aspartyltranscarbamylase

system

on limiting

increase

and Plant

11, 1966

systems

deals

Singh

Laboratory and Department of Botany Michigan State University East Lansing, Michigan, 48823

of investigators

in cell-free

M.M.

of the mutant.

acids

are added

and is

not by puromycin. EXPERIMENTAL

The mutant containing all

limiting

subsequent

washed

was grown

aerobically

amounts

operations

in a standard

buffer

at 37'

of uracil performed

to early

log

phase

(8 pg/ml)'.

The cells

at 0 to 5'.

The harvested

containing

0.01 g potassium

in a medium

were cells

phosphateL

chilled

and

were pH 7.4,

*This investigation was supported in part by the U.S. Atomic Energy Commission, Contract No. AT(ll-l)-1338 and in part by research grant AM 05916 from the National Institutes of Health to Dr. R. S. Bandurski. 1 I thank Dr. .I. C. Gerhart for cating unpublished observations

providing regarding

the mutant of E. coZi and communisuitable growth conditions.

n

'Use of phosphate buffer during preparation tion medium more than doubled the increas.e observed with tris(hydroxymethyl)aminomethane

of the "enzymd'and in the incubain ATCase activity above that buffer at the same pH.

Vol. 22, No. 6, 1966

0.014

g magnesium

passage

through

centrifuged the

acetate

at 10,000

fluid

was dialyzed

a change

of buffer

with

"enzyme",

liquid

caused

centrifuged fluid natant.

stored

aliquots

the

Both

at -20'

after

of standard

enzyme was not

was raised

stored

to 0.03

About

quick

discarded,

and

The superfor

designated

quick

freezing

again

since

were

separated

fourths

sedimented

x g supernatant

freezing

in liquid

in

thawing

of the

and used as the 105,000

105,000

8 hours

from

the

5 and the preparation

three

was resuspended,

the

was

buffer

after

by

suspension

preparation,

When ribosomes

by aspiration

cell

30 minutes.

at -20'

2 hours.

x g pellet,

fraction.

x g for

and disrupted

pellet

The dialyzed

of activity.

removed

The 105,000

the ribosome

4 hours.

x g for

The broken

100 volumes

in small

in buffer

the resultant

at 30,000

i-t of Mg

at 105,000

was carefully

were

after

the concentration

psi.

30 minutes,

against

loss

suspended

at 16,000

Once thawed

and refreezing

g KCl,

centrifuged

was stored

nitrogen.

enzyme,

Press

x g for

fluid

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

and 0.06

a French

supernatant

natant

as,

BIOCHEMICAL

supernatant

x g super-

again

and used as

enzyme and ribosomes

nitrogen.

RESULTS AND DISCUSSION TablIe

I shows the requirements

The increase

is

dependent

and to a lesser degree

extent

of dependence

natent

is

shown here.

The increase inhibited REase ATCase is

II)..

by about

incorporation per

cent

OS a mixture inhibits

optimal

and the

14C labeled

of

cent.

widely

cent

amino

with

both

A)

The

different and a typical

the 105,000

of amino

respectively)

x g super-

of amino

acids

in activity

acids

of RNase which acids

into

are

by 5 ug of

the increase

incorporation

685

B).

to no effect

A concentration

14C labeled

(experiment

(experiment

incorporation

incorporation

L-leucine

in ATCase activity.

activity.

100 vg puromycin

even though 60 per

varies

C shows that

for

with

acids

dependency

(23 and 25 per

Flowever,

inhibited

acids

complete

in ATCase activity

is not

inhibited

amino

are required

to the same extent (Table

of amino

Experiment

increase

of ATP, GTP and Mg*

on the addition

from nearly

and ribosomes

an in vitro

on the addition

on exogenous

enzyme preparations experiment

for

protein

by more than

into

of

protein

inhibits by only

25

90 per

cent.

BIOCHEMICAL

Vol. 22, No. 6, 1966

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE I Requirements

for

an in vitro

increase

in ATCase activity milli-units

Experiment

A

Endogenous Complete 11 ,I

("enzyme"

I,

Experiment

buffer)

117 240

minus

ATP

125

GTP +I-

140 139

Mg

B

Endogenous Complete I,

plus

system II minus 11 minus

11 Experiment

ATCase

(~'enzyme" system 11

plus

122

buffer)

