Purine ribonucleotide biosynthesis in gravid Angiostrongylus cantonensis (Nematoda: metastrongyloidea)

Comp. Biochem. Physiol., Vol. 65B, pp. 303 to 308

0305-0491/80/0201-0303502.00/0

© Pergamon Press Ltd 1980. Printed in Great Britain

PURINE RIBONUCLEOTIDE BIOSYNTHESIS IN GRAVID A N G I O S T R O N G Y L U S C A N T O N E N S I S (NEMATODA: METASTRONGYLOIDEA) PATRICK C. L. WONG j and RONALD C. KO 2 ~Departments of Biochemistry and 2Zoology, University of Hong Kong, Hong Kong (Received 29 May 1979) Abstract--1. The pathways of purine ribonucleotide synthesis and intereonversion that are operative in

the gravid rat lung worm, Angiostronoylus cantonensis, were identified by radioisotope tracing in intact whole worms and by measurement of enzyme activities in cell-free extracts 2. Ribonucleotides were rapidly formed by utilization of adenine, hypoxanthine and guanine and by direct phosphorylation of adenosine. 3. AMP and GMP were interconvertible but these reactions were very slow. 4. The synthetic capacities of purine ribonueleotide metabolism were much higher than the catabolic functions in the parasite at this particular stage of the life cycle.

(erythro-9(2-hydroxy-3-nonyl)adenine), was a gift from Dr F. F. Snyder, University of Calgary, Canada.

INTRODUCTION

The metastrongyloid nematode, Angiostrongylus cantonensis, develops from the third-stage to young adults in the brain of the rat host and then migrates via the circulatory system to the heart and pulmonary arteries. During a period of about 1 month, the worm can grow from approx 300/tm to almost 16-22 mm in length. In the lung stage, oviposition occurs and large number of eggs are produced by the female worms. Egg production can continue for 1-2yr. Such a remarkable biosynthetic capacity definitely requires a large amount of purine nucleotides both as a source of energy (ATP) and for purines supply as required in D N A and R N A synthesis. Recently, our earlier study has demonstrated that the worm may undergo de novo purines synthesis (Wong & Ko, 1979). Although the preformed purines ingested by this tissue-dwelling parasite during the course of development most likely may represent a substantial source of purine nucleotide precursors, the pathways of their utilization for ribonucleotide synthesis are virtually unknown. The present study is an attempt to identify these pathways in the metastrongyloid parasite. MATERIALS AND METHODS Chemicals

Maintenance of worms The strain of A. cantonensis was originally isolated from local wild Rattus sladeni in 1974. The worms were main-

tained in the laboratory by methods described previously (Wong & Ko, 1979). Worms recovered from lungs of infected rats usually remained active for several hours in saline at room temperature or in Krebs-Ringer phosphate solution (pH 7.4) at 37°C. Whole worm studies

About 15-30 mg of mixed male and female worms were used to study the metabolism of various radioactively labelled purines and purine nucleosides. The worms, firstly cleaned of all lung tissues and thoroughly rinsed in saline were incubated in 1 ml of Krebs-Ringer phosphate solution (pH 7.4) supplemented with 5.5 mM glucose as described previously (Wong & Ko, 1979). Aliquots of 0.01 ml of the medium were removed at various intervals to analyse the uptake of radioactive precursors and external metabolites. For studies of the rates of synthesis of intracellular nucieotides, approx equal amounts of worms were incubated separately for various intervals. At the end of each incubation period, the worms were removed, rinsed and a trichloroacetie acid-soluble fraction was obtained (Wong & Ko, 1979). The freeze-dried residue was taken up in 0.5 ml of water just prior to the analysis for purine metabolites.

[8-14C]adenine (59 mCi/mmol), L r8- 14,-,~, ~lnypoxanthine (60mCi/mmol), [8-14C]guanine (56mCi/mmol), [8-14C]adenosine (47mCi/mmol), and [8-~4C]inosine (57mCi/ mmol) were obtained from The Radioehemical Centre, Amersham, U.K. The purity of these radioactive compounds was established by the use of the t.l.c, systems described below. Adenine and hypoxanthine contained virtually no contaminants, adenosine contained 2% adenine, inosine contained 3.4% hypoxanthine and guanine contained 1.5% xanthine. Non-radioactive purine bases, ribonucleosides, ribonucleotides, phosphoenolpyruvate and pyruvic kinase were obtained from Sigma Chemical Co. (St Louis, MO, U.S.A.). Other chemicals of analytical grade were obtained from various commercial sources. EHNA

Analysis of radioactive purine metabolites in the incubation medium and in the acid-soluble fraction

