A non-functional arginine biosynthetic pathway in polyoma infected-mouse embryo cells

Vol. 47, No. 5, 1972

BIOCHEMICAL

A NON-FUNCTIONAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ARGININE

BIOSYNTHETIC

IN POLYOMA INFECTED-MOUSE

Division Department

Received

April

EMBRYO CELLS

R. A. Consigli,'

A. L. Winters,'

PATHWAY

and Q. R. Rogers

of Biology, Subdivision of Molecular Biology and Genetics Kansas State University, Manhattan, Kansas and of Physiological Sciences, School of Veterinary Medicine University of California, Davis, California

24, 1972

The normal pathway for the biosynthesis of arginine was non-functional in polyoma infected-mouse cells. During viral replication, equimolar concentration of citrulline substituted for arginine but equimolar or ten times equimolar concentration of ornithine did not replace arginine. Purified polyoma virus obtained from infected cultures that were allowed to incorporate citrulline-14C revealed upon amino acid analysis that the label was found exclusively as arginine-14C. When the experiment was repeated using ornithine-14C the label was found as proline-14C and glutamate-14C and not arginine. Both normal and polyoma infected-mouse embryo cells possessed an active ornithine carbamoyltransferase. The inability of ornithine to serve as an arginine precursor appears to be due to the fact that exogenous ornithine is utilized by ornithine-&transaminase to synthesize proline and glutamate and not by ornithine carbamoyltransferase to synthesize citrulline. Successful virus

(5),

medium. the

infection

SV40 (6) It

has been

inhibition

the reason(s) cultures 1

2

of tissue

and polyoma

virus

suggested

(5,7,8)

of an arginine-rich for

have not

cultures

th$ strict

(7)

by herpes requires

that

arginine

been demonstrated.

Thus,

adeno-

in the maintenance

deprivation

protein

requirement

(l-4),

arginine

arginine

structural

virus

effected

of the virus.

of virus

infected

an investigation

However, tissue

of arginine

Recipient of an NIH Predoctoral Fellowship: 5-Fl-GM-30,342. address is: Department of Microbiology, School of Medicine, Pennsylvania, Philadelphia, Pennsylvania.

His present University of

Recipient of USPHS Career Development Award: ported by a grant from the USPHS: CAo7139.

work

Copyright01972,by

Academic Press,Inc.

1044

CA12056.

This

was sup-

BIOCHEMICAL

Vol. 47, No. 5, 1972

biosynthesis to gain

in polyoma

a fuller

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

virus-infected

understanding

mouse embryo

of the

arginine

MATERIALS Primary

mouse embryo cell

(MEM) medium

containing

penicillin,

200 units/ml;

Experiments

were

adsorption

of virus

in Eagle's

medium

on occasion

virus

cultures

that

1 pc/ml

medium

used for

were

(sp.

was purified

virus

from

with

6N HCl (nitrogen

HCl,

the sample

using

a three

amino

acids

column cell

meter

Ornithine

by suspending

acid

standard

with

the harvested the cells

cells

for

After

days. (9).

removal

The

of the

acid

analyzer

exchange

anthracene.

spectrometer

which

meter

recorder, for

The

was

recorder thus

was set

facilitating

quantitation

of

14 C and arginine-

citrulline2.1.3.3)

Cell

activity free

in Tris-acetate

in a Sorvall

five

was hydrolyzed

the ion

crystalline

cultures.

or

of individual

from

The factor

with

Schwarz/Mann)

120 B amino

effluent

(OTC; EC No. infected

calf

infected

supplemented

gradient

19 hr.

The rate

peak.

from

Radioactivity

analyzer

After

fetal

described

density

Beckman

scintillation

using

and polyoma

and homogenizing

for

the

packed

carbamoyltransferase in normal

chloride

and recorder.

as the amino

was determined

as previously

system.

of each radioactive

radioactivity

10 nmoles)

in a Packard

medium

SchwarzfMann)

by passing

mg/ml.

maintained

5% dialyzed

5 mcfmmole,

at 121'

cell

were

8 mcfmmole,

buffer

a 2.0 ml flow

cultures

act.

cesium

0.01

confluent.

was purified

Eagle's

in a modified

gradient

to a rate

identification

prepared

second

was determined

to the same speed

determined

the

antibiotics:

or ornithine.

analysis

C (sp.

with

appeared

containing

and quantitated

was analyzed

was monitored

connected

act.

atmosphere)

step

through

acid

14

the

citrulline,

in Eagle's

and kanamycin,

cultures 3 hr,

in complete

of DL-ornithine-5

virus

purified

with

maintained

L-citrulline-ureido-14C Polyoma

the

arginine)

amino

and maintained

0.2 mg/ml;

for

or without

grown

serum and supplemented

(100 p.f.u./cell)

serum supplemented Polyoma

calf

72 hr after

(with

requirement.

were

streptomycin,

initiated

was performed

AND METHODS

cultures

5% fetal

cultures

Omnimixer

was lysates

buffer for

14 C.

were

(pH 8.0, 0.5 min

Vol. 47, No. 5,1972

BIOCHEMICAL

at maximum rheostat

setting.

and the supernatant

assayed

taining

1.0 ml of lysate;

0.1 Pmoles

The homogenate for

of cold

citrulline

was centrifuged

OTC activity.

