Precursors of pyrimidine nucleotide biosynthesis for gravid Angiostrongylus cantonensis (Nematod́a: Metastrongyloidea)

PRECURSORS OF PYRIMIDINE NUCLEOTIDE BIOSYNTHESIS FOR GRAVID ~~G~~~T~O~GY~U~ CA~~~~ENS~~ (NEMATODE METASTRONGYLOIDEA) NELLIE N. C. So,* PATRICK C. L. WONG*~

and RONALD C. Ko$

Departments of *Biochemistry and $Zoology, Faculty of Medicine, University of Hong Kong, Hong Kong (Received 201Augmt I991; accepted 27 January 1992) N. C., WONG P. C. L. and Ko R. C. 1992. Precursors of pyrimidine nucleotide biosynthesis for gravid Angiostrongyha cantonensis (Nematoda: Metastrongyloidea). International J5wnal fm Pamsit&gy 22: 427-433. Gmvid Angiosirungylur cantonens& can utilixe radiolabelled bicarbonate, orotate, uracil, uridine and cytidine but not cytosine, thymine and thymidine for the synthesis of RNA and DNA. In cell-free extracts of the worm, a phospho~bosyltransfer~ was shown to convert orotate to OMP and uracil to UMP. A similar reaction was not observed with cytosine and thymine. Uridiae was readily phosphorylated by a kinase but a similar reaction for thymidine and deoxyuridine was not found. Cytidine could be phosphorylated by a kinase or be deaminated by a deaminase to uridine. No deaminase for cytosine was detected. There was also no phosphotransferase activity for pyrimidine nucleosides in the cytosolic or membrane fractions. Pyrimidine nucleosides were, in general, converted to the bases by a phosphorylase reaction but only uracil and thymine could form nucleosides in the reverse reaction. The activity of th~idyla~ synthetase was also measured. These results indicate that the nematode synthesizes pyrimidine nucleotides by de 110~0synthesis and by uti~~tion of uridine and uracil and that cytosine and thymine nucleotides are formed mainly through UMP. The thymidylate syntbetase reaction appears to be vital for the growth of the parasite. Aim&a&--SoN.

INDEX KEY WORDS: Angiastrongyius cantonensis; pyrimidine; lie nova biosynthesis; salvage; DNA; RNA; thy~dylate synthetase; phospho~~syltransfe~e; kinase; deaminase; phosphorylase.

INTRODUCTION ANGIOSTRONGYLIMIS is an

important zoonotic disease in South-east Asia and the south Pacific region. This nematode develops from the third-stage larvae to young adults in the brain of the rat host, which then migrate to the pulmonary vessels and heart where they become gravid and oviposition occurs. More recently, the parasite has been reported in rodents in New Orleans (Campbell & Little, 1988). In humans, the parasite has been implicated as the causative agent of eodnophihc mening~n~phalitis (Alicata & Jindrak, 1970). Active biosynthesis of DNA and RNA requires a balanced supply of purine and pyrimidine nucleotides. Some of these nucleotides also take part in sustaining and regulating various metabolic activities. In general, parasitic organism5 can be divided into three categories according to their ability to synthesize py~midine nucleotides from different precursors (Wang, 1989). The plasmodia, such as

7 To whom all correspondence should be addressed.

Plasmodium knowlesi and P. falciparum,

were unable to utilize preformed pyrimidines by salvage pathways and must depend on de nova synthesis (Gutteridge & Trigg, 1970; Gero, Brown & O’Sullivan, 1984). Members of the Kinetoplastida can make pyrimidine nucleotides both by salvage and de now synthesis (Hammond & Gutteridge, 1984), whereas in trophozoite extracts of Tritrichomonas foetus and Giardia lamb&a the activities of some of the enzymes in the de nova pathway could not be detected (Jarroll, Lindmark & Paolella, 1983; Lindmark & Jarroll, 1982). Among parasitic helminths, Qhistosoma mans& was found to contain all the enzymes of de nova UMP biosynthesis (Hill, Kilsby, Rogerson, McIntosh L Ginger, 1981) and incorporate preformed pyrimidines, especially cytidine, into nucleic acids (el Kouni & Naguib, 1990). Very little is known about py~midine nucleotide biosynthesis in nematodes. We have previously studied the pathways of purine nucleotide metabolism in Angiostrongylus cantonensis and reveabd some unusual features (Wang & Ko, 1979, 1980). Since the biosynthetic process for 427