The purine bases, ribonucleosides and uric acid were analyzed by two chromatographic systems. (1) They were separated by thin layer chromatography using Eastman Kodak unsubstituted cellulose thin layers on Mylar sheets (Crabtree & Henderson, 1971). (2) Adenine, adenosine, hypoxanthine and inosine were sometimes separated by paper chromatography using water-saturated butan-l-ol (100 ml) and cone. NH,,OH (l ml) as solvent (Senft et al., 1973b). The purine ribonueleotides were separated on Baker-Flex polyethyleneimine-eellulose (PEI) thin layers by the method of Crabtree & Henderson (1971). 303

304

PATRICK C. L. WONG and RONALD C. Ko

The results represent the radioactivity in each metabolite rather than the total amount of each metabolite. Enzyme extract

The worms were collected from rats which had been infected for 6-12 months. They were washed in 20raM Tris-HCl, pH 7.5, containing 0.5 mM EDTA and were homogenized in 10 vol of the same buffer. The homogenate was centrifuged at 20,000 g for 60min at 4°C. The clear supernatant was used as the source of enzymes. Enzyme assay

Adenine, hypoxanthine and guanine phosphoribosyltransferases (APRT, EC 2.4.2.7; HPRT and GPRT, EC 2.4.2.8) were assayed by the method of Senft et al. (1973a). The reaction mixtures consist of, in a total vol of 1 ml, Tris-HC1 buffer pH 7.5 (0.ZM), MgC12 (5 mM), 5-phosphoribosyl-l-pyrophosphate (PRPP, 0.5mM), [8-14C] adenine (0.1 mM; sp. act. = 10 #Ci/#mol), [8-~4C]hypoxan thine (0.1 mM ; sp. act. = 10 #Ci/#mol) or [8-14C]guanine (0.02 mM; sp. act. = 27 #Ci/#mol) and 0.02 ml of worm extract (156 #g of protein). Adenosine kinase (EC 2.7.1.20) was assayed by the formation of nucleotides in the presence of the powerful adenosine deaminase inhibitor, EHNA. The reaction mixture contained, in a final vol of 0.2 ml, 100 mM Tris-HC1 pH 7.5, 0.ZmM MgC12, 50mM KC1, 0.TmM ATP, 3raM phosphoenolpyruvate, 25 #M EHNA, 18.5 units of pyruvic kinase, 70 #M [8 -14C]adenosine and enzyme extract containing 84#g of protein. The mixture was incubated at 37r'C and 0.01 ml was transferred to Whatman 3 M M paper at various times up to 40 min. The chromatogram was developed in water-saturated butan-l-ol (100 ml) and conc ammonium hydroxide (1 ml) using adenine, adenosine, hypoxanthine, inosine and AMP as markers. The nucleotides remained at the origin and the radioactivity associated with this area was measured. Inosine kinase (EC 2.7.7.73) was assayed by the same method except that [8-~4C]inosine, at a final concentration of 0.112 mM (sp. act. = 17.9 #Ci/#mol) replaced adenosine. Adenosine deaminase (EC 3.5.4.4) was measured by the formation of inosine and hypoxanthine. The reaction mixture contained in 0.2 ml, 100 mM Tris-HC1 pH 7.5, 0. l mM [8-14C]adenosine and enzyme extract containing 84 #g of protein. Aliquots of 0.01 ml of the mixture, incubated at 37°C, were analysed for radioactivity in inosine and hypoxanthine by the chromatographic method described for adenosine kinase. AMP deaminase (EC 3.5.4.6) was assayed by the method of Smiley et al. (1967). The asaay was performed at 30°C, using a mixture containing 100mM potassium succinate, pH 6.5, 0.1 mM ATP and extract containing 156 #g protein in a final vol of 1 ml. The change in optical density at 265 nm was measured by a Cary 17D spectrophotometer. Inosine phosphorylase (EC 2.4.2.1) was assayed at 37°C in 30mM potassium phosphate, pH 7.5, containing 0.42 mM [8-14C]inosine (2.4 #Ci/#mol) and worm extract (containing 156 #g protein) in a final vol of 0.5 ml. Aliquots of 0.02 ml, taken at intervals over a period of 40 min, were analysed for radioactivity in hypoxanthine using the chromatographic system described for adenosine kinase. Adenosine phosphorylase was assayed by the same method except that [8-14C]adenosine, at a final concentration of 0.34 mM (sp. act. = 3.0 #Ci/#mol) replaced inosine. Guanine deaminase (EC 3.5.4.3) activity was measured by following the rate of formation of xanthine at 37°C. The reaction mixture contained 200mM Tris-HCl pH 7.5, 100mM KC1, 7 m M MgClz, 0.04mM [8-14C]guanine (27 #Ci/#mol) and 0.02 ml worm extract (84 #g protein) in a final vol of 0.5 ml. Aliquots of the reaction mixture were removed at various time intervals and the amount of radioactive xanthine formed was measured by the chromatographic system described previously for adenosine kinase.