25 pmoles

MgC12 was incubated

by addition

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TCA (5% final)

The 2.0 ml assay

L-ornithine;

at 37"

1500 x g for

for

15 pmoles

60 min.

carbamyl

The reaction

and the supernatant

mixture

fluid

10 min con-

phosphate;

was terminated assayed

for

(10). RESULTS AND DISCUSSION

In viral for

arginine

replication whereas

equimolar equimolar

ornithine

did

not

ornithine

are

constituents

interest

to allow

replace

concentration

or

ten times

arginine

(Fig.

equimolar

1).

in the arginine

infected

5.d

cells

to separately

20 HOURS

Since

I

substituted

concentrations both

biosynthetic

I 0

of citrulline

incorporate

citrulline pathway, citrulline-

I

40

60

AFTER

INFECTION

Fig. 1. Growth of polyoma virus in Eagle's medium. M, complete medium; H, medium without arginine; O------O, medium with 0.6 mM L-citrulline substituted for arginine; @------ @, medium with 0.6 mM or 6.0 mM L-ornithine substituted for arginine. 1046

of and it

was of 14 C

Vol. 47, No. 5, 1972

BIOCHEMICAL

and ornithine-14C if

the label

acid

was ultimately

analysis Table

Amino

and to analyze

of these 1.

the purified

acid

acid

viral

analysisa

Moles

preparations of purified

acid

progeny

to determine

arginine.

revealed polyoma

per 100 Moles

range aspartic

polyoma

as 14 C labeled

incorporated

purified

Amino

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

that

Amino radioactive

virus. % total

radioactivity

average

8.4-10.9

10.0

threonine

9.5-10.4

9.9

serine

5.2-8.3

6.5

7.7-9.6

8.7

7.1C

proline

8.5-10.6

9.6

74.4c

glycine

7.9-10.9

9.3

alanine

2.6-5.1

4.1

valine

8.9-9.2

9.0

glutamic

acid

cystine

trace

methionine

trace

isoleucine

4.3-4.9

4.7

leucine

8.6-9.4

8.9

tyrosine

trace

phenylalanine

2.2-2.8

2.4

lysine

8.5-10.4

9.7

histidine

1.5-2.9

2.1

ammonia

not

arginine

4.7-5.7

tryptophan

not

a

average

b virus

of three was grown

c;;~;;e;;ofrYP determined.

amino

calculated 5.1

100b

determined

acid

analyses

in L-citrulline-ureido-

14 C labeled

medium

in DL-ornithine-5-14C labeled medftm. The lack of 100% C input counts was probably due to CO2 which was not 1047

Vol. 47, No. 5, 1972

incorporation

from

exclusively amino

with

acid

analyses

obtained

and Kass

(12),

tyrosine

content

this

14 C label

the

(74.4%) did

not

were except

14 C labeled

was not

acid

any unusual

similar

1).

low

preparation

arginine,

but

The amino

acids.

acid

In general,

by Murikami

tyrosine

of the polyoma

associated

However,

viral

with

reported

the consistently

may be characteristic

(Table

(7.1%). amino

to analyses

was found

purified

associated

and glutamic

reveal

for

cultures

peak of the analysis

of the ornithine-

proline virus

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

14 C labeled

citrulline-

the arginine

that

with

of polyoma

the

analysis

demonstrated marily

BIOCHEMICAL

analysis the

et al.,

content. virus

pri-

(11)

The low

strain

used

in

investigation. The above data

catalyzes

the

may be lacking pqlyoma

(Fig.

synthesis

1, Table

1) raised

of citrulline

in the polyoma

infected-mouse

from

the possibility ornithine

infected-mouse

embryo cells

were

OTC which

and carbamyl

embryo cells. then

that

assayed

for

phosphate

Normal OTC activity

20.0

i

IS.0\f “0 c 10.0z= z3 E 5.0 i 2

(1

S

I2 HOURS

I5

IO AFTER

21

24

30

0.0

INFECTION

Fig. 2. Ornithine carbamoyltransferase in normal and polyoma infected-mouse O----O infected; M normal; p.f.u.

1048

27

activity embryo cells. and O------O

and over

BIOCHEMICAL

Vol. 47, No. 5, 1972

a 30 hr multiplication infected

cycle.

cells

possessed

enzyme activity

observed

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

It was found

OTC activity

that

both

2).

The three-fold

(Fig.

9 hr post

infection

normal

and polyoma increase

can not be explained

in

at this

time. The reason

for

the failure

to be at the intracellular OTC appeared thesize acid

via

but

ornithine

that

of arginine

infectious

progeny

non-functional required

arginine

it

is

pathway

phosphate

into

one for

Apparently,

of ornithine total

since

suppression

might

result

phosphate

arginine

of

in a synthetase

biosynthesis.

in eucaryotic

pyrimidine

14).

cells

This

that

two carbamyl

biosynthesis

and one for

the arginine

biosynthetic

(15 - 21).

concluded

from

the data

is non-functional a stringent

of carbamyl

was demonstrated

existed,

biosynthesis

It

ducing

carbamyl

source

which

and glutamic

and endogenous

(13,

medium produced

may be due to a lack

since

synthetases

biosynthesis

possibility

to syn-

was demonstrated

ornithine

an intracellular

An alternate

(7).

channeling

may be a reality

lack

It

exogenous

arginine

from maintenance

pathway

for

phosphate

cells

pathway.

proline

appears enzyme

was not used

to synthesize

handled

system

The initial

ornithine

(OTA)

to OTC for

in this

level.

was utilized

the OTA pathway

infected-mouse

the removal

exogenous

6 transaminase

was channeled

arginine

(ornithine)

since instead

the ornithine

in Neurospora

polyoma

substrate

non-functional

citrulline

to synthesize

presented

in polyoma

requirement

for

that

infected-mouse exogenous

embryo

cells,

thus

pro-

arginine.

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