428

N. N. C. So, P. C. L. WONG and R. C. Ko

pyrimidine nucleotides in parasites may differ substantially from that in their mammalian host, identification of key metabolic differences is useful for the targeting of selective drugs. We have therefore investigated the ability of A. cantonennsis to utilize various precursors for pyrimidine nucleotide synthesis. MATERIALS

AND METHODS

Chemicals. Na[‘4C]HC0, (2.07 TBq mall’), [2-‘4C]uracil (2.00 TBq mall’), [2-‘4C]thymidine (1.90 TBq mall’), [2-14C]uridine (1.87 TBq mall’) and deoxy[5- ‘H]UMP (740 TBq mall’) were obtained from Amersham International plc, U.K. [6-?]Orotate (1.74 TBq mall’) was from New England Nuclear Corp., Boston, MA. [5-‘H]Cytosine (736 TBq mall’), [5- ‘Hlcytidine (851 TBq mall’), 2-deoxy[2Y$tridine (2.15 TBq mall’), and [f-‘%]thymine (2.09 TBq mall’) were supplied by Sigma Chemical Co., St. Louis, MO and so were 5-phosphorylribose-I-pyrophosphate (PRPP) and non-radioactive pyrimidines. Polyethyleneimine cellulose sheets (300 PEl) were supplied by Brinkman, Westbury, NJ and Silica gel u.v.~~ sheets by Schleicher & Schnell, Germany. All other reagents were of analytical grade from various sources. Source and maintenance of parasites. A. cantonensis was maintained as describe.d by Wong & Ko (1979). Briefly, the first-stage larvae from the lungs of infected Wistar albino rats were injected into Achatinajiilica. After 5-6 weeks, the thirdstage larvae were obtained by digesting the snail with 0.6% pepsin in 0.1 M-HCl at 37°C for 3 hand fed to rats by stomach intubation. The worms used in these experiments were obtained from the pulmonary vessels and hearts of sacrificed rats which had been infected for at least 4 weeks. Incubation. The collected worms were rinsed in 0.9% NaCl at room temperature, blotted lightly and weighed. About 40 mg of worms, irrespective of sex, were incubated with shaking for 10 mm at 37°C in 2 ml of Krebs-Ringer phosphate solution pH 7.4 (110 mM-NaCL4.9 mM-KCl, 1.2 mr+MgSO,, 25 mM-sodium phosphate) containing 5.5 mMglucose. Various radiolabelled precursors were added and the incubation continued for 2 h. The worms were then removed, rinsed in 50 ml ofKrebs-Ringer phosphate, blotted with filter paper and homogenized in 1 ml of 5% (w/v) ice-cold TCA. An aliquot of 50 ~1 of the homogenate was counted for radioactivity (total uptake) in Aquasol 2. A control experiment was performed in parallel in which worms were removed and homogenized immediately after addition of precursor (zero-time control). All worms remained lively with wriggling motion throughout the incubation period. Incorporation of precursors into nucleic aciak After incubating with various radiolabelled precursors as described above, the worms were washed and homogenized in 1 ml 0.25 M-NaOH containing 0.5 mg of calf thymus DNA and 0.05 mg of the corresponding unlabelled precursor. The contents were incubated at 37°C for 16 h. DNA and proteins were precipitated by addition of 2 ml ice-cold 5% TCA. The precipitate was sedimented by centrifugation, washed once with 5% TCA and collected onto a cellulose nitrate disc (0.45 pm in diameter). The radioactivity in the filter disc was used

