Porasirology Vol 20,No.

lnternationulJournal/or Primed in Great Britain

I. pp. 37-44

1990 0

PYRIMIDINE

OOM7Sl9/90 $3.00 + 0.00 Pergamon Press p/c Socre~y for Parasirology

1990 Australian

SALVAGE PATHWAYS IN ADULT

SCHISTUSOMA MAHMOUD H. EL KOUNI*

MANSONI and FARDOS N. M. NAGUIB

Division of Biology and Medicine, Brown University, Providence, RI 02912, U.S.A. (Received 13 April 1989;accepted 12 August 1989) Abstract-EL KOUNIM. H. and NAGUIBF. N. M. 1990. Pyrimidine salvage pathways in adult Schistosoma

~ansani. internat~anal Jaurnal for Parasitalogy 20: 37-44. Adult Schistosoma mansoni can utilize radiola~lled cytidine, uridine, uracil, orotate, deoxycytidine and thymidine for the synthesis of its nucleic acids. In this respect, cytidine is the most efficiently utilized pyrimidine precursor. Cytosine, thymine and orotidine are transported into the parasites but not metabolized. High performance liquid chromatography analysis of the nucleobase, nudeoside and nucleotide pools from in vivo metabolic studies and assays of enzyme activities in cell-free extracts indicate the presence of nucleoside and nucleotide kinases which phosphorylate the various nucleosides to their respective nucleoside mono-, di- and triphosphates. Uridine, thymidine and deoxyuridine can also be cleaved to their respective nucleobases by uridine phosphorylase. Uracil can be converted directly to UMP by orotate phosphoribosyltransferase or by the sequential actions of uridine phosphorylase and uridine kinase. Nucleoside 5’-monophosphates were dephosphorylated by active phosphohydrolases. Ail enzymes tested were found in the cytosoi fraction with the exception of the phosphohydrolases which were associated mainly with the particulate fraction. No deam~nation of cytosine, cytidine, deoxycytidine, CMP or dCMP was detected either in viva or in vitro. The active metabolism of cytidine and absence of deamination and phosphorolysis of cytidine derivatives in schistosomes raise the possibility of using cytidine analogues for the selective treatment of schistosomiasis. INDEX KEY WORDS: ~ch~sta~a~a m~~onj; pyrimidine; salvage; kinase; deaminase; phospho~bosyltransferase; phosphorylase; phosphohydrolase.

above; Huang et al., 1984; el Kouni, Naguib, Niedzwicki, Iltzsch & Cha, 1988). In the present study, we investigated the in vivo metabolism of pyrimidine nucleobases and nucleosides by S. mansoni and the activities of the enzymes involved in their metabolism in cell-free extracts. Comparison between pyrimidine metabolism in this parasite and that of its host revealed differences which could lx manipulated as possible targets for the chemotherapy of ~histosomiasis.

LIVING organisms

can satisfy their pyrimidine requirements by the de novo pathway and/or the salvage pathways. The de now pathway leads to the synthesis of pyrimidines from simple precursors such as CO,, amide nitrogen of glutamine, ATP and Sphosphoribosyl-lr-l-pyrophosphate (PRPP). All six enzymes of de now UMP biosynthesis were shown to be present in extracts of Schistosoma mansoni by Aoki & Oya (1979), Hill, Kilsby, Rogerson, McIntosh & Ginger ( 198 I), et Kouni, M .H., Niedzwicki, J.G., Lee, K.-H., Iltzsch, M. H., Senft, A. W. & Cha, S. (1983. Abstract in Federation Proceedings 42: 2207), Iltzsch, Niedzwicki, Senft, Cha & el Kouni (1984) and in extracts of S. jff~Qnjcu~ by Huang, Chang & Chen (1984), indicating that schistosomes are capable of de novo pyrimidine biosynthesis. This is in contrast to the inability of this parasite to synthesize purines de novo (Senft & Crabtree, 1983). Enzymes of the salvage pathways are involved in the reutilization metabolism of preformed pyrimidines from either exogenous or endogenous sources. Little information is available on the pyrimidine salvage pathways in schistosomes (el Kouni, M. H., Niedzwicki, J. G., Lee, K.-H., Iltzsch, M. H., Senft, A. W. & Cha, S., 1983, Abstract cited l

