Feedback regulation in the ribopyrimidine “salvage” pathway

Experimenlal Cell Research 41, 338-349 (1966)

338

F E E D B A C K REGULATION IN THE R I B O P Y R I M I D I N E "SALVAGE" P A T H W A Y 1 A. ORENGO Stazione Zoologica, Naples, Italy

Received June 29, 1965z THE feedback by which an enzyme catalyzing an early step in a reaction sequence has its activity modulated by intermediates or end products of the sequence [19, 28] m ay prove to be an important mechanism for regulating cell metabolism generally. The immediate precursors of DNA and RNA-the nucleoside t r i p h o s p h a t e s - - a p p e a r to be involved in such regulatory mechanism controlling the rate of their own synthesis [2, 3, 5-7, 9-11, 13-15, 17, 20, 22-241. In addition, the triphosphates impinge upon other metabolic pathways not directly involved in nucleic acid synthesis, and appear to serve also as regulators of these pathways [8, 16, 21, 271. Thus the nucleic acids, apart from their established role in the storage and flow of genetic information, m a y also have to be considered in the role of sequestering agents of these (allosteric) regulators [4]. T h e y would then be having a passive influence on cell metabolism and perhaps cell differentiation bringing about activation or inhibition of metabolic pathways. Developing embryos of the sea urchin P a r a c e n l r o t u s lividus are k n o w n to incorporate added cytidine and uridine into RNA and DNA [18]. As shown by Anderson and B r o c k m a n Ill the formation of nucleotide derivatives by phosphorylation of cytidine and uridine in extracts from acetone powders of tumor cells is inhibited by U T P and CTP. The aim of this work is to find out whether in developing embryos of P a r a c e n t r o t u s lividus the level of cytidine and uridine is controlled by a feedback regulation. A uridine-cytidine kinase has been partially purified from the same material. Evidence is presented that the activity of this enzyme is regulated by UTP, CTP, GTP and dGTP. Kinetics studies of these regulations are to be described elsewhere. EXPERIMENTAL

PROCEDURE

Materials and Melhods

Eggs of Paraeentrotus lividus were collected, fertilized and the embryos grown as described by Tocco el al. [26]. 1 "Salvage" pathway indicates the formation of nucleotides from bases and nucleosides. 2 Revised version received September 30, 1965. Experimental Cell Research 41

Feedback in ribopgrimidine "saloage" palhu,ay

339

The nueleosides and nucleotides used were products of P a b s t or Sigma Chemical Co. ~4C-Cytidine (U.L.) and ~4C-2-uridine were purchased from t h e R a d i o c h e m i c a l Center, A m e r s h a m , B u c k i n g h a m s h i r e , England. ~4C-Cytidine-5"-monophosphate (U.L.) was obtained from Schwarz BioReseareh, Inc., U.S.A.