to represent the amount incorporated into DNA of the worms. For measurement of incorporation into RNA, worms incubated with various radiolabelled precursors described above were collected, washed and homogenized in 1 ml of 1% (w/v) SDS containing 0.5 mg of baker’s yeast transfer RNA and 0.25 mg of the corresponding unlabelled precursor. The homogenate was mixed immediately with 1 ml of 5% ice-cold TCA and kept at 4’C for 16 h. The resulting RNA precipitate was washed with 5% TCA and treated with 1 ml of 0.3 M-KOH at 37°C for 16 h. After that, 0.1 ml of 3 Mperchloric acid was added and the radioactivity in an aliquot of 0.1 ml of the supernatant was determined. Separation of nucleobases of RNA. The nucleobases containing radiolabel in the RNA precipitate obtained as described above were identified. The RNA fraction was hydrolysed at 100°C in 1 ml of 12 M-perchloric acid for 1 h. After neutralizing with KOH, the mixture was kept in ice for 2 h and centrifuged. The supernatant was freeze-dried and the contents redissolved in 100 ~1 of 0.05 M-HCl. A sample of 10 ~1 was chromatographed on thin layer silica gel plates (t.1.c.) using a mixture of chloroform and methanol (85:15). The R, values for cytosine, uracil and thymine were 0.02.0.2 and 0.32, respectively. The bases were visualized under u.v., cut out from the chromatogram and the radioactivity determined directly. Alternative methodsfor isolating DNA and RNA. For some preparations, a method used to prepare ‘genomic’ DNA was employed. Worms (40 mg) incubated with radiolabelled precursors (as before) were suspended in 400 ~1 of a solution containing 20 mmTris-HCl pH 7.5, 1 mM-EDTA, 2% (w/v) SDS, 0.5 mg mll’ proteinase K and then digested at 50-55°C for 16 h. The digest was extracted twice with 400 ~1 of phenol and then once with 400 ~1 of a mixture of phenol, chloroform and isoamylalcohol (50:49:1). Absolute ethanol (1 ml) was then added followed by sodium acetate pH 5.6 to a final concentration of 0.2 M. The precipitated DNA, referred to as genomic DNA, was washed with 70% ethanol, dried and suspended in 100 ~1 of H,O for the determination of radioactivity. A method different from the one described above was used to prepare RNA in some instances. Radiolabelled worms (40 mg) were mixed with 200 ~1 of guanidinium thiocyanate containing 0.75 M-sodium citrate pH 7.0, 10% (w/v) N-lauroylsarcosine (sodium) and 14.2 M2-mercaptoethanol. After digesting at 2o’C for 10 min, 20 ~1 of sodium acetate pH 4 was added. The mixture was extracted once with 100 ~1 of phenol followed by 20 ~1 of chloroform/isoamylalcohol (49 1). The final aqueous layer was transferred to 240 ~1 of propan-2-01. The precipitated RNA was collected by centrifugation and washed in 70% ethanol. This RNA was then treated with 40 units of ribonuclease-free deoxyribonuclease (DNase; in 40 mM-TrisHCl pH 8.5 and 6 mM-MgCl,) for 1 h at 37’C, precipitated with 1 ml of absolute ethanol and washed with 80% ethanol. The radioactivity in this RNA fraction (DNase-treated RNA) was determined. Enzyme extract. Freshly collected worms were washed in 0.9% NaCl, and homogenized in 4 vol of a solution containing 0.1 M-Tris-HCl pH 7.5, 0.1 mM-EDTA and 5 m&r-2-mercaptoethanol. After centrifugation at 3O,C@Og for 30 min, the supernatant was further centrifuged at 105,000 g