MATERIALS AND METHODS Chemicals. [2-‘%Z]Uridine (2.07 TBq mol-‘), 12-“‘Cl thymidine (2.07 TBq-mol- ‘), [2-‘%I deoxyuridine (2.67 TB4 mol-‘1. 12-L4Cldeoxvcvtidine (2.07 TBa mol-‘1. and 17-‘4C1 orotidine (I .85 TBq- n&l’) were obtained from Moravek Biochemicals Inc., Brea, CA; [2-14C] cytosine (2.02 TBq mall ‘), [2-‘%I cytidine (1.89 TBq mall ‘) and [U-14C]CMP (16.65 TBq mall’) from Research Products International Corp.; [2-‘4C] uracil(2.04 TBq mol- ‘). [2-‘“Cl thymine (2.05 TBq mall’), [6-‘4C]orotate (2.08 TBs moi-‘), 12-14C1 dCMP (2.0? TBq mall’), and [2-i‘%?]dUMP (2.07 TBq &i-l), [214C1dTMP (2.07 TBa mol- ‘). and Omnifluor scintillant from New England Nuclear Corp., Boston, MA, silica gel G/ u.v.ss4, and polyethylen~imine cellulose 300 PEI/u.v.,,, thin layer chromatography (t.1.c.) plates from Brinkmann, Westbury, NJ; Bio-Rad protein assay kits from Bio-Rad Laboratories, Richmond, CA; Fischer’s medium was from GIBCO and penicillin/streptomycin mixture from M. A.

‘To whom all correspondence should be addressed. 37

38

M. H. EL KOUNI and F. N. M. NAGUIB

Bioproducts. All other chemicals were obtained from the Sigma Chemical Co.. St. Louis, MO. Source of worms. The life cycle of S. mansoni (Puerto Rico strain) was maintained as described in detail by el Kouni, Diop & Cha ( 1983) and el Kotmi, Messier & Cha (I 987). Live worms were collected by portal perfusion of 4S- to 60-daysinfected female CD-I mice (Charles River Laboratories, Wilmington, MA) infected with approximately 400 cercariae as described previously (el Kouni et al., 1983, 1987). Metabolism of pyrimidine nucleobases and nucleosides by whole worms in vitro. For these experiments Fischer’s medium supplemented with a penicillin (1 unit ml-‘)streptomycin (1 pg ml-‘) mixture and buffered with sodium bicarbonate was used as the perfusing fluid to collect the worms as well as the incubation medium (el Kouni et nl., 1983; el Kouni & Cha, 1987). The worms were captured on nylon mesh (200 mesh), quickly rinsed in situ -and then incubated in Fischer’s medium at 37°C. Those found in copula were transferred in groups of 10 pairs into test tubes containing 1 ml of Fischer’s medium with the [“C]nucleobase or I’~Cl-nucleoside (45 UM z 1.85 TBa mol ‘) and incubated at 3?d. Under these conditions, the uptake of all compounds tested was linear with time for at least 6 h. Worms were then washed three times with normal saline (0.9% NaCl) and homogenized in 0.5 ml ice-cold perchloric acid (4%) (el Kouni er al., 1983; el Kouni & Cha, 1987). The acid-insoluble fraction (nucleic acids) was removed by centrifugation and washed three times with ice-cold 4% perchloric acid and filtered through Whatman GF/A filters. Filters were heat dried in counting vials. Hemoglobin pigment was then decolorized with I ml toluene and 0.5 ml hydrogen peroxide (30%). The vials were left uncapped for at least 5 h in order for the hydrogen peroxide to evaporate. Radioactivity on the filters (nucleic acids) was counted in 10 ml Omnifluor using a Packard 460C scintillation counter. The supernatant fluid (acid-soluble fraction) was neutralized with 50 ~1 KOH (5 N) and 50 ~1 potassium phosphate (OS M, pH 7). Insoluble potassium perchlorate was removed by centrifugation and the supernatant fluid was collected and stored frozen at -20°C until analyzed for nucleoside and nucleotide contents by High Performance Liquid Chromatography (HPLC). HPLC analysis of nucleobase, nucleoside and nucleotide conrents. Nucleobase and nucleoside contents were analyzed using a C,, FBondapack (30 cm x 3.9 mm) column (Waters Ass.) on a Waters LCS III liquid chromatograph system equipped with an on-line Flo-one radioactive flow detector (Radiomatic Instruments and Chemical Co., Tampa, FL). Fifty microliters of the extract was coinjected with 5 ~1 of 1 mM (or higher) standard solution of cytosine, uracil, thymine and their various nucleosides and nucleotides. The column was eluted with water (@- I5 min) followed by 50% methanol in water (IS-30 min) at a flow rate of 1 ml mm’. The eluent was monitored at 254 nm, then mixed with Flo-Stint III (Radiomatic Instruments and Chemical Co, Tampa, FL) at 2 to measure simultaneously the radioactivity ml min-’ (c.p.m.) in the eluent. U.V. absorbance and radioactivity were synchronously recorded and the data were analyzed with a Hewlett-Packard 3390A integrator. When the nucleotide content was analyzed, a Reeve-Angel Partisil-10 SAX (25 cm x 4.6 mm) column (Whatman, Inc.) was used. The column was eluted with a linear gradient (l-500 mM) of potassium phosphate (PH 4.0) for 45 min at a flow rate of 1 ml min-‘. ?ompounds were identified by retention time and coelution with authentic samples. Bufirs. The following buffers were used: Buffer A (20 mMpotassium phosphate, pH 8.0; 1 rnM-dithiothreitol (DTT); I