Preparation of the enzyme The em b r y o s were collected at the end of gastrulation or at the pluteus stage by low speed eentrifugation in a Sharples super centrifuge. A f t e r two washes w i t h 8 vol of 3.7 per cent NaC1, t h e y were suspended in 2 vol of 0.1 M tris-HC1 buffer p H 7.2. The suspension was h o m o g e n iz e d in a P o t t e r - E l v e h j e m h o m o g en i zer with a m o t o r - d r i v e n pestle. F o r e x p e r i m e n t s with crude e n z y m e p r e p a r a t i o n the h o m o g e n a t e was centrifuged in a refrigerated I n t e r n a t i o n a l centrifuge at 24,000 • 9 for 45 min, and the s u p e r n a t a n t was used as enzyme source. For t h e p r e p a r a t i o n of the purified e n z y m e the h o m o g e n a t e was centrifuged in a Spinco p r e p a r a t i v e u l t r acen t r i f u g e for 30 rain at 105,000 9 and th e nucleic acids were r e m o v e d from t h e s u p e r n a t a n t b y p r e c i p i t a t i o n with s t r e p to m y c in . 45 ml of 5 per cent s t r e p t o m y c i n solution per each 100 ml of e x t r a c t were ad d ed over a period of 20 nfin with continuous stirring. The suspension was left o v e r n i g h t in a refrigerator. A f t er centrifugation in the Spinco at 105,000 x 9 for 15 rain t h e s u p e r n a t a n t was b r o u g h t to 33 per cent s a t u r a t i o n with a m m o n i u m sulphate. Solid a m m o n i u m sulp h a t e was added at a rate of 1 g/rain with continuous stirring. The suspension was centrifuged for 15 rain and the precipitate was discarded. The s u p e r n a t a n t was t h e n b r o u g h t to 38 per cent s a t u r a t i o n with a m m o n i u m sulphate and allowed to stand for 1 hr. The precipitate was collected as before, t h e n dissolved in tris buffer (onet e n t h of the original volume). The fraction is referred to as first a m m o n i u m sulphate t r e a t m e n t or F r a c t i o n III. To each 10 ml of e x t r a c t 1.53 g of a m m o n i u m sulphate were added and after 30 min under stirring, the suspension was eentrifugated. To t h e s u p e r n a t a n t more a m m o n i u m sulphate was slowly added: 392 mg per each 10 ml of fluid. A ft er centrifugation, the precipitate was dissolved in one t w e n t i e t h of its original v o l u m e of tris buffer. The fraction is referred to as second a m m o n i u m sulphate t r e a t m e n t or Fraction IV. The procedure was carried out at 0-2~ The R.C.F. values are r e p o r t e d for n m x i m u m radii. Enzymalic assays. The s t a n d a r d assay m i x t u r e (final v o l u m e 60 /~l) contained: 5 /~moles of t r i s - m a l e a t e - t ( O H buffer p H 7.35, 200 m/~moles of MgC12, 400 m/,moles of A T P , 53 m/ramies of x4C-cytidine (U.L.) or a~C-2-uridine (specific a c t i v i t y , 1 ttC/ /,mole) and 0.015 to 0.060 units of enzyme. An enzyme unit in the a m o u n t of protein which phosphory]ates one /mmle of uridine or eytidine to uridylic or cytidylic acid in 1 hr, at 37:C under the conditions of the assay. A t intervals 25 / d aliquots of t h e reaction m i x t u r e were t a k e n in duplicate and subjected to descending c h r o m a t o g r a p h y on a I ) E A E cellulose paper ( W h a t m a n n No. I ) E 20). P ap er strips 2.9 /, 14 cm were used and the samples were applied as spots at 5 cm from one end, t o g e t h e r with 0.1 /mmle uridine or cytidine as carriers. The solvent used was ethanol 85 per cent. Whereas the nueleosides m o v e with the solvent and are found near the front, 23 - 661801

E.rperimenlal Cell Research41

A. Orengo

340

the nucleotides remain at the origin. These were located by a u.v. lamp; the spots were cut out, placed in aluminum planchets and counted to a 5 per cent statistical error in a Tracerlab windowless flow counter. By this procedure 98 per cent of cytidine and 98.6 per cent of uridine are washed out while 100 per cent of the phosphorylated derivatives remain adsorbed to the ion exchange paper. The purified enzyme preparation was also assayed for its nucleotide monophosphate kinase, cytidine deaminase and CMP nucleotidase activities. Larger volumes (300 pl)of the incubation mixture were used and ATP was omitted when cytidine deaminase and CMP nucleotidase activities were measured. The dephosphorylation of nucleoside monophosphates was measured using 14C-cytidine-5"-monophosphate (U.L.) as substrate. The reaction mixture was incubated at 37~ After 30 min the samples were instantaneously frozen and 400 m/~moles of the appropriate carrier were added. Sixty #1 aliquots were applied in duplicate to a Whatmann paper No. 3 M and subjected to electrophoresis in 0.05 M citrate buffer pH 3.4 for the eytidine derivatives and in the same buffer pH 6.2 for the uridine derivatives at a potential gradient of 38 volts per cm. The buffer pH 3.4 was also used for separating cytidine from uridine in the assay of cytidine deaminase. Under these conditions, cytidine migrates toward the cathode while uridine remains on the origin. The spots were located with an ultraviolet lamp, cut out, immersed in a scintillation solution (4 g of 2,5-diphenyloxazole and 100 mg of 1,4-bis-2-(4-methyl-5-phenyloxazolyl) benzene per liter of toluene) and counted in a Packard Tricarb liquid scintillation counter. For the phosphatase assay the chromatographic procedure described for uridine and cytidine kinases was used. The ratio: radioactivity of the nucleoside over that of the nucleotide represents the percentage of CMP dephosphorylated under the conditions of the assay. Protein was determined by the method of Lowry el al. [12].