429

Pyrimidine nucleotide precursors in A. cantonensis for 1 h. The activities of enzymes in the supernatant (cytosol) and pellet (microsome) obtained in the second centrifugation step were determined. The pellet was suspended by sonication in 200 ~1 of extraction buffer. Proteins were assayed by the method of Bradford (1976) using bovine serum albumin as standard. Enzyme aways. All enzyme activities were determined at 37’C using the linear portion of a plot of product formation against time. Unless stated otherwise, reactions were. started by addition of enzyme and terminated by rapidly applying 5 ~1 of the reaction mixture onto the t.1.c. plate. After the chromatograms were developed, radioactivities associated with the spots containing both product and substrate were determined by counting the cut-out areas directly. For nucleoside kinases, the reaction mixture contained 0.1 MTris-HCl pH 7.5,s mM-MgCl,, 5 mM-ATP, either 200 PM of [2-“Cluridine (5000 d.p.m. nmol- ‘), or 40 fly of [2-14C] 2-deoxyuridine (28,600 d.p.m. nmol-‘), or 400 FM of [S- ‘Hlcytidine (1250 d.p.m. nmol-‘) or 200 PM of [2-‘Qthymidine (4730 d.p.m. nmolV’) and enzyme extract in a total volume of 100 ~1. The nucleotides (remaining at origin) and nucleosides were separated in a sheet of PEI cellulose developed in methanol/H,0 (1:l). Redevelopment of the chromatogram in 0.55 M-LiCl containing 0.2% (v/v) formic acid separated the mono-, di- and triphosphates of the nucleosides. For nucleoside phosphorylase measured in the direction of nucleobase formation (catabolic), the reaction mixture contained 0.1 M-Tris-HCl pH 7.5, 5 mM-MgCl,, 5 mM-Na,HPO, either 200 pM-[2-Y]uridine or 200 PM-[~‘Qthymidine, or 400 PM-[5-‘Hlcytidine all having specific radioactivities similar to the nucleoside kinase reaction, or 200 flM-[2-14C]2’-deoxyuridine (20,000 d.p.m. nmol-‘) together with enzyme extract in a final volume of 100 ~1. The nucleosides and bases were separated by chromatography in silica gel thin layers. For uridine and thymidine, the chromatograms were developed in chloroform/methanol/ glacial acetic acid (44:3:3 by volume), the respective R, values for uracil, uridine, thymine and thymidine being 0.28, 0.03, 0.45 and 0.14. For cytidine, the developing solvent was chloroform/methanol/acetic acid in proportions of 28: 12:2. For this system, the R, values were cytosine, 0.23; cytidine, 0.15; uracil, 0.58 and uridine, 0.40. For the anabolic direction, the reaction mixture contained 0.1 M- Tri-HCl pH 7.5, 10 mM-o-ribose-l-phosphate, 5 mM-MgCl,, either 200 j&+[2-14C]thymine (21,200 d.p.m. nmol-‘), or 200 PM-[2“C]uracil (4352 d.p.m. nmol-I), or 400 PM-[5-‘Hlcytosine (4672 d.p.m. nmol-‘) and enzyme extract in a final volume of 100 ~1. The chromatographic systems were the same described for the catabolic reaction. For the phosphoribosyltransferases, the reaction mixture contained 0.1 M-Tris-HCI pH 7.5, 5 mM-PRPP, 5 mM-MgCl,, either 20 pM-[2-‘4C]uracil (40,700 d.p.m. nmol- ‘), or 20 PM-[2‘Qhymine (240,000 d.p.m. nmol-‘), or 20 p~-[5‘Hlcytosine (3 1,800 d.p.m. nmol-I), or 40 pM-[6-‘4C]orotic acid (34,500 d.p.m. nmol-‘) and enzyme extract in a total volume of 100 ~1. Substrate and products were separated as described for the nucleoside kinases except for the orotate assay in which the chromatogram was developed in 0.2 MLiCl pH 5.5. R, values were OMP, 0.00; UMP, 0.11 and orotate, 0.35. The nucleoside phosphotransferase activity

