J. Nutr.

Effect

of Dietary

Protein

Sci.

Vitaminol.,

37, 517-528,

1991

on Pyrimidine-Metabolizing

Enzymes

in Rats

Masae KANEKO, Shigeko FUJIMOTO, Mariko KIKUGAWA, Yasuhide KONTANI, and Nanaya TAMAKI* Laboratory of Nutritional Chemistry, Faculty of Nutrition, Kobe-Gakuin University, Nishi-ku, Kobe 651-21, Japan (Received February 25, 1991) Summary

The

enzymes

was

dehydrogenase free

diet

effect

studied

were

significantly

was

of ƒÀ-ureidopropionase.

unaffected.

increased

the

depended

of

liver,

also

hand,

a

diet

amount

of

and

given

decreased

the diet

activities

of ƒÀ-

aminotransferase) ((R)-3-amino-2which

by

a protein

high-protein

The

protein

fed

dihydropyrim

in

are the

injection

present

diet. did

enzymes

in

mi

Ammonium not

affect

the

(dihydropyrimi

dihydropyrimidinase, ƒÀ-ureidopropionase, ƒÀ-alanine and

Dietary but

of

(aminobutyrate

aminotransferase

notransferase).

(5%) other

pyrimidine-metabolizing

dehydrogenase,

- oxoglutarate

rats

activity

of ƒÀ-ureidopropionase.

the

the

liver

dihydropyrimidine

of

aminotransferase

on in

rat

the

aminotransferase),

supplemented

activities

the

the

of

livers

deficiency

D-3-aminoisobutyrate-pyruvate

ions

the

while

aminotransferase

methylpropionate-pyruvate tochondria,

pyrimidine-metabolizing

activities in

On level

on

The

Protein

alanine-oxoglutarate

dine

protein

rat.

decreased,

activity

and

dietary

the

and ƒÀ-ureidopropionase

idinase

(60%)

of in

did

uridine

not

affect

D-3-aminoisobutyrate-pyruvate

resulted the

in

the

activities

of

accumulation

ami of

uracil

in

pyrimidine-metabolizing

enzymes. Key

Words

dihydropyrimidine

dehydrogenase,

ureidopropionase, ƒÀ-alanine-oxoglutarate isobutyrate-pyruvate

dihydropyrimidinase, ƒÀaminotransferase,

aminotransferase,

D-3-amino

pyrimidine

The de novo biosynthesis of uridine monophosphate (UMP) involves six enzymatic reactions and appears to be encoded by only three structural genes in animals. The active sites of the first three enzymes, carbamoyl-phosphate synthase, aspartate transcarbamylase, and dihydroorotase, are on a single large polypeptide *

To

whom

correspondence

Abbreviations: ƒÀ-AlaAT - aminoisobutyrate-pyruvate

should

be

addressed

I, ƒÀ-alanine-oxoglutarate

. aminotransferase; ƒÀ-AlaAT

aminotransferase.

517

II,

D-3

518

M. KANEKO

that

aggregates

the

fifth

to

and

form

sixth

orotidylate

the

enzymes

decarboxylase,

- biosynthetic and

the

atectomy, On

post-natal growing

is

by

with other

time

and

of

are to

transported

acetyl-CoA

aminotransferase (ƒÀ-AlaAT

mitochondrial

matrix

1.

Degradation

[EC

1.3.1.1];

3.5.1.6];

4,

pathway 2,

(9-11).

aminobutyrate

dehydrogenase

I),

pyrimidine.

rat

aminotransferase aminotransferase 1.2.1.27].

of

6).