RESULTS Adenine as precursor

Adenine was found to be utilized for purine ribonucleotide synthesis by the intact worms. As shown in Fig. 1, the rate of synthesis was approx linear for the first 60 min. At this time, the total ribonucleotides synthesized represented a b o u t 8 ~ of the initial a m o u n t of adenine added to the medium. Analysis of the purine ribonucleotide composition showed that adenine ribonucleotides were mostly formed. The ratios of the a m o u n t of guanine ribonucleotides to that of adenine ribonucleotides were 0.007, 0.016 and 0.03 after incubation for 30, 60 and 90 min respectively. Analysis of the adenine ribonucleotide composition showed that A M P was favourably converted to A T P ; the ratio of A T P : A D P : A M P being approx 30:5:1. Neither I M P (inosinate) nor X M P (xanthylate) was present in any of the acid-soluble extracts. Analysis of the incubation medium showed that apart from the added adenine, no other radioactively labelled purine derivative was present. Hypoxanthine as precursor

The rate of purine ribonucleotide synthesis using hypoxanthine as precursor was approx linear for 90 min (Fig. 2). At that time only a b o u t 7~o of the total a m o u n t of hypoxanthine was converted to ribonucleotides. Approximately equal a m o u n t s of adenine a n d guanine ribonucleotides were synthesized. Of 8°

21 .Jz

e

_o

,o

n

$

30

60

90

Time ( m i n i

Fig. I. Rate of formation of radioactive purine ribonucleotides in gravid A. cantonensis. About 10rag of male and female worms were incubated at 37°C with shaking in 1.0 ml of Krebs Ringer medium containing 25 mM sodium phosphate buffer pH 7.4 and 5.5 mM glucose. [8-14C] Adenine was added to a final concentration of 8.5 #M. At the end of the incubation period, the worms were removed and rinsed in cold Krebs-Ringer medium. An acid-soluble extract was made and the radioactivity associated with various purine ribonucleotides analysed.

Purine nucleotide synthesis in A. cantonensis

or hypoxanthine indicating that adenosine deaminase was not released under these conditions. In one experiment, one of the worms was damaged at the end of a 30 rain incubation period, but after the removal of worms, inosine and hypoxanthine still continued to accumulate in the medium until all the adenosine was exhausted. Under these circumstances, the total amount of inosine and hypoxanthine present accounted for all the adenosine which has disappeared. Further analysis of the medium incubated with intact worms showed that adenosine, inosine and hypoxanthine were the only purine derivatives which were radioactively labelled.

20

"~

GTP÷

°-

m 1G E

"--~

~

ATP*ADP*AMP

O

E

Q.

305

Guanine as precursor

s. J~.~.~.---.-6 I

0

30

I

60 Time (min)

iMP I

90

/

120

Fig. 2. Formation of radioactive purine ribonucleotides when the worms (16-20 rag) were incubated, as described in Fig. 1, with 8.33 #M I-8-~4C]hypoxanthine. these, the ribonucleoside triphosphates were again found to predominate. A considerable amount of IMP which reached a steady state in 60min was found to accumulate. No XMP was detectable under these conditions. Apart from inosine, no other radioactively labelled derivative of hypoxanthine was detectable in the incubation medium which was analysed for purine derivatives. The formation of inosine in the medium appeared to increase linearly over a period of 60 min. At this time, an amount equivalent to 8~o of the initial hypoxanthine was present as inosine.

Guanine was found to be utilized for ribonucleotide synthesis. Guanine ribonucleotides were formed proportionately with time over a period of 120rain (Fig. 4). At that time, the amount formed represented about 5~ of the guanine added initially. The ratio of GTP: GDP :GMP was about 8 :1.4:1. There was also a small but measurable amount of radioactive ATP formed in worms which had been incubated for longer than 60 min. At 120 min, the ratio of adenine to guanine ribonucleotides was about 0.07. The acidsoluble extracts did not contain any radioactive IMP or XMP. Except for xanthine, which accumulated to about 5 ~ of the total radioactivity added initially, no other detectable radioactively labelled metabolite of guanine was detected in the medium after an incubation period of 60 min. Enzymes o f purine metabolism in cell-free extracts

Some of the key enzymes in the synthesis of purine ribonucleotides as well as those responsible for interconversion between these nucleotides were measured in cell-free extracts of the worms (Table 1). Among the phosphoribosyltransferases, the specific activity of 2O