was assessed by a method modified from Nelson, LaFon, Tuttle, Miller, Miller, Krenitsky, Elion, Berens & Marr (1979). The reaction mixture contained 0.1 M-sodium acetate pH 5.4, 50 mi+MgCl,, 10 mw-p-nitrophenyl phosphate, either 200 pM-[2-‘4C]thymidine, or 200 pM-[2-14C]uridine, or 400 phi-[S’H]cytidine with specific radioactivities described for the nucleoside kinase reaction, together with enzyme extract in a total volume of 100 ~1. Products and substrate were separated by t.1.c. as described for the kinase. The deaminases were determined by incubating the enzyme extract in 0.1 M-Tris-HCl pH 7.5, 5 mM-MgCl, with either 400 PM-[5-‘Hlcytosine or 400 pM-[5-‘Hlcyytidine (specific radioactivity described for the phosphorylase reaction) in a total volume of 100 ~1. The products were separated in silica gel developed in chloroform/methanol/acetic acid (28: 12:2) as described before. Thymidylate synthetase was determined by the method of Hunston, Jones, McGuigan, Walker, Balazarini & De Clercq (1984). The reaction mixture contained 0.1 M-Tris-HCl pH 7.4, 15 mh+Zmercaptoethanol, 5 mM-formaldehyde, 1 mM-tetrahydrofolate, 10 PM-~'deoxy[S-‘H]UMP (11,200 d.p.m. nmol-I) and enzyme extract in a total volume of 100 ~1. The reaction was terminated by addition of 150 ~1 of a suspension of activated charcoal (100 mg in 1 ml of 20% TCA). After centrifugation, 50 ~1 of the supernatant was counted for radioactivity. All enzyme activities were expressed as pmol of product formed per min per mg of protein.

RESULTS

Uptake and incorporation into nucleic acid The uptake of radiolabelled precursors into A. cantonensis was determined by measuring the total radioactivity in intact worms. In Table 1, the amounts accumulated after 2 h of incubation are described. Radioactivities with associated the worm homogenates in zero-time control samples were < 2% of that at 2 h. Approximately 10-20, 9-l 1, 7-8, G-8 and 4-S’/& respectively of the added bicarbonate, orotate, uracil, uridine and cytidine were taken up whereas ~2% of the added cytosine, thymine or thymidine were incorporated. Radioactivities incorporated into DNA and RNA fractions are also shown in Table 1. These results represented the lower limits of the rates of the biosynthetic processes as no attempt was made to determine the specific radioactivities of the end products. About 27% of the radioactivity associated with the bicarbonate that was taken up was found in the ‘nucleic acid’ fractions while the figure for orotate was about 13%. Approximately 12% of the radiolabelled uridine taken up was incorporated into nucleic acids compared with only about 6% for uracil. A small amount of the radioactivity associated with cytidine was found incorporated into RNA. Very little radioactivity was found in DNA or RNA when cytosine, thymine or thymidine was presented to the worms. The results for the incorporation of uridine

430

N. N. C. So, P. C. L. WONCand R. C. Ko TABLE I-INCORPORATION

OF RAD~OLABELLED PRECURSORS INTO NUCLEIC ACIDS OF GRAVID CcPltOnWlSiS

Precursor

A.

AFTER2HlNCUBATlON Incorporation(nmol

Concentration

per g fresh weight)

(flM

Bicarbonate Orotate Uracil Uridine Uridine Cytosine Cytidine Thymine Thymidine Thymidine

;; 4 4 4 4 4 4 4

Total uptake

DNA fraction

RNA fraction

57.83 f 26.76 i 10.10 f 16.90 f 13.93 + 2.07 f 5.95 f 2.24 f 2.74 f 1.61 f

1.50 * 0.13 0.90 f 0.10 0.03 f 0.01 0.30 f 0.03 0.68 f O.IO*
13.96 f 2.63 2.44 f 0.95 0.56 f 0.17 1.47 f 0.07 1.86V
6.99 2.80 1.05 2.08 2.75’ 0.61 0.92 0.33 0.39 0.15*

* Experiments in which genomic DNA and deoxyribonuclease (DNase)-treated RNA were obtained. t Value represents mean of two experiments. All others were means of three to six determinations f standard deviation. All values had been corrected for zero time control.

TABLE

~-IN~~R~~RATIoN

OF RADIOLABELLED PRECURSORS INTO PYRIMIDINE NUCLEOBASESOF

RNA

% of total radioactivity in RNA hydrolysate Precursor

Bicarbonate Orotate Uridine

Cytosine

Uracil

Thymine*

65-90 85-99 73-90

IO-27 I-15 IO-20

O-8 0 o-7

* Probably due to presence of DNA. All values represent range of two to three determinations.

