Acetyl phosphate utilization with animal and bacterial enzymes

Vol. 7, No. 5, 1962

PHOSPHATE UTILIZATION

ACETYL

WITH ANIMAL

S. Grisolia University

Received

of Kansas

January

and P. Harmon Center,

recent

studies

et al,

19621,

with

has become

AP,

evident

replacing

kinase

the

cocci

about

yield

after

petitive

to test

+ ATP +

postulating for

the

* Supported by a Grant

activity CP,

more

from

induction

(Slade

inhibition indeed

of ATP from glutamate,

partial

to the

acetyl not

the

activities

1958)

reversibility

of for

CP

Strain

DlU Group

D strepto-

and Slamp,

1952).

Com-

also

indicate

that

These

findings

led

perhaps

CP and ADP catalyzed

et al,

due to

as

AP could

This

phosphate,

purification,

acetokinase.

AG.

1:

identity

with

experiments

in which

kinase

reaction

with

likely

activities

both

carbamate

is probably

constant

(Metzenberg

reactions

Kansas

CP + ADP,

and remain

is

acetyl

marked

but

reactions

Synthesis

ing

12,

catalyzes

equal

arginine

other

that

since

for

kinase

requires

City

purified

and acetokinase,

substrate

carbamate

highly

phosphate,

contamination

and AP are does

that

carbamyl

acetokinase carbamate

Kansas

an enzyme

1) NH2cooit

ENZYMES*

26, 1%2

During (Grisolia

Medical

AND BACTERIAL

has been

replace

us

CP.

by CP synthetase the

two enzymatic

of CP synthesis

basis

for

sites according

accountto

2 and 3:

by Grant A-1855 National from the American Heart 357

Institutes AsSOCiatiOn.

of Health

and

Vol. 7, No. 5, 1962

BIOCHEMICAL

2) ATP + CO2

AG,

3) ATP + "active Carbamyl-P phosphate requires enzyme the

date

acetyl

form

uses AP about shown in

the

ATP and in

this

this

3 times

Table

+ NH3 s

catalyzes

glutamate;

RESEARCH COMMUNICATIONS

ADP + Pi + "active

C02"

synthetase

and ADP to

AND BIOPHYSICAL

is

faster

I and from

ADP + CP interaction case,

that

of as with

illustrated than

C02"

in

CP, in

acetyl CP,

it

I.

The

Table

as calculated

the

from

literature

(Caravaca

TABLE I ACETYL GLUTAMATE REQUIREMENT WITH FROG LIVER

FOR ACETYL PHOSPHATE UTILIZATION

CARBAMYL PHOSPHATE

Additions Enzyme

Protein

SYNTHETASE

poles Acetyl

mg

poles

0.4

0

0.4

10

0.8

0

0.8

10

1.6

0

1.6

10

Glutamate

AP transferred

Exp.

1

Exp.

2

0 0.9 0

0

2.4

1.6

0

0

4.0

2.2

Each tube contained the following in poles: Tris-ClpH 100; MgS04, 20; acetyl phosphate, 10; ADP, 10 and the indicated amount of carbamyl phosphate synthetase (Raijman and Grisolia, 1961). Tubes from experiment #l contained also 50 units of hexokinase and 50 poles of glucose (Mokrasch et al, 1960). 10 min incubation at 38O. 1.0 ml final volume. The above findings were confirmed in other experiments by measuring ATP formation (Mokrasch et al. 19601, and with rat liver CP synthetase. There 7.4,

was no demonstrable AP formation with the above system when AP was replaced by 100 poles of K acetate and the ADP by ATP, even when including 100 umoles of NH20H.

Vol. 7, No. 5, 1962

BIOCHEMICAL

and Grisolia, present

1960;

findings

mechanism of

containing

supplemented not

documented

0.6% the

rate

respectively, other

than

in Table ornithine

with

1958).

further

II,

the

It

appears

understanding

here with

However,

show that CP for

AP is

a substrate

transcarbamylase

ornithine.

indicating the

to

et al,

RESEARCH COMMUNICATIONS

that

the

of the

of CP synthetase.

As illustrated tions

Metzenberg

may serve

action

AND BIOPHYSICAL

rat that

AP is liver,

when they

parallel

frog

present

preparaare

experiments

used at about liver

AP utilization

transcarbamylase

with

is

12,

3 and

and bacteria due to

an enzyme

in the preparations.

e

TABLE II ORNITHINE RF,QUIREMENT FOR ACETYL PHOSPHATE UTILIZATION

WITH

ANIMAL AND BACTERIAL PREPARATIONS

poles

Enzyme source

and poles

Ap used

Ornithine Added 0 10

Rat Liver

Frog

Liver

Bacteria

0

0

0

4.1

7.4

5.4

Each tube contained the following in junoles: Tris-Cl-pH 7.4, 50 (100 for the experiments with frog liver enzyme); acetyl as indicated. 0.8 mg phosphate, 10; MgS04, 20 and L-ornithine protein from frog liver (Raijman and Grisolia, 1961), 0.9 mg rat liver mitochondria (Grisolia and Cohen, 1951), and 1.0 mg of bacterial ornithine transcarbamylase (Caravaca and Grisolia, 1960) was used when indicated. The incubation volume was 1.0 ml. phosphate utilization was measured as 10 min at 38O. Acetyl indicated in Table I. ,359

Vol. 7, No. 5, 1962

BIOCHEMICAL

The present utilization

findings

of AP for

The possibility acetylated

that with

demonstrate synthetic

aminoacids

evidence

demonstration the

recognition

case

for

AG and CP synthesis.

catalytic,

in for

enzymes

it

is

of AP synthesis

only

aspartate

other

is negative

However,

by CP synthetase.

brain other

of

for

purposes

AP by animal

our preliminary

the

AND BIOPHYSICAL

acetyl

with

first

ornithine

remains

open.

Indeed,

to

cofactor, the

presence

may be metabolic

aminoacids.

may be As indicated,

speculate

tissues

the

enzymes.

synthesis

attractive animal

time

animal

regarding

in

there

the

than

an unsuspected

suggests

RESEARCH COMMUNICATIONS

of AP that

may await as has been

the

of acetyl roles,

perhaps

e

REFERENCES Caravaca, J., and Grisolia, S., J. Biol. Chem., 235, 684 (1960). Grisolia, S., and Cohen, P. P., J. Biol. Chem., 191, 189 (1951). Grisolia, S., Caravaca, J., and Joyce, B. K., Biochim. et Biophys. Acta, 29, 432 (1958). Grisolia, S., Harmon, P., and Raijman, L., Biochim. et Biophys. Acta, in press (1962). Lipmann, F., and Tuttle, L. C., J. Biol. Chem., 159. 21 (1945). Metzenberg, R. L., Marshall, M., and Cohen, P. P., J. Biol. Chem., 233. 1560 (1958). Mokrasch, L.,C., Caravaca, J., and Grisolia, S., Biochim. et Biophys. Acta, 37. 422, (1960). Raijman, L., and Grisolia, S., Biochem. and Biophys. Research Communs ., & 262 (1961). Slade, H. D., and Slamp, W. C., J. Bact. 64. 455 (1952).

360