mM-EDTA) and Buffer B (100 mM-Tris-Cl, pH 7.5; 10% glycerol; 2 mM-D’I”I; 1 mM-MgCl,). Prepararion of extracts. Buffer A was used to determine nucleoside phosphorylase and deaminase activities and Buffer B was employed when the activities of all other enzymes tested were dete~ined. Live S. mansoni were homogenized (1:3 v/v) in the appropriate buffer using a Teflon pestle. In general the homogenate was centrifuged at 105,000 x g for 1 h at 4°C in a Beckman L8-M ultracentrifuge and the supernatant fluid (cytosol) was collected. When the activity of phosphohydrolases was tested the homogenate was first centrifuged at 30.000 x gfor 30 min at 4°C in a Sorvall RCZ-B centrifuge. The 30,000 x g supernatant fluid was recentrifuged at 105,000 x g. The 30,000 x g and 105,000 x g pellets were resuspended in homogenizing buffer and sonicated using a Heat Systems Ultrasonics sonicator. Enzyme assays. All assays were run at 37°C under conditions where activity was linear with time and enzyme concentration. Reactions were started by the addition of extract and stopped, unless otherwise mentioned, by boiling in a water bath for 2 min followed by freezing. Precipitated proteins were removed by centrifugation. In radioactive assays, 5-10 ~1 of the supernatant fluid was spotted on the appropriate t.1.c. plate prespotted with 5-10 ~11from a 10 mM solution of unlabelled carriers and developed in the applicable system. Spots containing substrate and product(s) were visualized by U.V. quenching, cut out and the radioactivity was counted in 20 ml Omni~uor using a Packard 460C scintillation counter. The amounts of substrate and product(s) were calculated on a percentage basis. Pyrimidine nucleoside and nucleotide kinases. Nucleoside and nucleotide kinase activities were determined isotopically as the sum of nucleotides formed from their respective radiola~lled nucleosides or nucleotides. The assay mixture contained 50 m&r-Tris-Cl. DH 7.4. 125 EM-radiola~lled nucleoside or nucleoside 5’-monophosphate (14.8 GBq mol-‘), 2.5 mM-ATP, 5 mM-MgCl,, 10 mM-NaF, and 30 ~1 of cytosol extract in a final volume of 60 ~1. After termination of the reaction and centrifugation, 10 ~1 of the supernatant was spotted on PEI-cellulose t.1.c. plates. The plates were first developed with water to separate the nucleosides which migrate with the front from the nucleotides which remain at the origin. The plates were dried and redevefoped in I .O MLiCl to separate the nucleoside mono- di- and triphosphates from each other. The R, values were; uridine, 6.93; UMP. 0.68; UDP. 0.44: UTP. 0.16: deoxvuridine. 0.85: dUMP. 0.51: dUDP, 0.35; dUTP, 0.131 thymlrdine, 0.93; dTMP, 0.73; dTDP, 0.48; dTTP, 0.14: cytidine, 0.93; CMP, 0.57; CDP, 0.33; CTP, 0.09; deoxycytidine, 0.86; dCMP, 0.46: dCDP, 0.29; dCTP, 0.10. The presence of nucleoside diphosphate kinases was inferred from the formation of nucleoside triphosphates. Uridine phosphorylase. Nucleoside cleavage was measured isotopically by following the formation of nucleobases from their respective nucleosides as previously described (el Kouni et al., 1988). The assay mixture contained Buffer A, 125 FMradiolabelled nucleoside (66.6 GBa mol. ‘) and 30 ul of enzyme in a final volume of 60 ~1. The incubation was terminated after 1 h. Uridine, thymidine and deoxyuridine were separated from their respective nucleobases on a silica gel t.1.c. plate developed with chloroform:methanol:acetic acid (90:5:5 v/v/v). The R, values were: uridine, 0.07; deoxyuridine, 0.1; uracil, 0.43; thymidine, 0.14; thymine, 0.62. Cytidine or deoxycytidine was separated from cytosine by developing the plates with chloroform:methanol:acetic