and thymidine were confirmed by experiments using methods designed for the isolation of genomic DNA and DNA-free RNA (Table 1). When the individual pyrimidine bases of the RNA fractions were analysed, it was found that most of the radioactivity incorporated associated with cytosine (Table 2). A small percentage was found in thymine indicating that the RNA preparations were contaminated with DNA. Interestingly, when bicarbonate was used, a small but significant amount of radioactivity was also found in adenine and guanine of the RNA preparation (results not shown). Enzymes

of ~yri~idine salvage

The activities of several enzymes known to be involved in utilizing preformed pyrimidines for nucleotide biosynthesis in mammalian systems were measured in cell-free extracts of A. cantonensis (Table 3). Orotate and uracil were readily converted to the

respective nucleotides by the phosphoribosyltransferase reactions. About 50% of the products of the reaction with orotate were present as UMP indicating that OMP decarboxylase was also present in the cytosolic fraction. The amount of UMP formed was proportional to the time of reaction for as long as 30 min (result not shown). No phosphoribosyltransferase activity was found for cytosine or thymine. Uridine could be converted rapidly to nucleotides by enzymes in the cytosolic fraction. Upon further analysis of the products of the reaction, it was found that UTP, UDP and UMP were present in the ratio of 55:30:15, indicating that besides uridine kinase, the kinases for UMP and UDP were also present in the worm extract. For cytidine, the kinase activity was demonstrated by measuring the total amount of CMP, CDP and CTP formed during 30 min, over which time the rate was linear. A strong deaminase activity was also found for cytidine. Thymidine and deoxyuridine

Pyrimidine nucleotide precursors in A. cantonensis

431

TABLE~-A~I~IT~~OFVARIOUSENZYMESOFPYR~MID~NEMETABOLISMINCELL~FREEEXTRACTSOF GRAvlD

Enzyme

Phosphoribosyltransferase

Nucleoside kinase

Phosphoryla~ (catabolic direction) Phosphorylase (anabolic direction) Phosphotransferase

Deaminase Thymidylate synthetase

A.

CU~fO~enS~S

Substrate

Activity (pm01 mm ‘mg protein-‘)*

Orotate Uracil Cytosine Thymine Uridine Cytidine Thymidine Deoxyuridine Uridine Cytidine Thymidine Deoxyuridine Uracil Thymine Cytosine Uridine Cytidine Thymidine Cytosine Cytidine 5’-DeoxyUMP

* Means of three to six dete~inations t Not determined

were, however, not substrates for the kinase reaction. All the pyrimidine nucleosides tested can be converted to the corresponding bases. Bases were not formed if phosphate was not present in the assays, indicating that a phosphorylase rather than a nucleosidase was responsible. In the anabolic direction, nucleosides were formed from uracil and thymine but not from cytosine. No deaminase activity was detected which could convert cytosine to uracil. The activity of a phosphotransferase, measured using p-nitrophenol phosphate as the phosphate group donor, was assayed in the cytosolic and microsomal fraction as well as in a suspension of a fraction containing membranes and cell wall debris obtained by sedimenting the worm homogenate by centrifugation at 30,000 g. No significant activity was detected in any of these subcellular fractions. A fairly substantial activity of thymidylate synthetase was found in the cytosol of the worms. DISCUSSION Among parasitic hel~nths, detailed studies on the uptake of pyrimidines have only been made for some cestodes. For S. manson& cytosine, thymine and uracil

Cytosol

Microsome

1127 i 101 45.1 f 5.8

f standard deviation.

can enter by diffusion and uridine by a mediated system which is shared with adenosine (Levy & Read, 1975). The cestode also utilizes exogenous pyrimidine nucleosides as well as orotate for nucleic acid synthesis (d Kouni & Naguib, 1990). Thymine and uracil can be transported into Hymenolepis diminuta by a common carrier (Pappas & Read, 1975). In contrast, the uptake of orotate, cytoine, thymine and thymidine into adult worms of Brug~upahff~g~are limited, and only uracil is utilized to any significant extent (Chen & Howells, 1979-1981). Not much is known about the ability of parasitic nematodes to accumulate pyrimidine nucleotide precursors. In the present study, A cantonensis was shown to take up bicarbonate, orotate, uracil, uridine, cytidine and to a lesser extent, cytosine, thymine and thymidine. The route for uptake into the intact worm is, however, not clear. The ability for A. cuntonensis to form pyrimidine nucleotides by de novo synthesis and by utilization of pyrimidine bases and nucleosides was studied by measuring the incorporation of various radiolabelled precursors into nucleic acids of the intact worm. The possible metabolic routes are shown in Fig. 1. Bicarbonate and orotate were incorporated into both