RESULTS

AND

DISCUSSION

Uridine kinase activity at various stages of embryonic development of Paracenlrolus lividus. T h e u r i d i n e - e y t i d i n e kinase is present in the ripe unfertilized eggs. T h e rate of p h o s p h o r y l a t i o n of uridine at v a r i o u s stages of e m b r y o n i c d e v e l o p m e n t is r e p o r t e d in T a b l e II. After fertilization a s h a r p increase of the specific activity is o b s e r v e d followed b y a progressive decrease w h i c h r e a c h e s its m i n i m u m v a l u e at the stage of the m e s e n c h y m e blastula. During the f o r m a t i o n of the pluteus the activity increases again.

Properties of the purified enzgme.--The p r e p a r a t i o n o b t a i n e d f r o m e m b r y o s of Paracenlrotus lividus is able to p h o s p h o r y l a t e cytidine a n d uridine. As s h o w n in Fig. 1 the specific activity of uridine kinase is one a n d half times higher t h a n cytidine k i n a s e w h e n the e m b r y o s are h o m o g e n i z e d at the gastrula stage. This is true for b o t h c r u d e a n d partially purified e n z y m e p r e p a r a tions, a l t h o u g h the specific activity of the e n z y m e is 40 times higher as

Experimental Cell Research41

Feedback in ribopgrimidine "sah,age" p a t h w a y compare(1

w i t h t h a t of t h e e t u d e

does not dealninate

cyfidine or de|)hosl)horylate

of t h e n u c l e o s i ( i e k i n a s e a s s a y . S m a l l a l n o u n t s of n u e l e ( ) s i ( l e enzyme

to

UI)P,

u s e d as s u b s t r a t e , and

m()nophosphate

enzyme

CMP

under

kinase

preparation

the con(litions

are l)resent

in t h e

preparation.

A f t c r 30 r a i n of i n c u b a t i o n , verted

extract. The purified

341

whereas

no

0.7 p e r c c n t of t h c U M P UTI ) couhl

9 p e r e e n ! ot' t h e C M P s y n t h e s i z e d

o n l y 1.7 h) C T P .

These

aclivities cannot

tions since the rate measurements

synthesized

be detected.

of u r i ( t i n c

When

was con-

eyti(line was

w a s c o n v e r t e ( t to C D P

interfcrc with our determinacyfidine kinase werc madc

over

a p e r i o d of 5 ()r 1() l n i n of i n c u b a t i o n .

TA I~I,v: I. Purifica lion o[' uridim,-c,qtidin e kin ase.~ 1 hilts Fraction and slep

pei' ml

Total

Protein rag/nil

1.13

113

16.6

1.37

236

.

1. Supernatant extract II. After treatment with streptomycin III. First ammonium sulphale treatment IV. Second ammonium sulphate treatment

.

.

Specific activity units/ mg protein 0.068 .

~. ~

88.2

8.66

0.96

6.0

31.6

2.24

2.68

a Uridinc was used as substrate. T A B L E I I . l'ridine l~'inase aclivil!t al various slages o[" embryonic developmenl.

l'mbryonic slag(,

Time after fertilization hr

Unfertilized egg 1-2-cell stage I latching blastula Mesenchyme blastula Gastrula [,ate gastrula I 'risma l'luteus Pluteus

0 1.15 10 13 16.30 22.30 35.30 40 58.30

Specific activity ttpmoles UMP/ rain/rag protein 520 1590 900 700 80O 960 1780 1660 1020

Experimental Cell Beseareh 41

A. Orengo

342 1.5

~ t

.

~

U

M

P

/f:

1.0 d

o 0.5

, ~0

20 ~9

, 80

proceln

Fig. 1 . - - E n z y m a t i c formation of UMP and CMP from uridine and cytidine respectively. The assay procedure used was the same as described in Table III.

As for m e t a l r e q u i r e m e n t s m a l l differences w e r e f o u n d b e t w e e n cytidine a n d u r i d i n e kinase. Both k i n a s e r e a c t i o n s are d e p e n d e n t on A T P a n d Mg 2§ Mn 2§ c a n r e p l a c e Mg 2§ to a certain extent. W h e n Ca 2§ w a s p r e s e n t as the o n l y d i v a l e n t cation, the p h o s p h o r y l a t i o n of u r i d i n e o c c u r r e d at a r e d u c e d r a t e (20 p e r cent) while that of cytidine w a s a l m o s t nil. Na + a n d K + s e e m not to be required (Table Ill). TABLE I I I .

