Protection and reactivation of carbamyl phosphate synthetase with sulfate

Vol. 1, No. 4

BIOCHEMICAL

PROTECTION

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Oct. 1959

AND REACTIVATION OF CARBAMYL PHOSPHATE SYNTHETASE WITH SULFATE

Joanne

M. Ravel, Clayton

Roberta

F. Sund,

and William

Shive

Foundation Biochemical Institute Department of Chemistry University of Texas

Received September 20, 1959 Purified mammalian

preparations liver

concentrated dilute

have been reported ammonium

buffer

synthetase

from

ammonium

sulfate

also

prevents

treatment; extent

sulfate sible

for

carbamyl

destructive

the ammonium

on a DEAE-cellulose

appears

cent

against

0.04

1 Abbreviations: Tris, adenosine triphosphate.

of the

and elution

cent

M Trisl

buffer,

with

0.08

active

from with

adsorption M phosphate

fractions followed

pH 8.5,

tris(hydroxymethyl)aminomethane; 186

synthetase

of saturation);

of saturation)

dialysis

The

by precipitation

of the

(90 per

treatment. to be respon-

phosphate

and precipitation

sulfate

to a large

restoration

were obtained

to 75 per

ammonium

but

to preparations

and heat

of carbamyl

column

storage

and of heat

activity

ion

that

activity.

lactis

(50

was noted

in restoring

and the

synthetase

phosphate

of dialysis

by dialysis

in

and Cohen,

enzyme upon

synthetase

stabilization

of 2.

the

it

in

unstable

carbamyl

is effective

preparations

sulfate

pH 8;

than

very

from

stable

Marshall,

8039,

effects

phosphate

the

extracts

buffer,

protects

inactivated

phosphate

ammonium

lactis

rather

Purified sonic

Streptococcus

carbamyl

both

Hall,

of purified

the

ion

but

investigation

only

synthetase

to be reasonably

(Metzenberg,

not

have been

phosphate

solutions

and furthermore, the

which

sulfate

solutions

In this

1957).

of carbamyl

for

with by

2 hours

at 4'. ATP,

BIOCHEMICAL

Vol. 1, No. 4

This

purified

preparation

(moles

of product

diluted

with

protein.

ing

0.04

and after

amount

containing

poles;

magnesium

various

of 1 ml.

380,

(Archibald,

As indicated enzymatic buffer

activity

2 minutes.

than

This

I,

of ammonium

sulfate.

chloride

to the

buffer

not prevent

the

Trls

in Table

by allowing sulfate

the

the

presented

synthetase

for

upon the

sulfate

solution

to which

amount

of sulfate

present

previously 1959).

loss

in

by high of 0.04

Tris

at 60' for concen-

M magnesium

sulfate

is inactivated

activity

1 hour

The degree

is dependent

'Purification and Shive,

on heating

of ammonium

preparation

tivated

previously

loss

cent

at 4' against

Is prevented

enzymatic

II.

2 Preparation

incuba-

does

due to dialysis.

pH 8.5,

in Table

in a

15 minutes

18 hours

in lieu

inactivated

solution,

trans-

77,0003,

The addition

phosphate I,

and ornithlne

is an 80 per

for

destruction

When carbamyl cribed

there

in activity

trations

50,umoles;

calorimetrically

an 80 per cent

loss

10

aliquots.

on dialysis

and better

buffer,

After

was determined

in Table

ATP,

activity

at a pH of 8.8.

limit-

a reaction

250poles;

20pmoles;

of

a rate with

Tris

specific

was then

was determined

of protein)

on appropriate

1944)

hour)

to 1 mg. per ml.

carbamate',

protein,

cltrulline

per

activity

lOmoles;

Oct. 1959

of 1200

by incubating

(10 to 4O~g.

monohydrochloride,

volume at

pH 8.5,

treatments

chloride,

total

activity

synthetase

ammonium

5 w.

tion

buffer,

phosphate

=DL-ornlthine carbamylase,

a specific

per mg. of protein

M Tris

of enzyme

mixture

with

formed

Carbamyl

before

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

can be largely to stand

at 4'.

the

concentration

during

described 187

results

enzyme

are

is reac-

of the ammonium

is exposed

the enzymatic

described

restored

in an ammonium

These

to which

the enzyme

as des-

and not

upon

conversion

of

(Gmelin,

1936).