432

N. N. C. So,

DNA

RNA

RNA

dCl-P

cl-P4

UTP

CDP

UDP - -)

P. C. L. WONG and R. C. Ko DNA

f dTlT

3 I

dC!DP f--

4+

cytosine

4ir4] lmcil

dUDP

orotate+ f

dTDP

HCO;

&no”* pathway

FIG. 1. Possible routes by which pyrimidine precursors are incorporated into nucleic acids in A. cantonensis. The enzymes involved are (1) orotate phosphoribosyltransferase (2.4.2. lo), (2) orotidylate decarboxylase (4.1. I .23), (3) uracil phosphoribosyltransferase (2.4.2.9), (4) uridine phosphorylase (2.4.2.3), (5) uridine kinase (2.7.1.48). (6) cytidine deaminase (3.5.4.5), (7) thymidylate synthetase (2.1.1.45), (8) nucleoside monophosphate kinase (2.7.4.4) and (9) nucleoside diphosphate kinase (2.7.4.6). Reactions indicated by dashed lines are only assumed to occur.

RNA and DNA fractions. Analysis of the RNA fraction showed that most of the radioactivity was associated with cytosine residues. This result indicates that the enzymes of the de novo pathway of pyrimidine nucleotide synthesis established for mammalian systems may also exist in the parasite. All of the six enzymes of de nova UMP biosynthesis were shown to be present in S. mansoni and S. japonicum extracts (Iltzsch, Niedzwick, Senft, Cha & el Kouni, 1984; Huang, Chang & Chen, 1984). Little is known about these enzymes in nematodes, but the activities of orotate phosphoribosyltransferase and OMP decarboxylase have been found in A. cantonensis. Although the assay system employed was intended to measure the former enzyme, about 50% of the OMP formed was readily converted to UMP. The formation of UMP was linear with time of reaction. This observation is consistent with the view that, in mammalian tissues, the two enzymes form a complex and OMP is ‘channelled’ quickly to the next reaction (Keppler & Holstege, 1982). Of the pyrimidine bases and nucleosides studied, uridine was the best source for nucleic acid synthesis. RNA prepared by conventional methods was found to be contaminated with DNA (as shown in Table 2) and vice versa. By using more refined methods, uridine was confirmed to be incorporated into both RNA and DNA. It is noted that in the RNA fraction, most of the uridine incorporated was found in the cytosine units indicating that the formation of cytosine nucleotides

from uracil nucleotides is an important reaction in this parasite. Incorporation into DNA also means that the nematodes were capable of forming deoxyribonucleotides from the ribonucleotides of uracil. Compared to uridine, uracil was a poor substrate for nucleotide formation. This was reflected by the relatively poor rate of incorporation into RNA and DNA and the low activity of uracil phosphoribosyltransferase in comparison to uridine kinase. This is in contrast to S. mansoni in which uracil was thought to feature more prominently than uridine for UMP formation (el Kouni & Naguib, 1990). The utilization of cytosine and cytidine was limited by the low uptake of these pyrimidines into the intact worm. In the worm, cytosine cannot form nucleotides by a phosphoribosyltransferase reaction or via a phosphorylase reaction in the anabolic direction. A deaminase was not demonstrable in the extracts for cytosine so the metabolic fate of this pyrimidine is not clear. A small amount of cytidine, on the other hand, was shown to be incorporated into RNA. The nucleotide could be formed by reaction with a nucleoside kinase but because the cytidine deaminase activity was very high in the cytosol, it appears more likely that UMP was formed from cytidine which was later aminated to form cytosine nucleotides as discussed before. This again is in contrast with S. mansoni which lacks the deaminase and cytidine was more efficiently utilized than uridine (el Kouni & Naguib, 1990). In some parasitic protozoans, such as T. foetus, pyrimidine nucleotides can be formed by a phosphotransferase present only in the particulate fractions of cell extracts (Wang, Verham, Tzeng, Aldritt & Cheng, 1983). The enzyme transfers a phosphate group from p-nitrophenol phosphate (or, more naturally, a nucleoside monophosphate) to a Neither nucleoside. the membrane-containing fractions nor the cytosol of A. cantonensis contained such an enzyme activity as tested with uridine, cytidine or thymidine. The inability of A. cantonensis to utilize thymine and thymidine was shown by the poor uptake into intact worms, the lack of incorporation into DNA and the lack of thymidine kinase activity in cell extracts. This in fact is not surprising since developing Ascaris lumbricoides (see Farland and MacInnis, 1978) and B. pahangi (see Chen & Howells, 1979) were also known to be unable to utilize thymidine and a phosphoribosyltransferase activity for thymine has never been detected in animal tissues. For these nematodes therefore, dTTP, which is essential for DNA synthesis, could only be formed by the de novo pathway. The key enzyme in this pathway is thymidylate synthetase which converts 5’-deoxyUMP to dTMP. The activity of this enzyme in extracts of