227 minus

amino

196

acids

C

Supernatant

enzyme plus

buffer

Supernatant

enzyme plus

incubation

Ribosomes

plus

buffer

Ribosomes

plus

incubation

135 mixture

172 18

mixture

42

Supernatant

plus

ribosomes

plus

buffer

Supernatant

plus

ribosomes

plus

incubation

,152 mixture

267

The complete system contained in pmoles: potassium phosphate 50 (pH 7.4); magnesium acetate 60; ATP 10; GTP 10; (all experimentally determined as being the optimal concentrations) phosphoenolpyruvate 5; 50 ngrams of each of the 20 amino acids and in experiments A and B about 1 mg of enzyme protein (Lowry et In experiment C 105,000 x g supernatant aZ., 1951) in a final volume of 1 ml. enzyme or ribosomes or the 105,000 x g supernatant plus ribosomes were incubated in buffer alone or in the complete reaction mixture. Incubation was for 10 minutes at 37O and the reaction was terminated by chilling the tubes in an ice From each tube 0.5 ml of the incubated reaction mixture was removed and bath. dialyzed for 3 hours against 100 volumes of O.OlM potassium phosphate buffer pH 7.4 containing 0.06 g KC1 with two changes of the buffer. The dialyzed incubation mixtures were then diluted to 5 ml with the above buffer and an aliquot assayed for ATCase by the method of Gerhart and Pardee (1962). One milli-unit of ATCase represents 1 mpmole of carbamylaspartate synthesized per minute under the conditions of the assay. Each experiment was repeated at least twice and assays run in duplicate. The duplicates rarely varied by more than 10 per cent.

686

Vol. 22, No. 6, 1966

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE II Effect

of RNase and puromycin

incorporation

of a mixture

of

on the in vitro 14C amino

increase ATCase

Complete

system

II

8,

II

II

acids

increase

of ATCase and on the

and 14C leucine

in

amino acids incorporated into protein

into

protein.

14C &-leucine incorporated into protein

milli-units

mm

56

57

43

42

3.3

56

22

3.5

plus 5 ug RNase

plus 100 ug puromycin

uumoles 56

Conditions as in Table I except that 5 pg of each of the 20 amino acids were used. For studies of the incorporation of labeled amino acids a mixture of 5 ug each of fifteen 14C amino acids ("Reconstituted protein hydrolysate" from Schwarz Bioresearch Inc., Orangeburg, N.Y.) and l'+C histidine were added to give 9.6 x lo5 cpm per tube. Five ug each of unlabeled aspargine, cysteine, For the studies of incorcystine, glutamine and methionine were also added. poration of labeled leucine, 5 pg of 14C Irleucine (specific activity 9 mc/tiJ and 5 pg each of the other 19 unlabeled aGino acids were added. In studies of inhibition of ATCase increase, 3 to 4 replicates were run as well as controls containing equivalent amounts of RNase or puromycin to rule out any possible inhibition of ATCase activity by these compounds. Control tubes for amino To measure the incorporaacids or leucine incorporation were incubated at O". tion of the label into protein the reaction was terminated by adding 5 ml of 5% trichloroacetate (TCA) and heated for 20 minutes at 90'. The precipitated protein was washed once with 5% TCA, then with 3:l ethanol:ether and then 5 times the filter containing the washed on a milliipore filter with 5% TCA. Finally precipitate was counted with 15 ml of scintillation fluid (10% napthalene, 0.7% 2,5-diphenyloxazole and 0.03% 2,2-p-phenylenebis(5-phenyloxazole) in reagent grade dioxane) in a liquid scintillgtion spectrometer. Endogenous values for ATCase and the control values (0 ) for the incorporation experiments have been subtracted. The conclentration amino that

acids

of puromycin

by about

inhibits inhibits

incorporation

cent

also

&-leucine

system

for

an in vitro

for

protein

of a mixture incorporation

of by more

90 per cent. The requirements

are

60 per

which

identical

of the

to the requirements

667

increase

synthesizing

in ATCase activity systems.

Therefore,

Vol. 22, No. 6, 1966

one is

inclined

ATCase.

to attribute

However,

hypothesis other

BIOCHEMICAL

is

not

processes.

conversion

the ohserved

results

obtained

entirely

adequate

The increase

heating

for

do

cause

1965)

puromycin

and a part

treatments

at 60'

an increase

with

of the

does not

known

or treatment

in our

to cause

with

synthesis

of

suggests

that

this

increase

must represent

seem to be due to the

form of the enzyme to its

since

4 minutes

to de nova

increase

in activity

of the oligomeric

and Schachman,

not

the

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

monomeric this

form

conversion,

ATP (Gerhart

(Gerhart such as

and Pardee,

1962),

system. ACKNOWLEDGEMENT

I wish

to thank

Professor

R. S. Bandurski

for

helpful

discussions.

REFERENCES Gerhart,

J.C.,

and Pardee,

Gerhart,

J.C.,

and Schachman,

Lowry, O.H., 193, 265,

Roseborough, (1951).

A.B.,

N.J.,

J.

H.K., Farr,

Nisman, B., and Pelmont, J., in J.N. in Nucleic Acid Research and Mol. 1964, p. 235.

Biol.

m.,

237,

Biochemistry, A.L.,

891,

(1962).

5, 1054,

(1965).

and Randall,

R.J.,

J.

Biol.

m.,

Davidson, and W.E. Cohn (Editors), Progress Biol., Academic Press Inc., New York,

688