Detection of the feedback control of pyrimidine biosynthesis in slices of several rat tissues

BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS

Vol. 55, No. 4, 1973

DETECTION

OF

THE

BIOSYNTHESIS

Peter

FEEDBACK IN SLICES

C. S m i t h * * ,

Received

OF

PYRIMIDINE

SEVERAL

RAT

TISSUES*

C h a r l o t t e E° K n o t t , a n d G e o r g e C. T r e m b l a y

Department University

OF

CONTROL

of B i o c h e m i s t r y

of R h o d e I s l a n d , K i n g s t o n , R h o d e I s l a n d

October

02881

31,1973

S U M M A R Y : M e a s u r e m e n t s of t h e r a t e of i n c o r p o r a t i o n of l a b e l e d p r e c u r s o r s into o r o t i c a c i d in s l i c e s of s e v e r a l r a t t i s s u e s r e v e a l t h a t t h e r a t e of i n c o r p o r a t i o n of b i c a r b o n a t e , b u t n o t c a r b a m o y l p h o s p h a t e o r c a r b a m o y l a s p a r t a t e , is s i g n i f i c a n t l y i n h i b i t e d b y t h e a d d i t i o n of u r i d i n e to t h e r e a c t i o n mixture. T h e s e e x p e r i m e n t s d e m o n s t r a t e t h e o p e r a t i o n of a f e e d b a c k c o n t r o l m e c h a n i s m g o v e r n i n g t h e r a t e of p y r i m i d i n e b i o s y n t h e s i s in t h e i n t a c t cell and provide evidence that the reaction catalyzed by carbamoylphosphate s y n t h e t a s e is t h e s i t e of e n d - p r o d u c t i n h i b i t i o n . We have recently developed a simple and reliable method for isolating o r o t i c a c i d w h i c h h a s b e e n s y n t h e s i z e d d e n o v o f r o m l a b e l e d p r e c u r s o r s within tissue slices.

This method permitted

r e g u l a t i o n of p y r i m i d i n e b i o s y n t h e s i s d e t e c t e d in t h e i n t a c t c e l l .

u s to t e s t d i r e c t l y w h e t h e r t h e

by end-product

inhibition couid be

U s i n g t h e r a t e of i n c o r p o r a t i o n

into o r o t i c a c i d in t i s s u e s l i c e s a s a m e a s u r e

of N a H 1 4 C O 3

of t h e a c t i v i t y of t h e o r o t a t e

p a t h w a y , w e f o u n d t h e a d d i t i o n of u r i d i n e t o t h e r e a c t i o n m e d i u m t o a p p r e ciably inhibit the de novo biosynthesis experiments

of p y r i m i d i n e s .

In t w o s e p a r a t e

d e s i g n e d t o t e s t t h e s e n s i t i v i t y of t h e o r o t a t e p a t h w a y in l i v e r

slices to feedback control,

we observed an average

i n h i b i t i o n of 40% a t

3 mM uridine and a maximum

i n h i b i t i o n of 65% at 10 m M u r i d i n e ( F i g .

Considering

of u r i d i n e ,

that a metabolite

rather

than uridine itself,

1).

is m o r e

l i k e l y t o b e t h e a c t u a l i n h i b i t o r , t h e s e d a t a s u g g e s t t h a t t h e a c t i v i t y of t h e o r o t a t e p a t h w a y is s e n s i t i v e t o s m a l l p e r t u r b a t i o n s more

in t h e p o o l s i z e of one o r

of i t s e n d - p r o d u c t s .

*This work was supported by a grant (AM-15186) from the National Institute of A r t h r i t i s a n d M e t a b o l i c D i s e a s e s . * * P o r t i o n s of t h i s w o r k w a s d o n e in p a r t i a l f u l f i l l m e n t of t h e r e q u i r e m e n t s f o r t h e d e g r e e of D o c t o r of P h i l o s o p h y in B i o l o g i c a l S c i e n c e s .

Copyright © 1973 by Academic Press, lnc. All rights of reproduction in any form reserved.

1141

Vol. 55, No. 4, 1973

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

r.., (..) (..)