Pyrimidine salvage in S. munsoni acid (17:2:1 v/v/v). The RF values were: cytidine, 0.02; deoxycytidine, 0.02; and cytosine, 0.14. ~e~in~t~~n of qtosine and itsnu~ieos~des and~u~~eoti~s. Deamination of radiolabelled cytosine, cytidine, deoxycytidine, CMP and dCMP to their respective uracil compounds was determined isotopically. The assay mixture contained Buffer A and 125 JJM-[W] substrate (66.6 GBq mol-‘) and 30 ~1 of enzyme in a final volume of 60 ~1. The incubation was terminated after 1 h. Cytidine compounds were separated from their respective uracil derivatives on silica gel t.1.c. plates developed with chloroform:methanol: acetic acid (146: I v/v/v). The R, values were: cytidine, 0.46; deoxycytidine, 0.29; cytosine, 0.21; uridine, 0.78; deoxyuridine, 0.82; and uracil, 0.86. CMP and dCMP were separated from their respective uridine derivatives on PEI-

cellulose t.1.c.plates developed with 0.25 M-aceticacid. The R, values were: CMP, 0.37; UMP, 0.11; dCMP, 0.61; and dUMP, 0.04. Orotate phosphoribosyltransferase.This activity was measured by following nucleoside S-monophosphate and nucleoside formation from [‘“Cl labelled nucleobase. Nucleoside formation resulted from dephosphorylation of the nucleotide by phosphohydrolase activity. The standard assay mixture contained 50 mM-Tris-Cl (pH 7.5), 1 mt+ DTT, 5% glycerol, 3.5 mM-Mgcl,, 2.5 mM-PRPP, 800 PMnucleobase (0.16 TBq mall’) and enzyme extract, in a final volume of 150 pl. Products were separated from substrates on silica gel plates which were developed with chloroformmethanol (9:l v/v) when uracil or thymine was used as a substrate or with chloroform:methanol:acetic acid (204~1 v/v/v) when cytosine was used as a substrate. R, values were: UMP, 0.02; uridine, 0.12; uracil, 0.52, dTMP, 0.02; thymidine 0.42; thymine, 0.71; CMP, 0.02; cytidine, 0.12 and cytosine, 0.20. Phosphohydrolases. The activity was measured by determining liberated P, from OMP, UMP, CMP, dCMP, dUMP or dTMP as was previously described (el Kouni & Cha, 1982). The assay mixture contained 100 mki-Trisxl, pH 8.5, 1 m&r-substrate, IO mmMgcl, and 25 ~1 of enzyme preparation in a final volume of 0.5 ml. The reaction was carried out for 30 min and terminated by the addition of 0.5 ml of ice-cold trichloroacetic acid (10%). The precipitated proteins were removed by centrifugation and the liberated phosphate was determined by adding 1 ml color reagent (1 vol. each of 6 N-sulfuric acid, 2.5% ammonium molybdate, 10% ascorbic acid and 2 vol. of water), and incubating at 45’C for 30 min. The color densitv was determined at 820 nm in a Gilford Model 240 spectrophotometer. Protein determination. Protein concentrations were determined by the method of Bradford (1976) using bovine gamma-globulin as a standard. RESULTS Uptake and metabolism ofpyrimidine nucleobases and nucleosides by who& worms incorporation into nucleic acids. Table 1 shows that incubation of adult S. mansoni worms with radio-

labelled orotate, uridine, cytidine, deoxycytidine, thymidine and uracil resulted in the incorporation of the radiolabel into the nucleic acids of the intact parasite. These results indicate that the aforementioned pyrimidines are precursors for nucleic acid synthesis in S. mansoni. On the other hand, no incorporation was observed when orotidine, thymine or cytosine were used, suggesting that these

TABLE

39

~-INCORPORATION

OF

RADIOLABELLED PYRIMIDINE

NUCLEOBASES AND NUCLEOSIDES INTO NUCLEIC ACIDS 01" s. ~~SOniA~ER 4 HINCUBATION

Compound (45 W) Orotate Uracil Cytosine Thymine Orotidine Uridine Cytidine Thymidine Deoxycytidine