(Ravel,

Grona,

Hmphreys,

the

Vol. 1, No. 4

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Oct. 1959

TABLE I STABILITY OF PURIFIED PREPARATIONS CARBAMYL PHOSPHATE SYNTHETASE

OF

Per Cent of Control

Treatment None

100

Stored

at -20'

for

1 week

Stored at -20' sulfate

for

1 month

Dialyzed

40.

in 0.5 M ammonium 100

18 hours

at 4’ against

0.04

M Tris,

pH a.5

Dialyzed 18 hours at 4' a ainst ammonium sulfate, pH 8 .5

0.04

M Tris,

0.5 M 98

Heated

2 minutes

at 60'

Heated

2 minutes

at 60~ in 0.5 M ammonium

Heated

10 minutes

carbamate sulfate

above

without

sodium matic

can be stored losing

potassium

1 hour

phosphate are without

at 60°.for

protein)

fate

a period

effect

77

of ammonium

of several

to be reactivated.

are effective

and the

sulfate

more effective.

for

ammonium

86

in restoring

carbonate,

residue under

ammonium

from

the

Ignition

the

same conditions

synthetase

is inactivated

Inactivamonths

at

Both enzychloride, of ammonium (pH 8.5

for

at 4').

by ammonium

the

sulfate

Concentrations

ability

sulfate

whereas

When carbamyl ing

their

and potassium activity

sulfate

phosphate.

1 M are *not appreciably

ted preparations -20'

14

at 60’ in 0.5 M ammonium

to carbamyl

20

2 minutes, sulfate.

enzyme

activity

phosphate

If,

solution

cannot

the activity however,

the

is heated

be restored

can be partially

for

dilute longer

by standing

solution. 188

by heatrestored

(1 mg. per ml. periods

of

of time,

In an ammonium

sul-

Vol. 1, No. 4

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Oct. 1959

TABLE II REACTIVATION

Treatment I.

OF CARBAMYL PHOSPHATE SYNTHETASE BY SULFATE

Following

Inactivated

Per Cent of Control

Inactivation

by dialysis* 20

None Ammonium Ammonium Ammonium Sodium

sulfate, sulfate, sulfate, sulfate, sulfate,

Ammonium

carbonate,

Ammonium

chloride,

$5 51

M

40

0.5 M

19

0.35

25

2 M

phosphate,

Inactivated

M M M

0.5 M

Potassium

Potassium II.

0.25 0.5 1.0

0.5

by heat

8

M

treatment*

None

14

Ammonium *As in Table

These ammonium

sulfate, I.

data Ion

Indicate

of the

though

concentrations

high ionic

do not

sulfate

sulfate

for

might

introduce

or perhaps

exert

both

than

stabilization

other

activity.

salt

reactivation salts.

High

a sulfate

moiety ionic

189

into

effect

which

ionic

presence

concentrations

other

solutions

same concentration in the

Al-

factors

are of equivalent

and the

the and the

are required,

since

which

rather

synthetase

of sulfate

a specific

ion

the

phosphate

effect;

of other

actually

for

but

are without is required

sulfate

are involved

concentrations

cule

the

carbamyl

strength

contain

strengths

that

is responsible

restoration

than

46

1 M

of of high of sulfate

the enzyme in forming

moleand

Vol. 1, No. 4

BIOCHEMICAL

stabilizing

a specific

structure

and the biological

sulfate

phosphosulfate, tion.

of the

sulfate

of interest

lability

as on the

would

of mammalian enhanced

phosphate

synthetase

Grisolia,

1959).

be the

A comparison

of

3-phosphoadenosine-5reactivation

reac-

effect

of sulfate

on the

phosphate

lability

by substrates

Oct. 1959

in the

carbamyl

thermal

enzyme.

donor,

would be of interest

Also

extreme well

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

synthetase

of frog

and cofactors

liver

as

carbamyl

(Caravaca

and

References Archibald,

R. M.,

J. Biol.

Caravaca, J. and Grisolia, comm., 1, 94 (1959). "Handbuck Gmelin, Verlag Chemie Metzenberg, J. Biol.

Chem., S.,

156,

Biochem.

der anorganischen G.m.b.H., Berlin,

R. L., Hall, Chem., 229,

Ravel, J. M., Grona, Biol. Chem., a,

121 (1944). and Biophys.

Chemie", 8th ed., 1936, p. 348.

L. M., Marshall, 1019 (1957).

M. L., Humphreys, 1452 (1959).

190

J.

Research No.

23,

M.,

and Cohen,

P. P.,

S.,

and Shive,

W.,

J.