7-

ii0

m

O O

IO0

O I-

t

_z~

90

@

~_- 1z- 0 7 0 ~,o~° 6 0 0 :E 0

5O

I-

4O

0 EL n." 0 (J

30

I

I

I

5

o

I0 CONCENTRATION

URIDINE

15 (rnM)

FIGURE 1. THE EFFECT OF URIDINE ON THE INCORPORATION OF NaHI4co3 INTO OROTIC ACID IN SLICES OF RAT LIVER-Slices of 500 mg of rat l[ver were incubated at 37.5°C for 3 hrs in 20 ml of Krebs Improved Ringer II Solution (I) adjusted to 30 mM in NaHI4co3 (300 pCi) and made I0 mM in 6-azaur[dLne to inhibit the further metabolism of orotic acid (2). Each reaction was terminated by homogenization in an equal volume of icecold IN HCIO 4 and the precLpitate removed by centr[fugat[on. The acidsoluble fraction was neutralized with KOH, the precipitate of KCIO 4 removed by centrifugat[on, and the supernatant fluid dtluted with water to 50 ml to which 375 mg carrLer monosodium orotate was added and dissolved at the boiling point. The 14C-orotate synthesized during the incubation period was isolated by co-crystallization with carrLer orotate as the solution cooled slowly to 4°Co Aliquots of 25 mg of crystals were dissolved in 2 ml 0.25N KOH and diluted with 6.5 rnl of Aquasol (New England Nuclear Corp. ) to make a gel for measuring the content of radioisotope in a liquid scintillation spectrometer. The crystals so obtained were recrystall[zed from water to constant specific activity, which rarely required more than two recrystall[zations. The data from two separate experiments are presented.

We orotate

were

pathway

further

~nterested

and its regulation

by feedback

~n slices of extrahepatic

tLssues°

complement

eatalyzLng

of enzymes

Ln slices of several

extrahepatLc

the liver in TABLE

I.

in determ~nLng

Evidence

whether

control

could also be detected

for the occurrence

the de novo biosynthesis

tLssues

In this experiment

1142

the act[vLty of the

is compared

of the entire of orot[c acid

with that obtained

the activity of the orotate

for

pathway

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 55, No. 4, 1973

in the liver was about 4 times that observed in the kLdney and 1/5 and 1/2 that observed in the spleen and lactating m a m m a r y Evidence that the metabolite of N a H I 4 c o 3

gland, respectively (Column I).

which eo-crystallLzes w~th carrier

orotate during our isolation procedure is, ~n fact, 14C-orotate was obtained by eompar[ng the quant~t.y of radioisotope Lsolated wLth carrier orotate before and after treatment of the acid-soluble fractLon of the reaction mLxtures with phosphoribosylpyrophosphate ( P R P P ) and the mixed e n z y m e s O M P pyrophosphorylase and O M P verts orotate to U M P .

deearboxylase, the combination of which con-

M o r e than 80% of the metabol~te of N a H I 4 c o 3

isolated

f r o m all four tissues with carrier orotate was r e m o v e d by treatment of the acid-soluble fraetLons with P R P P

and the mixed e n z y m e s prior to co-crystal-

lization (Column III); however, only a small loss, probably at the expense of endogenous P R P P ,

w a s observed w h e n P R P P

was not added during treat-

m e n t wLth the m i x e d e n z y m e s (Column II). These data establish the LdentLty of the metabolite of N a H I 4 c o 3

which ~s isolated by eo-erystall~zatLon with

carrier orotate as 14C-orotate. W h e n the activity of the orotate pathway was tested for sensitivity to end-product inhibition, w e observed the addition of uridLne to inhibit the ~ncorporat~on of N a H I 4 c o 3 (TABLE

into orot[c acid in all four tissues exar~ned

If). In order to localize the site of feedback inhibition, w e

m e a s u r e d the ~nfluenee of ur[dine on the ~neorporat~on of sequential ~ntermediates [nthe de novo biosynthesis of orotLc acid° T h e additLon of ur~dLne appeared to cause a slight inhibition of the ~neorporation of 14Cearbamoylphosphate ~nto orotie aeLd only in liver slices. N o s~gnLf~eant effect of uridine on the incorporation of ureido-14C earbamoylaspartate into orot[e ae[d was observed ~n any of the four tissues studied.

T h e possibility

that ur~d~ne acts by antagonizLng 6-azaur[d~ne, thereby preventLng the a c e u m ulatLon of orot[c aeid, was excluded by repeating the experiments in the absence of 6-azauridLne.