Specific activity (TBq mol-‘)

Incorporation (pm01 per 10 worm pairs)

2.08 2.04 2.02 2.05 1.85 2.07 I .89 2.07 2.07

18.4 69.3 _* _* _* 56.3 210 19.4 18.9

Ratio

I 3.8

3.1 11 1.1 1.0

* Below the sensitivity of the assay (< 1 pmol per 10 worm pairs).

pyrimidines are poorly or not utilized as precursors for nucleic acid synthesis in the parasite. Table 1 also shows that cytidine, uridine and uracil are better precursors than orotate for the synthesis of nucleic acids in adult S. mansoni. In this respect, cytidine is the most efficiently utilized pyrimidine precursor. HPLC analysis of nucleobase, nucleoside and nucleotidepools. To determine the pathways by which

pyrimidine nucleobases and nucleosides were salvaged and incorporated into nucleic acids, the incorporation profiles of radiolabelled pyrimidine nucleobases and nucleosides into nucleobase, nucleoside and nucleotide pools of whole worms were analyzed by HPLC, as shown in Figs. l-5. Uridine was synthesized from uracil, and uridine was cleaved to uracil (Frg. 1). In contrast, neither the synthesis of thymidine from thymine nor the cleavage of thymidine to thymine could be detected in vivo (Fig. 2). Cytosine, cytidine and deoxycytidine were not deaminated to their corresponding uracil derivatives and neither cytidine nor deoxycytidine were cleaved to cytosine (Fig. 3). Figure 4 shows the distribution of radioactivity in the nucleotide pool after incubation with radiolabelled uridine. It indicates that UMP, UDP and UTP, and UDP-hexose are formed from uridine. A similar pattern of incorporation was obtained when radiolabelled uracil was used (data not shown). dTMP, dTDP and dTTP were formed from thymidine (Fig. 5). Similarly cytidine and deoxycytidine were converted to their mono-, di- and t~phosphate nucleosides as well as to hexose nucleotides. No inco~oration from radiolabelled cytidine or deoxycytidine was detected in uracil nucleotides (data not shown). Enzyme activities in ceil-free extracts To elucidate further the various steps of the pyrimidine salvage pathways in S. mansoni suggested

by the HPLC analysis of the acid-soluble fractions from the in vivo metabolic studies, enzyme activities catalyzing the various steps involved in these pathways

M. H.

and F. N. M.

ELKOUNI

NAGWB

0.4

D z z 0.2

-

, IO

20

IO

Retention

Time

20

IO

20

Retention

(min)

FIG. 1. HPLC profiles of pyrimidine nucleobases and nucleosides from S. munsoniafter 4 h incubation with [2-“C] uracil or [I~-‘~C] uridine.

/

/

IO

20

Time (min)

FIG. 2. HPLC profiles of pyrimidine nucleobases and nucleosides from S. mansoni after 4 h incubation with [2-“Cl thymine or [Z-“‘Cl thym~dine.

l-[

leoxycytidine

Cytosine

14 9 6 2 3 j.2 T

IO ?etention

FIG. 3. HPLC profiles of pyrimidine

20

Time (mid

nucleobases and nucleosides from S. mansoniafter [2-“%] cytidine or [2-‘“Cl deoxycytidine.

4 h incubation

or

Thymidine

Uridfne

n t,

with [2-“Cl cytosine

50-

i x ?j_

25-

Retention

Time

(min)

FIG. 4. HPLC profiles of pyrimidine nucleotide from S. ~ff~~~o~~after 4 h incubation with [2-W] uridine.

Retention

Time

(mln)

FIG. 5. HPLC profiles of pyrimidine nucleotide from S. ~~n~o~i after 4 h incubation with [2-?I) th~idine.

~~midine

41

salvage in S. ~Q~so~~

TABLE~--ACTIVITIESOFVARIOUSENZYMESOFTHEPYRIMIDINESALVAGEPATHWAYS 1NCYTOSOLEXTRACTSOFs. mansoni

Activity* f sot

Enzyme

Substrate

Nucieoside kinase

Uridine Cytidine Deoxyuridine Thymidine Deoxycytidine

16.2 9.53 7.51 7.32 5.66

i i i f f

1.32 0.67 1.05 1.35 1.36

UMP CMP dUMP dTMP dCMP

23.7 * 192 f 0.35 f 319 f 116 f

8.4 33 0.08 8.5 2.4

Nucleoside diphosphate kinaset

UDP CDP dUDP dTDP dCDP

7.18 f 116 f 0.35 f 297 f 101 f

5.64 21 0.03 8.1 5.6

Uridine phosphorylase

Uridine Cytidine Deoxyuridine Thymidine Deoxycytidine

118 f 3.8 n.d. 25.9 f 1.6 19.9 Zt 1.1 n.d.