Weber after

et

time

and

in

after with

rapidly

to ƒÀ-alanine

and ƒÀ-

, dihydropy

in Fig. 1. ƒÀ-Alanine (8 , 9), where they

semialdehyde

ami dehydrogenase

in has

dihydropyrimidine

2 .6.1.40];

Abbreviations: ƒÀ-Ala

been

dehydrogenase

3, ƒÀ-ureidopropionase 2.6 .1.19];

are

by ƒÀ-alanine

dehydrogenase

[EC

(7) hep

increased

, respectively, D-3-aminoisobutyrate-pyruvate

[EC

al.

partial

over

CO2

dehydrogenase

3.5.2.2];

(4) UMP

triphosphate

livers to

, as ilustrated mitochondria

1, [EC

[EC

in

novo

uridine

hepatectomy

Dihydropyrimidine

of

(5,

metabolized

methylmalonate

dihydropyrimidinase

methylpropionate-pyruvate semialdehyde

and

sites

chain de

propionyl-CoA

(ƒÀ-AlaAT II)

are

into

and

active

and

the

inceased

thymidine

partial

cytosol

in by

DNA

dihydropyrimidine in

The

polypeptide

inhibition

of

after

by

single

decreased

thymine

.

enzyme

into but

inactivated and

a

1-bisphosphate

catabolism

Uracil

of

feedback

thymidine

and ƒÀ-ureidopropionase

notransferse

-

site

(1-3)

phosphoribosyl-transferase

rate-limiting

growth,

respectively,

- oxoglutarate

Fig.

the

the

was

metabolized

, orotate

5-phosphoribose

hand,

and ƒÀ-aminoisobutyrate

the

the

protein

composed

is

of

(7).

aminoisobutyrate,

further

also

hepatoma

the

neoplasms

rimidinase

pathway

the

incorporation

and

birth.

and

activation

that

multienzymatic

of

synthetase pathway

(UTP) found

native

are

Carbamoyl-phosphate

et al.

5, 6,

[EC

(R)-3-amino-2 methylmalonate , ƒÀ-alanine; ƒÀ-

AIB, ƒÀ-aminoisobutyrate.

J Nutr.

Sci.

Vitaminol.

.

PROTEIN

LEVEL

ON

PYRIMIDINE

METABOLISM

519

identified as the rate-limiting enzyme of pyrimidine catabolism (12, 13). In the spectrum of hepatomas, dihydropyrimidine dehydrogenase activity decreased in parallel with increased growth rate (7, 14). In the livers of rats fed a protein-deficient diet, DNA synthesis and activity of thymidine kinase decreased to less than half of control values (15). In contrast, incorporation of [6-14C]-orotate into RNA and uridine kinase activity significantly increased (15). Ammonium ions have been shown to stimulate pyrimidine bio synthesis as a result of carbamoyl phosphate synthesis by mitochondria carbamoyl phosphate synthetase (16-18). In order to define the relevance of normal and abnormally increased uracil for hepatic pyrimidine catabolism, we investigated the effect of dietary protein, ammonium ions and uridine on pyrimidine-catabolizing enzymes in the rat liver. MATERIALS

Chemicals. from

All

Nacalai

Sigma

Chemicals.

uracil

by

Ltd., Animals.

Male

under fed

to

ammonium

casein

shown

M and

was

used

-

in

of hour assays:

10vol.

after After Vol.

to

of

5,6-dihydro from

obtained

from

centrifugation, 5, 1991

for

(12h

the

the

body

New

Oriental

cycle).

libitum

and and

housed

with

for

in

50%

The

1 week

environment.

selected

hu

animals

before

the

Acclimatized

separated

noon

compositions

animals

uridine

except

and

dissolved

weight the

was last

(10mM

2.5mM 1min,

and

20g

the

rats

into

groups.

those

used

of

low

diets

and

feed

ammonium

uridine

per

All in

the

in

saline

to

high-protein

day.

acetate

is shown

in

physiological

intraperitoneally

injection,

the

then

animals

injection. Table

The

2.

The

20%

After

was was

livers

7.4,

centrifugation, to

4•Ž. adjusted

neutralized

The to with

prepared or

were

to

3.7

at

1-h

the

4.85 0.5M

homog

containing

5mM

supernatant

precipitate pH

8 times

sacrificed.

The pH

be 4

were

phosphate,

cooled

supernatant

supernatant

injected

dehydrogenase. potassium

MgCl2). and

the

of

received

diets,

dihydropyrimidine

the

were

23•}1•Ž

control.

after

and

at light-dark

ad

new

were

chloride

A

130-150g)

water

The

The

was

and 50•Ž

centrifugation

37, No.