Although the quantity of 14C-orotate isolated under

these conditions was less than that observed in the presence of 6-azauridLne, s~gnLf~cant inh[bLt~on of the incorporation of N a H I 4 C O 3

into orot~e aeLd was again

detected in all four tissues (3). These data are in agreement with a prevLous eommun[eation f r o m our laboratory reporting end-produet LnhLbLt~on of the incorporation of N a H I 4 C O 3

~nto orotie acid ~n liver slLees, but they are at var[-

1143

Vol. 55, No. 4, 1973

BIOCHEMICAL AND BIOPHYSICAL RESEARCHCOMMUNICATIONS

TABLE

PYRIMIDINE

BIOSYNTHESIS

EXTRAHEPATIC

I

IN SLICES TISSUES

OF

OF

LIVER

THE

AND

RAT

I n c o r p o r a t i o n of N a H 1 4 C O 3 I n t o O r o t i c A c i d Tissue

I n moles*

II cpm+

cpm+

III loss

cpm+

(%)

Ioss

(%)

Liver

9.3

896

720

20

68

92

Kidney

2.6

252

237

6

38

85

Spleen

44.0

4232

3951

7

613

86

Mammary Gland

20.5

1972

1868

5

338

83

* n a n o m o l e s NaH14CO3 i n c o r p o r a t e d into orotic a c i d / g i n t i s s u e • hr + c p r n / 2 5 m g o r o t a t e i s o l a t e d u p o n r e e r y s t a l l i z a t i o n to c o n s t a n t s p e c i f i c a c t i v i t y . The r e a c t i o n c o n d i t i o n s w e r e the s a m e as t h o s e d e s c r i b e d in the l e g e n d to F i g . 1 e x c e p t t h a t t h e c o n t e n t of r a d i o i s o t o p e w a s d o u b l e d to 600 p C [ and m a m m a r y glands w e r e m i n c e d r a t h e r than sliced. E a c h n e u t r a l i z e d a c i d - s o l u b l e f r a c t i o n w a s f r e e z e - d r i e d , r e c o n s t i t u t e d in 6 . 5 m l of w a t e r , a d j u s t e d to pH8 a n d d i v i d e d into 3 a l i q u o t s of 2 m l e a c h . One a l i q u o t w a s d i l u t e d w i t h 0. 80 m l w a t e r ( C o l u m n t), the s e c o n d w i t h a n e q u a l v o l u m e of s o l u t i o n c o n t a i n i n g 3 u n i t s of the m i x e d e n z y m e s O M P p y r o p h o s p h o r y l a s e and O M P d e c a r b o x y l a s e ( S i g m a C h e m i c a l Co. ), ( C o l u m n II)0 a n d the t h i r d a l i q u o t w i t h a n e q u a l v o l u m e of s o l u t i o n c o n t a i n i n g 3 u n i t s of t h e m i x e d e n z y m e s a n d P R P P to g i v e a f i n a l c o n c e n t r a t i o n of 5 m M ( C o l u m n III). T h e a l i q u o t s of the r e a c t i o n m i x t u r e s w e r e t h e n i n c u b a t e d f o r 4 h r s a t r o o m t e m p e r a t u r e a n d t h e r e a c t i o n s t e r m i n a t e d b y h e a t i n g in a b a t h of b o i l i n g w a t e r for 5 minutes. The insoluble matter was removed by centrifugatton, washed w i t h w a t e r , a n d the w a t e r - w a s h a d d e d to the s u p e r n a t a n t f l u i d s w h i c h w e r e t h e n d i l u t e d to 50 m l w i t h w a t e r a n d s a t u r a t e d at the b o i l i n g p o i n t w i t h 400 m g of c a r r i e r m o n o s o d i u m o r o t a t e . T h e 1 4 C - o r o t a t e w a s i s o l a t e d f r o m e a c h d i l u t i o n b y c o - c r y s t a l l i z a t i o n w i t h c a r r i e r o r o t a t e a s the s o l u t i o n c o o l e d s l o w l y to 4 ° C . T h e c r y s t a l s so o b t a i n e d w e r e r e c r y s t a l l t z e d f r o m w a t e r to constant specific activity.

ance with the e a r l i e r data i n d i c a t i n g a s p a r t a t e c a r b a m o y l t r a n s f e r a s e

to b e a

s i t e of f e e d b a c k i n h i b i t i o n (4); n o n e of u s h a s b e e n a b l e to d e t e c t i n h i b i t i o n of t h e i n c o r p o r a t i o n of 1 4 C - c a r b a m o y l p h o s p h a t e

into o r o t t c a c i d b y the a d d i t i o n

of u r i d i n e to a n y e x t e n t b e y o n d t h e m a r g i n a l v a l u e s p r e s e n t e d in T a b l e II of this report.