Orotate phosphoribosyltransferase

Uracil Orotate Thymine Cytosine

92.2 + 2.9 171 f 21 nd. n.d.

Deaminase

Cytosine Cytidine Deoxycytidine CMP dCMP

Nucleoside monophosphate

kinase

n.d. n.d. n.d. nd. n.d.

* Activity = pmol min-’ mg protein’. t S.D.= standard deviation from at least three determinations. 1 Because this activity was measured as the accumulated nucleoside Striphosphates formed from their respective nucleoside S-monophosphates and nucleoside S-monophosphate kinase seems to be a rate limiting step, the values reported here are likely to be conservative estimates. n.d. = not detected.

TABLE ~-PHosPHoH~L~~~L~~E

ACTIVITIES(PMOL MIN-') IN v.4RlOUSFRACTIONS OF Schistosoma SUBSTRATES

30,000 x g Substrate (1 mM) CMP UMP OMP dTMP dCMP dUMP /3-Glycerophosphate

Homogenate (39.61 mg)* 1.90 5 1.79 * 1.50 f 1.71 f I.77 f 1.78 f

O.l5(lOO)t 0.15(94) 0.14(79) 0.07(~) 0.25(93) 0.32(94)

1.45 f 0.20(76)

Supernatant (17.75 mg) 0.26 0.25 0.22 0.22 0.24 0.25

+ f f + i f

0.09(100) 0.09(93) 0.07(83) 0.~(84) 0.08(89) 0.08(95)

0.20 f 0.06(75)

0.19(100) O.OO(106) 0.03(80) 0.15(93) O.lO(98) O.lO(lO3)

1.08 f 0.08(96)

* Total amount of protein from 10.8 ml of packed worms. t Per cent activity relative to that towards CMP in parentheses.

TOWARDS DIFFERENT

105,000 x g

Pellet (19.35 mg) 1.12 f 1.19 f 0.09 f 1.04 f 1.10 f 1.16 f

mansoni

Cytosol (12.40 mg) 0.10 0.06 0.08 0.09 0.08 0.10

l

f f f f f

0.03(100) 0.01(60) 0.03(80) 0.03(90) 0.03(85) 0.03(99)

0.09 f 0.02(91)

Microsomes (2.59 mg) 0.12 0.12 0.10 0.10 0.12 0.12

f * f f f f

0.02(100) 0.03(104) 0.02(88) 0.02(84) 0.03(100) 0.03(99)

0.11 f O.Ol(92)

M. H. EL KO~NZand F. N. M. NAGUIB

42

were determined in cell-free extracts. Table 2 shows the activities of pyrimidine nucleoside kinases, nucleotide kinases, uridine phosphorylase, orotate phosphoribosyltransferase, cytosine deaminase, cytosine nucleoside and nucleotide deaminases in cytosol extracts (~05,000 X g su~rnatant) of live worms. None of these activities could be detected in the particulate fraction (105,000 x gpellet). Extracts of S. mansoni can phosphorylate all pyrimidine nucleosides tested to their respective nucleoside S-monophosphates in the presence of ATP. Similarly, all pyrimidine nucieoside S-monophosphates tested were phospho~lated further to their respective di- and triphosphates by kinase activities (Table 2). Table 2 also shows that the nucleoside phosphorylase activity could be demonstrated towards uridine, deoxyuridine and thymidine but not cytidine or deoxycytidine. Phosphoribosyltransferase activity was detected towards uracil and orotate, but not thymine or cytosine. No deaminase activity could be detected towards cytosine, cytidine, deoxycytidine, CMP or dCMP, confirming the in viva studies (Fig. 3). Phosphohydrolase activity towards CMP, UMP, OMP, dUMP, dCMP and dTMP was tested in the various fractions of schistosome extracts. PGlycerophosphate was included as an indicator for the level of non-specific phosphatases. Worm homogenates exhibited activities towards all of the substrates. The distribution of phosphohydrolase activities in the various fractions of the homogenates is shown in Table 3. The highest specific activity was detected in