Requirements [br reaction.

The complete s y s t e m (60 pl) contained: 5/~moles of t r i s - m a l e a t e - K O H p H 7.4, 200 mpmoles of MgC12, 400/~moles of ATP, 53 mpmoles of 14C-2-cytidine (U.L.) or 14C-2-uridine and 20 #g of protein (fraction IV).

System Complete Complete +200 m#moles of Mn ~+ Complete + 200 mFtmoles of K + C o m p l e t e + 2 0 0 m/~moles of Na + Omit A T P Omit Mg 2+ Omit Mg~ + 200 m/~mo]es of Mn 2+ Omit Mg ~+ +200 re#moles of Ca ~+ Omit Mg 2+ + 200 m~moles of Na § Omit Mg'-++200 m~moles of K +

Experimental Cell Research 41

CMP formed m~moles/hr 14.8 12.8 14.0 15.0 0.0 0.0 7.4 0.4 O.0 0.0

U M P formed m/~moles/hr 24.9 20.0 27.1 25.1 1.7 0.0 17.5 4.7 0.0 0.0

Feedback in ribopgrimidine "salvage" pathway

343

Is the kinase reaction catalyzed by a single or by two i n d e p e n d e n t enzymes? By adding labeled cytidine in its usual a m o u n t along with increasing a m o u n t s of unlabeled uridine, anti vice versa, if there is a single e n z y m e a competition w o u l d l)e ,,bserved between the two substrates present. The results obtained s h o w a (Icerease of the labeled nucleotidc formed; they do not show, however, whether there is a single binding site for the substrates. It is worth noting that the e n z y m e shows a higher affinity for uridine than for cytidine (Table IX').

TABLI,; IV. Formalion of labeled CMP or UMP in the presence of cohl uridine

or cglidine respectively. T h e a s s a y p r o c e d u r e u s e d w a s t h e s a m e as d e s c r i b e d in T a b l e III. 46.6 ttg of p r o t e i n ( F r a c t i o n IV) were used. ~4(; CMP

formed m / m m h , s/lu"

Addi[ ion

None

67.5 135 270 337

m,amoles m/~moles re#moles n)/mmles

of of of of

laC-nridine a2C-nridine ~2C-uridine J2C-uridinc

16.25 13.15 5.51 1.3~ 0

Addition

None 45 m t o n o l e s 90 ln/zmolcs 180 m/~moles 270 m/*moles

of of of of

a2C-eytidine ~2C-eytidine azC-eytidine ~C-eytidine

a4C U M P formed m/*moles/hr

71.37 3260 23.32 11.69 8.20

q'm~l.E V. Specificily of the tdmsphale donor. T h e a s s a y p r o c e d u r e u s e d w a s the s a m e as d e s c r i b e d in T a b l e l l I e x c e p t t h a t A T P w a s rep l a c e d b y 400 m / t m o l e s of the i n d i c a t e d n u c l e o s i d e t r i p h o s p h a t e s .

1)hosphate d o n o r

None ATP dATP (;'FP

dGTP

CMP fornle(i m/gmoles/hr

() 14.8 11.2 3.8 8.1

dCTI )

0

(ITTI) dUTI )

6.,l 11.0

UMP formed m/gmoles/hr

0.1 17.5 14.6 3.8 20.9 1.0

6.9 18.0

Experimenlal Cell Research 41

A. Orengo

344 100

100

\ \

8 "~ so

"6 50 o

4-

o!i

o!3rnM CTP~0.5 ~

0.7

0.9

0!i

I

01,3

Fig. 2.

0,5 mM.UTP

I

0,7

1.25

Fig. 3.

Fig. 2 . - - T h e effect of C T P on t h e uridine (open circles) a n d cytidine (filled circles) k i n a s e activities. T h e a s s a y p r o c e d u r e used was t h e s a m e as described in Table III. Fig. 3 . - - T h e effect of U T P on t h e uridine (open circles) a n d cytidine (filled circles) kinase activities. T h e a s s a y p r o c e d u r e used was t h e s a m e as described in Table III.