Pyrimidine

nucleotide

precursors

A. cantonensis

was demonstrated simply by including S-deoxy UMP, tetrahydrofolate and formaldehyde in the assay. Thymidylate synthetase may represent an important target for selective inhibition of the growth of A. cantonensis, as mammalian tissues in general can form dTMP by the thymidine kinase reaction. Little is known about thymidylate synthetase in parasitic helminths. The enzyme purified from several parasitic protozoans exists as a bifunctional protein together with dihydrofolate reductase (Ivanetich & Santi, 1990). In animal cells, however, the two enzymes are distinct monofunctional proteins. In some tumour cells, thymidylate synthetase has been the target for the antineoplastic agent S-fluorouracil. It would therefore be important to characterize the enzyme from A. cuntonensis in full. In conclusion, A. cantonensis was shown to form pyrimidine ribonucleotides for nucleic acid synthesis by de novo pathway as well as by utilization of preformed pyrimidines mainly through the uridine kinase reaction and that dTMP originated from UMP and not by utilization of thymidine. Acknowledgements-This work was supported by funds from the Research and Conference Grants and the Medical Faculty Research Grant of the University of Hong Kong. RERERENCES ALICATA J. E. & JINDRAK K. 1970. Angiostrongylosis in the Pacijic and Southeast Asia. C. C. Thomas, Springfield, IL. BRADFORDM. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing of protein-dye binding. Analytical the principle Biochemistry 12: 248-254. CAMPBELL B. G. & LI*TLE M. D. 1988. The finding of Angiostrongylu cantonensis in rats in New Orleans. American Journal of Tropical Medicine and Hygiene 38: 568-573. CHEN S. N. & HOWELLS R. E. 1979. Brugia pahangi: uptake and incorporation of adenosine thymidine. and Experimental Parasitology 47: 209- 221. CHEN S. N. & HOWELLS R. E. 1981. Brugia pahangi: uptake and incorporation of nucleic acid precursors by microlilariae and macrofilariae in vitro. Experimental Parasitology 51: 296306. EL KOUNI M. H. & NAGUIB F. N. M. 1990. Pyrimidine salvage pathways in adult Schistosoma mansoni. International Journalfor Parasitology 20: 37- 44. FARLAND W. H. & MACINNIS A. J. 1978. In vitro thymidine kinase activity: present in Hymenolepis diminuta (Cestoda) and Monillformis dubius (Acanthocephala) but apparently lacking in Ascaris lumbricoides (Nematoda). Journal of Parasitology 64: 56+565. GERO A. M., BROWN G. V. & O’SULLIVAN W. J. 1984. Pyrimidine de novo synthesis during the life cycle of the intraerythrocyte stage of Plasmodium falciparum. Journal of Parasitology 70: 536-541. GUTTERIDGE W. E. & TRICG P. I. 1970. Incorporation of radioactive precursors into DNA and RNA of Plasmodium

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