the 100,000 x g pellet while the largest amount of activity was detected in the 30,000 x g pellet. These results indicate that phosphohydrolase activities in schistosomes are largely associated with membranes. This activity was also correlated with that towards @gIycerophophate. DISCUSSION The present studies indicate that although thymine, cytosine and orotidine are transported into S. mansoni, these pyrimidines are not metabolized by the parasite. Thymine and cytosine were reported to be transported into the worms by simple diffusion (Levy & Read, 1975a). In contrast, schistosomes can take up and metabolize orotate, uridine, cytidine, deoxycytidine, thymidine and uracil to the nucleotide level, and incorporate these pyrimidines into their nucleic acids. Uracil, uridine. thymidine and orotate were previousty shown to be inco~orated into nucteic acids of this parasite (Mattoccia, Lelii & Cioh, 1981; Mattoccia & Cioli, 1983; Huang et al., 1984). HPLC analysis of acid-soluble fractions from in viva metabolic studies and enzyme activity in cell-free extracts delineate the routes by which these compounds are metabolized. Figure 6 summarizes the pathways of pyrimidine salvage as concluded from the present investigation. All pyrimidine nucleosides tested (Table 2) were phosphorylated by cell-free extracts to their respective nucleotides in the presence of ATP. Further phosphorylation of nucleoside 5’-mono- and Sdiphosphates was also detected in the presence of dTTP

dC’DP~------

CD?

UtiP

Chip . . . . . . . . . .

Cytidlne-.-..-..* * ‘*

i Deoxicitidine

8

. . . . . . . . . ..

e...

. .

.

. .

*...*

---

t”

*dU’DP

. . . . .

diDP

.?*+,u

’

Urid;ne

i

Deox$dine

:

Thydi$ine 5

CytAine

..*...*

-

Thymine

T . . . .

. .

. . . . . . .

. . . * . . . . .

. . .

. .

. .

. . . . . . . . .

. .

* . . . .

. .

. . ..-

FIG. 6. Pyrimidine salvage pathways in S. mansoni. Solid lines indicate reactions that have been established; dotted lines, reactions that could not be detected; dashed lines, reactions that have not been studied yet. Numbers shown in the figure correspond to the enzymatic reactions catalyzing the various steps of the pathways. I, Nucleoside kinase; 2, nucleoside monophosphate kinase; 3, nucteoside diphosphate kinase; 4, phosphohydrolase; 5, uridine phosphorylase; 6, orotate phosphoribosyltransferase; 7, cytosine nucleobase, nucleoside or nucleotide deaminase; 8, dTMP synthetase; 9, ribonucleotide reductase; 10, CTP synthetase; 1 I, dUTP pyrophosphatase.

Pyrimidine salvage in S. mansoni ATP. This indicates that, in schistosomes, the phosphorylation of pyrimidine nucleosides and nucleotides observed in the in viva metabolic studies can be carried out by kinase reactions. Among the nucleosides tested only uridine, deoxyuridine and thymidine were subject to in vitro phosphorolysis to their respective nucleobases (Table 2). We have previously shown that a relatively nonspecific uridine phosphorylase is the only pyrimidine nucleoside phosphorylase present in schistosomes (el Kouni et al., 1988). The metabolic studies in whole worms show the formation of uracil from uridine and uridine from uracil (Fig. l), suggesting that the phosphorolysis (Table 2) and reverse phosphorolysis (el Kouni et al., 1988) of uridine demonstrated in vitro also occur in vivo and that endogenous ribose-lphosphate could be in adequate amounts for reverse phosphorolysis. In contrast, there is a discrepancy between the in vitro (Table 2) and in viva (Fig. 2) results with regard to thymidine. Despite the presence of both phosphorolysis and reverse phosphorolysis of thymidine in vitro (Table 2 and el Kouni et al., 1988) neither of these two activities was detected in vivo (Fig. 2). The lack of detectable thymidine cleavage in vivo could be due to the competition between the added thymidine and endogenous uridine since schistosomes have only one pyrimidine nucleoside phosphorylase (uridine phosphorylase) which reacts six-fold better with uridine then thymidine (el Kouni et al., 1988). Such competition by uridine, the better substrate, in addition to the relatively higher ratio of kinase/ phosphorylase activity towards thymidine (0.4) than uridine (0.1) (Table 2) could result in thymidine cleavage activity below the sensitivity of our assay. On the other hand, the lack of detectable synthesis of thymidine from thymine in vivo (Fig. 2) could be due to low intracellular concentration of deoxyribose-lphosphate. The metabolism and incorporation of uracil into the nucleic acids of schistosomes (Table 1) take place via the formation of UMP. The conversion of uracil to UMP can occur either in two steps, by the sequential reactions of uridine phosphorylase and uridine kinase, or by the direct conversion of uracil to UMP by a phosphoribosyltransferase reaction (Fig. 6). In contrast to mammalian systems, schistosomes have two orotate phosphoribosyltransferases (Iltzsch et al., 1984). The first is specific for orotate and the second is relatively non-specific as it can also utilize uracil as a substrate under physiological conditions (Iltzsch et al., 1984). The phosphoribosyltransferase reaction could be the principal pathway for the conversion of uracil to UMP in schistosomes because of its relatively higher activity with uracil as a substrate (92 pmol mini ’ mg protein-‘) over those of uridine phosphorylase (synthetic direction = 38 pmol mini’ mg protein -‘) (el Kouni et al., 1988) and uridine kinase (37 pmol min-’ mg protein -‘). The metabolism of uridine to UMP may also proceed via the formation of uracil and