TABLE VI.

Effecls of nucleoside triphosphates.

T h e a s s a y procedure used w a s t h e s a m e as described in Table III. To t h e s t a n d a r d i n c u b a t i o n m i x t u r e v a r i o u s nucleoside t r i p h o s p h a t e s were a d d e d at a final c o n c e n t r a t i o n of 1.25 • 10 -4 M . T h e m i x t u r e c o n t a i n e d 276/~g of F r a c t i o n I (a) or 20/~g of F r a c t i o n IV (b). CMP f o r m e d

UMP formed ^

Compound None dATP GTP dGTP CTP dCTP UTP dUTP dTTP

(a) m~tmoles/5 m i n

(b) m/~moles/hr

ia) m~moles/5 min

(b) mftmoles/hr

1.42 1.40 1.78 1.89 0.12 1.42 0.75 1.33 1.56

3.77 4.05 3.42 4.16 0 3.94 2.00 3.60 4.33

2.5 2.59 3.30 3.07 1.13 2.61 1.67 2.34 2.7

6.38 6.33 7.67 7.39 2.02 7.0 4.37 7.39 6.05

Experimental Cell Research 41

Feedback in ribopgrimidine "salvage" p a t h w a y Sk6ht also found of both

uridine

that one enzyme

anti cytidine

was responsible

using a puritied

Spe<'ific=ily of the ldmsphale donor.--Thc (co-substrales)

showetl further T*m,E VII.

345

for the phospht)rylation

l)reparation

from

tumor

specificity of the phosphate

differences between

('ells

(h)n()rs

the kinase reactions.

For

li~hibition by pyrimidine derivatives.

The assay procedure use(l was the same as described in Table III. 30/tg of Fraction IV were used. (iMP formed f--

Compound added None CTI ) 1.25 "-: 10 CDP 1.25 :< 10 (iMp 1.25. 10 UTP 1.25 :- 10 1;DI' 1.25 : It) I ; M P 1 . 2 5 . 10

4M 4 5I a,l/ 4M ~ 5I 4M

('TP

SM

1.48

10

UMP formed

.-~

%

m/tmolcs/hr

100.0 6.8 23.0 1()0.o 57.0 63.0 90.(}

%

26.56 1.80 6.23 26.73 15.22 16.74 23.81

66.(}

100 8.2 23.0 88.0 58.0 53.0 89.0

17.'15

CTP 2.97 .: 10 5 M 47.5 CTP 5.94 :,:10 -5 M -UTP1.5 - 10 -531 100.0 UTP 3.05 .: 10 -5 M 98.5 UTP 6.1 : 10 5 3I 95.5 CTt' 1 . 4 8 + U T P 1.5 xlO 5M 60.() (66) CTP 2.97 +:UTP 3.05:< 10 5M 39.0 (17) C T P 5 . 9 1 + U T P 6.1 .'10 5_3I -

m/tmoles/hr 40.32 3.31 9.21 35.49 23.25 21.45 36.00

--

12.58 -26.56 26.10 25.31 15.89 10.40 --

-

52.0 27.0 92.0 84.5 74.1) -35.0 (-14) 17.0 (20)

-

20.84 10.73 37.10 3.1.00 29.80 -14.26 6.78

The numbers in parentheses arc the inhil)itions predicted by summating the fractional inhibitions.

the cytidine

kinase reaction,

On the contrary, speciticity. while

UTP

V and

ATP

In bt)th reactions behaves

VI couhl

is m o r e

for the phost)horylation r

seems

as a potent

indicate

sub-site) of the enzyme

that

active than

dATP,

dUTP,

r

of uridine dGT1 ) shows the highest

inhibitor.

to b e a n The

excellent co-substrate

values

as for the co-subs(rate,

reported

in Tables

the binding

site (or

is n o t a b l e to d i s t i n g u i s h b e t w e e n t h e r i b o a n d d e o x v -

ribo moiety of the triphosphates. A specilicity, however, binding

f o r t h e r i b o m o i e t y s e e m s to e x i s t a s r e g a r d i n g

the

s i t e ( o r s u b - s i t e ) <>f t h e i n h i b i t o r s .