1144

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 55, No. 4,1973

TABLE FEEDBACK

II

INHIBITION OROTIC

ACID

OF

IN SLICES

Liver

Precursor

THE

BIOSYNTHESIS OF

RAT

Kidney

OF

TISSUES

Spleen

NaH14CO3, 30 rnM, 300pC[ cprn/25 rng o r o t a t e n m o l e s / g m t i s s u e . hr [nhLbitLon by u r [ d i n e (%)

n=ll 700 8.4~0.9 72 ~- 2.7

n=6 475 5.7~-0.6 56-+6.5

1 4 C - C P , 5.8 mM, 5.3 ~Ci cprn/25 m g o r o t a t e n m o l e s / g i n t i s s u e , hr Lnhib[tion by urtdine (%)

n=5 854 111-+14 14~2

n=3 885 115-+5 6±5

n=3 915 119 :~ 13 iI~4

14C-CA, 5 raM, 4.3 ~C[ cpm/25 mg orotate n m o l e s / g m t i s s u e , hr inhibition by ur[d[ne (%)

n=3 6336 887 ~- 26 5~-2

n=3 3500 490 ±40 4=L3

n=3 1121 157 ~: 13 -6~:9

*Experimental

conditions

altered

as described

n=8 3833 46 -+3.9 75-+1.8

in the legend

Mammary Gland*

n=8 738

I 16-+2.3 47-+1.4 n=3 284 6657 3f6 n=3 2329 782 :~ 123 _5-+5

below.

Experimental conditions and procedures were the same as those described in the legend to Fig. 1 except that radioactive intermediates in the incorporation of bicarbonate into orotie acid were substituted for NaHI4CO 3 where indicated and the initial concentration of urLd[ne was i0 rnM. In the experiments employing mammary glands, the glands were excised at various stages of lactation, tissue minces were employed [nplace of tissue slices, and the following modifications were made: NaHI4CO3, 15 mM (50ttC[); 14C-carbarnoylphosphate (14C-CP), 5 mM (I. 53 PC0; ureido-14C-earbamoyl aspartate (14C-CA), 5 rnM (I. 1 t~C[); the incubation period was extended to 4 hr; and the quantity of radioisotope contained in aliquots of 50 mg orotate was determined following the recrystallLzat[on of 600 mg carrier orotate from 75 rnl of the neutralized acid-soluble fraction of the incubation mixture. Values are given as averages ~ the standard error with the number of observations (n) entered at the head of each column of data.

We

interpret the data presented in this communication to establish the

occurrence of the complete c o m p l e m e n t of e n z y m e s catalyzing the de novo bLosynthesLs of orotic acid in three extrahepatic tissues and to demonstrate the operation of a feedback control m e c h a n i s m in the intaet cell which governs the rate of pyrim[dine biosynthesis by end-product inhibition of carbamoylphosphate synthetase°

This interpretation is consistent with reports of the

1145

Vol. 55, No. 4, 1973

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

inhibition of partially purified preparations

carbamoylphosphate appreciable aspartate

synthetase by UTP

end-product

of the glutamine-dependent

(5, 6) and the failure to detect any

inhibition of cell-free extracts of mammalian

carbamoyltransferase

(7-9).

REFERENCES i.

2. 3. 4. 5. 6. 7. 8. 9.

R.M.C. Dawson in Data for Biochemical Research, R.M.C. Dawson, D.C. Elliott, W.H. Elliott, and K.M. Jones (Eds.), Oxford University Press, New York and Oxford, 2nd Edition, 1969, p 507. Handschumacher, R.E. and Pasternak, C.A., Biochim. Biophys. Acta, 30, 451 (1958). SmLth, P.S., Knott, C.E., and Tremblay, G.C., unpublished observations. Bourget, P.A. and Tremblay, G.C., Biochern. Biophys. Res. Commun., 46, 752 (1972). Tatibana, M. and Ito, K., J. Biol. Chem., 244, 5403 (1969). Levine, R.L., Hoogenraad, N.J., and Kretchmer, N., Biochemistry, i0, 3694 (1971). Curci, M.R. and Donachie, W.D., Biochim. Biophys. Acta, 85, 338 (1964). Bresnick, E. and Mosse, H., Biochem. J., I01, 63 (1966). Koskimies, O., Oliver, I., Hurwitz, R., an(i Kretchmer, N., Biochem. Biophys. Res. Cornrnun., 42, 1162 (1971).

1146