43

the phosphoribosyltransferase pathway because the activity of uridine phosphorylase (catabolic direction = 118 pmol min-’ mg protein -‘) is higher than that of uridine kinase. This contention may be supported by the similarity in the pattern of incorporation of radioactive uracil and that of uridine into the nucleotide pool of these parasites (Fig. 1). The predominance of one pathway over the other for the salvage of uracil or uridine may depend on the efficiency (V,,,,,/K,,,) of the various enzymes as well as the availability and intracellular concentrations of the co-factors. The present results also show that schistosomes have high levels of phosphohydrolase activities associated mainly with the particulate fractions. These activities are capable of dephosphorylating all pyrimidine nucleoside S-monophosphates tested. However, the activities are also correlated with that towards Pglycerophosphate indicating that the dephosphorylation of pyrimidine nucleoside Smonophosphates in schistosomes is probably due to non-specific phosphohydrolases rather than the more specific 5’nucleotidases. Non-specific phosphohydrolases capable of these activities have been identified previously in schistosomes and were localized in the tegument of the parasite (NimmoSmith & Standen, 1963; Levy & Read, 1975b). In contrast to most other systems, S. mansoni is incapable of deaminating cytosine, cytidine, deoxycytidine, CMP and dCMP to their respective uracil derivatives. Cytidine and deoxycytidine are also not cleaved by uridine phosphorylase (Table 2 and Fig. 3). Therefore, cytidine and deoxycytidine appear to be exclusively incorporated into their respective nucleotides. The absence of cytidine deaminase and phosphorylase (Fig. 3 and Table 2) along with the fact that cytidine is the most efficiently utilized pyrimidine by the schistosomes (Table 1) can be manipulated for chemotherapy. Various available cytidine analogues may be used as antischistosomal drugs. These compounds could be phosphorylated efficiently to the nucleotide level in the parasites without being degraded by the sequential reaction of cytidine deaminase and uridine phosphorylase as is the case in the host. Deamination of cytosine nucleoside and nucleotide analogues is the major obstacle for their use in cancer chemotherapy. Finally, the utilization of orotate by the worms reconfirms the existence of at least the last two steps of the de now UMP biosynthesis (Iltzsch et al., 1984). However, the relatively better efficiency of cytidine, uridine, and uracil utilization for nucleic acid synthesis suggests that the contribution to pyrimidine metabolism by the salvage pathways may be of greater importance to the schistosomes than the de now pathway. This apparent reliance on the salvage pathway may have evolved from the selective adaptation of the parasites to their environment. Plasma concentrations of cytidine and uridine are in the micromolar range in mammals (Rustum, 1978;

At H. ELKOUNIand F. N. M. NAGUIB

44

Moyer, Oliver & Handschumacher, 1981; Pellwein, Jayaram & Weber, 1987) and can probably satisfy the schistosome requirements for pyrimidines. ~ckno~~e~gemenzs-We thank Dr Sun~aR Cha for his continuous support and critical reading ofthis paper, and Dr In-Soon Kim and MS Norma Messier for excellent technical help. This research was supported by Grants AI-22219 and CA-13943 from the NIH, DHHS, U.S.A. REFERENCES AOKIT. & OYA H. 1979. Glutamine-de~ndent carbamoylphosphate synthetase and control of pyrimidine biosynthesis in the parasitic helminth Schisfosonra mansoni. Comparative Biochemistry and Physiology 63B: 511-515.

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