Experimental Cell Research41

A. Orengo

346

Feedback inhibition by CTP and UTP.--As shown in Fig. 2, CTP as the end product of the "salvage" pathway of the ribopyrimidine nucleosides is an efficient feedback inhibitor of the uridine and cytidine phosphorylation. Fifty per cent inhibition was obtained using 5 • 10 -~ M CTP and 8.8 x 10 _4 M of uridine and cytidine. The sensitivity of the enzyme towards UTP is different. The regulation of the cytidine kinase reaction by UTP is less sensitive 10

o~o~

/

o--

10

,..,.,..~o~-

e + ' ~ C T P , 0.225 mM

/

d o~ control a~ c~

ef

+ UTP, 0.76 mM

/

E

l 1

I 3 ram dGTP

Fig. 4.

t

I

I

S

1

2 mM dGTP

Fig. 5.

Fig. 4 . - - R e v e r s a l of CTP i n h i b i t i o n b y d G T P . C y t i d i n e w a s us e d as s u b s t r a t e . The a s s a y proc e dur e u sed w a s t h e same as described in T a b l e I I I w i t h t h c e x c e p t i o n t h a t a n e x t r a c t from P l u t e i 46 h r old was used. The p r o t e i n c o n t e n t was 240/~g per each a s s a y. Fig. 5 . - - R c v e r s a l of U T P i n h i b i t i o n b y d G T P . U r i d i n e was used as s u b s t r a t e . The a s s a y p r o c e d u r e u s e d was t h e s a m e as described in T a b l e ] I I w i t h the e x c e p t i o n t h a t a n e x t r a c t from P l u t e i 46 h r old was used. The p r o t e i n c o n t e n t was 240 ~tg per each as s a y.

and it seems to be effective only above a threshold value. This effect was not evident when the kinase was using uridine as substrate (Fig. 3). UMP and CMP did not inhibit, whereas UDP and CDP however, did (Table VII). This is perhaps due to a nucleoside diphosphate kinase contamination. It seems that the entire molecule of the inhibitors is necessary for the inhibition. Experiments were carried out to determine whether a cooperative inhibition could be observed by comparing the inhibitions observed when (at equimolecular concentrations) UTP and CTP are present together or one at time. As m a y be seen in Table VII the inhibitions produced were always higher than those obtained by summating the inhibitions caused by Experimental Cell Research 41

Feedback in ribopgrimidine "salvage" pathway

347

e a c h i n h i b i t o r singly. T h i s suggests the possibility of two distinct sites for the b i n d i n g of the e n z y m e . Reversal <7["the inhibition by GTP and d(;TP.--lq'onl T a b l e VI it c a n be seen that a m o n g the v a r i o u s n u c l c o s i d e t r i p h o s p h a t e s used, (YFP a n d d G T P are able to e n h a n c e the rate of p h o s l ) h o r y l a t i o n of u r i d i n e anti cytidine u n d e r the d e s c r i b e d a s s a y p r o c e d u r e . T h i s ell'cot w a s m o r e evident when the c r u d e e n z y m e l)rel)aration was used. W h e n two l)y two c o m b i n a t i o n s of the nucleoside t r i p h o s p h a t e s were tested, it a p p e a r e d that (Y['I ~ a n d d(YFP c o u n t e r a c t the inhibition by UT1 ~ in the cytidine anti u r i d i n c k i n a s e reactions, d G T I ~ releases the ( ' T P inhibition in the cytidine reaction. Both the C T P i n h i b i t e d cytidine k i n a s e reaction anti the U T P i n h i b i t e d u r i d i n e k i n a s e r e a c t i o n were measurehosphates were not effective w h e n tested with purilied e n z y m e ( F r a c t i o n IV). ()n the basis of the results o b t a i n e d the l n a i n t e n a n c e ot' the level of the r i l m l ) y r i m i d i n e nucleosides in sea u r c h i n emt)ryo can I)e exl)lained as follows. T h e lirst e n z y m e ()f [he " s a l v a g e " I>athway of the rit>ol)yrimittinc nucleosi(les is sensitiw' to two r e g u l a t o r y c h e m i c a l signals. ()he of I h e m is inhihitory a n d m a k e s i>ossit)le Ihc m a i n t e n a n c e <)f tt fixed level of 1.1TP a n d C T P in the cellular sap. W h e n the hvo ribonuch, otide t r i p h o s p h a t e s accum u l a t e as a c o n s e q u e n c e o[' a d e c r e a s e d synthesis of RNA a n d I)NA, or b e c a u s e of a d e c r e a s e d c a r t m h y d r a l e a n d / o r lipid m e t a b o l i s m , the u r i d i n e cytidine k i n a s e is blocked, a n d therefore the entire p a t h w a y will be shut d o w n . T h e other regulah>ry signal is r e p r e s e n t e d b y G T P anti d G T P , two of the e n d p r o d u c t s of the p u r i n e biosynthetic p a t h w a y s , w h i c h are able to c o u n teract the inhibition of the r i b o p y r i m i d i n e n u c l e o s i d e t r i p h o s p h a t e s . T h i s p r o p e r t y of the s y s t e m m a y be used to m a i n t a i n a b a l a n c e b e t w e e n the p u r i n e a m l p y r i m i d i n e n u c l e o s i d e t r i p h o s p h a t e s a n d to p r e v e n t the cell f r o m a deticiency of i n t e r m e d i a t e s as UI)I ~ a n d / o r CI)P. This is outlined in Text-