1.

as

buffer

mercaptoethanol heated

from

experiments.

and

acetate

One

Enzyme enized

purchased a product

purchased

were

maintained

10:00a.m.

diets.

ammonium

3.7mol/kg

intervals.

Table

chloride

Ammonium

to

gain

protein

in

of

diet

were was

synthesized

diets

room

and

between

injection

high

Ammonium composition

diet

weight

acetate

a

them

sacrificed

and

are

and

Uridine

was

animals'

conditions

stock

acclimatize

were

Low

grade

stated.

(Sprague-Dawley, in

lighting

progressive

animals

analytical

was

the

rats cages

commercial

experiment

diets

for

albino

controlled

a

showing

of

otherwise

(19). ƒÀ-[2-14C]alanine

Materials

screen-bottom

midity

were

Tokyo.

individual

were

unless

bromination

Nuclear.

Yeast

used

(Kyoto)

METHODS

5-Bromo-5,6-dihydrouracil

direct

England

chemicals

Tesque

AND

was with KOH

2 was

discarded

5% and

acetate. treated

520

M.

Table

1.

Compositions

a Sucrose

e corn

c

80;

D-biotin

Table

0.066;

(in

IU/kg

folic

2.

NaCl

diet);

4;

10

diet):

CaHPO4•E2H2O 21,054;

,000;

menadione 16;

calcium

of

104;

pantothenate

100;

KI

cholecalciferol

0

1908;

0.018;

thiamin

cyanocobalamin

8 .736;

Fe-citrate

CuSO4•E5H2O

acetate

100;

hydrochloride

acid

g/kg

Ca-lactate 0 .072;

retinyl

acetate

chloride

(in

2 .796;

MnSO4•E4-6H2O of

pyridoxine

0.4; choline

were

5,610;

diet): ƒ¿-tocopheryl

riboflavin

120;

ZnCO3

were

mg/kg

b Minerals

NaH2PO4

4.302;

Vitamins (in

diets .

starch=2

15.432;

MgSO4

et al.

of the experimental

:1. KH2PO4

KANEKO

0.006.

2,000,

and

hydrochloride

.01;

24;

ascorbic

p-aminobenzoic

acid

acid

100;

600; niacin

4,000.

Compositions

of

the

diets

supplemented

with

ammonium

chloride

and

uridine.

1

Diets

used

for the experiments

experiments vitamin

with

on the

mixture

ammonium

dissolved

in

effect

are shown

sulfate.

the

mM

50mM

NADPH

in

of

gation,

phosphate, the

buffer

potassium a total

at

of

The pH

supernatant

7.0,

pH

livers

used

used for the mixture and

obtained

at

30-50%

for

enzyme

used was

(20) 7 .4,

followed

.

The

including

by

saturation analysis measuring

standard

. the

assay

0.15mM

was

uracil

rate

mixture and

0.15

3 .0ml. were

containing was

ions . 2 Diets b Salt

starch=2:1.

and

37•Ž

phosphate, volume

A activity

NADPH

Dihydropyrimidinase. sium

of

corn

1.

precipitate

volume

of ammonium

a Sucrose:

dehydrogenase

disappearance

contained

.

in Table

The

a minimum

Dihydropyrimidine of

on the effect

of uridine

homogenized

10mM for

analyses

in

2-mercaptoethanol of

10vol .

.

10mM

After

dihydropyrimidinase

J. Nutr.

potas centrifu and

Sci.

,ƒÀ-

Vitaminol.

PROTEIN

ureidopropionase

LEVEL

activity

was

was

- 5,6-dihydrouracil

at

pH

of

8.2,

in

in

the

rate

tained

of

0.1M

10mM

was

used

to

The

activity

min

at

37•Ž.

was

immediately

2ml

0.2%

stand

at

The

for

with of

Protein

al.

II except

were

using

acid.

KOH.