lig. 1. Experimental (;ell Resetu'ch41

348

A. Orengo

Finally the physiological significance of the "salvage" pat hw ay during the early embryonic development of sea urchin m a y be discussed. In sea urchin DNA increases from 20-30 • 10 -6 #g in unfertilized egg up to 7100 • 10 -e ~g in 40-hr-oht embryo while during the same period the total RNA remains constant. However, the RNA of the different subcellular fractions is not meta[.___

; ............ CO I + N H = / /

UR

/

Other pathways

~ , UMP

~ UDP

,'

/ I

/

~ UTP

.,

ii

-~

~. CTP .

CDP.

I ii

,' /

RNA

/

,7 7

CMP ~ C R

, -,.,, /

/ ii

dGTP . . . . . .

,; .... 7 .... f .............. ii DNA iI Other pathways

I~

1

i

Text-fig. 1.--Feedback regulation in the ribopyrimidine "salvage" pathway.

bolically inert. The most striking event is the increase of nuclear RNA from 1 per cent of the total in unfertilized eggs to 15 per cent in the pluteus stage. Incorporation of ~2I) orthophosphate suggests that the new nuclear RNA m a y be synthesized from the nucleosides derived from the b r e a k d o w n of cytoplasmic RNA [26]. Martin has also shown that a large am oun t of DNA pyrimidine m ay derive from exogenously supplied ribonucleosides. For example, in the transition from the 8- to the 16-cell stage exogenous cyti(tine can supply 45 per cent of the 4.8 • 10 -5 re#moles of DNA pyrimidine synthesized per embryo. F r o m the amo u n t of exogenous cytidine which is incorporated into DNA, an endogenous reservoir of 4 • 10 -4 re#moles in each unfertilized egg was calculated. A pool of this size can support DNA synthesis up to the 128cell stage without the necessity of a de nooo synthesis of pyrimidines [18]. Without excluding the possibility of a de nooo synthesis, the "salvage" p a t hway of the pyrimidine nueleotides can be operative during the early development of the sea urchin embryos to a greater extent than in the other system studied. SUMMARY A uridine-cytidine kinase has been partially purified from an extract of the sea urchin Paracentrotus lividus. It has been shown that the enzyme is under feedback inhibition by U T P and CTP. Th e requirements of the kinase reaction have been investigated. Experimental Cell Research 41

Feedb(wk

The

UTP

the CTP The pathway

inhibition inhil)ition

in ribopgrimidine

(.an be removed of the cytidine

I)rot)al)le physiological of the rit)opyrimi(line

"sr

by GTP

kinase

349

l)alhwag

and

dGTP.

(t(iTl' can remove

reaction.

significance

of such

nucleosi(les

control

in the "sr

in the early emt)ryonic

deveh)p-

l n c n t is d i s c u s s c ( t . T h e a u t h o r is i n d e b t e d t o Prof. J . R u n n s t r 6 m w h o k i n d l y r e v i s e d t h e m a n u s c r i p t , t o Prof. 1". G h i r e t t i a n d A. P a c k a r d for a s s i s t a n c e w i t h it, a n d t o Giulio P e r e z for excellent technical assistance. REFERENCES

l. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22.

23. ')].

25. 26. 27. 28.

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Experimental Cell Research 41