Uracil

gradient

from with

a

groups. 37, No,

5, 1991

were

C set

at

analysis. a

40ƒÊM

of

malonate to

methods

sodium

borate 1mM ƒÀ-

2-oxoglutarate

0.5ml

2M

for

and

the

30 tube

1M ƒÀ-alanine

mixture

the

a final

bath

HCl

0.02ml the

in

water

was

formed

and

allowed

was

to

extracted

radioactivity

Tri-Garb

were

of

liquid

by a 2-ml

scintillation

significant

a

analytical

by

after

method acceptor.

the

the

rats

method

homogenized

HPLC

to

in

(Waters,

a 20%

sliced

5vol.

of

with

linear

0.6 4M

methanol

methanol

5ƒÊm,

sac

The

neutralized

using 5.6,

were

razor.

was

pH

column

above

amino by

stainless-steel and

buffer,

the

the

determined

supernatant

separated

as

a standard.

removed

using

to

used

was as

quickly

the

according was

weighed,

phosphate 18

assayed

albumin

manually

potassium

reverse-phase

When

phos

supernatant

amount

50mM

adding

concentration

centrifugation,

uridine

was

37•Ž.ƒÀ

and The

a shaking

HCl)

pyruvate

freeze-clamped,

and

wavelength Statistical

was

serum

livers

sliced

After

50mM

in

a Packard

10mM

bovine

The

then

immediately

M perchloric

activity that

Protein

(24),

were

at

potassium

5•L-phosphate,

centrifugation,

with

30min

according

adding

After

brief

N-carbamoyl-ƒÀ-

for

the

l0mM

out

2M

con (0.1%),

type).

assays.

they

and

bath.

2mM

centrifuged.

dinitrophenylhydrazone

measured

measurement. et

Analytical

Vol.

CD

I activity

Lowry

The

ice

mixture

10mM

pyridoxal

by

respect

albumin

bath

contained

carried

terminated

After

was

reaction serum

2-oxoglutarate

GBq/mol)

with

activities.

mixture

was

the

II. ƒÀ-AlaAT

for ƒÀ-AlaAT

min,

and

measured

by

0.5mM

an

The

was

2-mercaptoethanol

II

(in

toluene.

extract (460

ƒÀ-AlaAT

tissues

15min

5.0ml the

spectrometer

rificed;

to

37•Ž.

5-bromo-5,6

10vol.

briefly

with

2,4-dinitrophenylhydrazine 37•Ž

shaking

transferred

at

0.17mM

and

determined

reaction

37

was

path

The

5-bromo

and

water

2mM was

was

incubation

reaction

(21). of

3,24•~103M-1•E

standard

in

EDTA,

I

et al.

was

bovine

I and ƒÀ-AlaAT

activity

The

The

a shaking

from ƒÀ-alanine

(specific 1.0ml.

8.2

including

in

1mM

The

light 225nm

2-mercaptoethanol,

2-mercaptoethanol,

[2-14C]alanine of

(22). 7.0,

homogenate

(23).

5mM

1.0cm

pH

Brooks

disappearance

at

homogenized

of ƒÀ-AlaAT

of of

activity

out

were

The

produced

8.8),

a

buffer

5mM carried

livers

described

volume

of

EDTA,

analyze ƒÀ-AlaAT

semialdehyde

aliquot

pH

containing

5•L-phosphate.

(pH

521

3.0ml.

ammonia

The

pyridoxal

previously

of

of

was

7.5,

method rate

with

Tris-HCl

phosphate,

1mM

I.

pH

cuvette

the the

. ƒÀ-Ureidopropionase

Incubation

phate,

a

volume

formation

-AlaAT

by

measuring

in

50mM

a total

sodium

MgCl2,

alanine.

METABOLISM

5-bromo-5,6-dihydrouracil

ƒÀ -Ureidopropionase to

by

225nm

coefficient

- dihydrouracil

measured

assessed

at

extinction cm-1,

PYRIMIDINE

activities.

Dihydropyrimidinase enzyme

ON

for

40

0.39•~15cm).

254nm. One-way difference

analysis

of (p