Adenosine as precursor

Adenosine was utilized for ribonucleotide synthesis by the intact worms. A linear rate of formation of ribonucleotide could be demonstrated (Fig. 3). In this experiment, the maximum amount formed at 30 min represented about 6~o of the amount of adenosine initially added to the medium. The rate of formation of ribonucleotides inside the worms closely paralleled the rate of adenosine disappearance in the medium. Adenine ribonucleotides accounted for most of the purine nucleotides formed. After 30 min of incubation, the amount of radioactivity in adenine ribonucleotides was about 17 times greater than that in guanine ribonucleotides. Compared with using hypoxanthine as the precursor (Fig. 2), the amount of guanine ribonucleotides formed from adenosine was rather small. A small but significant amount of IMP was also detected in each of the worm extracts. Both inosine and hypoxanthine were found in the medium. Their respective amounts were 2.4 and 1.3~ of the initial adenosine concentration (9.09/~M) after 28 mg of worms were incubated for 40 min. If the worms were removed at this time and the medium was allowed to incubate further for as long as 190rain, there was no further increase in the inosine

t-

E

15

10

n

5

GTP÷GDP÷GMP 0

10

! 20 Time (rnin)~

a 30

40

Fig. 3. Formation of radioactive purine ribonucleotides when the worms (26-28 mg) were incubated, as described in Fig. 1, with 9.09 #M [8-z4C] adenosine.

306

PATRICK C. L. WONG and RONALDC. KO detectable. This is consistent with the fact that adenylate deaminase activity was not detectable in the cellfree extracts even when the reaction was measured in the presence of 100mM K + and 0 . 7 m M ATP. The extracts, however, contained guanine deaminase.

10

DISCUSSION

r

2V / 0

30

60

90

120

Time (rain)

Fig. 4. Formation of radioactive purine ribonucleotides when the worms (18-23 mg) were incubated, as described in Fig. 1, with 5.8 #M [8J4C]guanine. adenine was many times higher than those of hypoxanthine and guanine. This order closely paralleled those measured in the intact worms. It can also be seen that the activities measured in the intact worms were only a small fraction of that measured in cellfree extracts. Adenosine kinase activity was found to be almost three times higher than that of the deaminase. This difference is consistent with the preference for adenosine to be incorporated into adenine ribonucleotides in the intact worms. Inosine kinase was not measurable whereas inosine can be converted to hypoxanthine as indicated by the presence of the phosphorylase. The latter enzyme can also use adenosine as substrate almost as efficiently as inosine. In the adenine phosphoribosyltransferase assay, A M P was the only nucleotide accumulated. As much as 45 nmols of this was present in the assay mixture after 30min. Under these conditions, no I M P was Table 1. Enzymes in purine metabolism in cell-free extracts of A. cantonensis Enzyme Adenine phosphoribosyltransferase Hypoxanthine phosphoribosyltransferase Guanine phosphoribosyltransferase Adenosine kinase Inosine kinase Inosine phosphorylase Adenosine phosphorylase Adenosine deaminase AMP deaminase Guanine deaminase

nmols/min/ mg protein

nmols/min/g wet wt

9.19

680

1.05

78

0.64

47

On the basis of the present study, a set of pathways for the synthesis and interconversion of purine ribonucleotides in A. cantonensis can now be established (Fig. 5). Adenine and adenosine were utilized mostly for ATP synthesis by the most direct routes. Both A P R T and adenosine kinase activities were demonstrable in the cell-free extracts. The activity of A P R T observed is higher than that in most rat tissues and is comparable to the high activity found in Ehrlich ascitestumour cells in the mouse (Murray, 1966). The adenosine kinase activity in the parasite is also higher than that of rat brain (Wong, unpublished data), Ehrlich ascites-tumour cells (Murray, 1968) and rat heart (Maguire et al., 1972). In most mammalian tissues, deamination of adenosine occurs at a high rate (Henderson & Paterson, 1973). In the mouse brain the reactions are dependent upon the concentration of adenosine (Wong & Henderson, 1972). In the malarial parasite, Plasmodium berghei, the only pathway of adenosine metabolism is by deamination and the inosine formed is then converted to hypoxanthine before nucleotide formation occurs (Manandhar & Van Dyke, 1975). This deamination pathway is also preferred by Schistosoma mansoni (Senft et al., 1973b). A. cantonensis is, therefore, unique among animal parasites because the deamination of adenosine, although demonstrable in cell-free extracts, does not occur to any appreciable extent in the intact worm. This parasite is capable of using adenosine directly for A M P synthesis by the kinase reaction. An alternative pathway for nucleotide synthesis from adenosine is the nucleoside phosphorylase reaction. This enzyme reacts equally well with adenosine and inosine. The Adenine

•

185 Nil

0.66 0.57 0.95

49 39 64 Nil

0.78

58

•

Lo.Jt / 'r- T/

Hypoxanthine

~ IMP

Xanthine

2.73

AMP

Guanine

'¢,--

ADP

,¢

~ ATP

De novo

( MP

•

GMP

~ GDP

~ GTP

Fig. 5. Proposed pathways of purine ribonucleotide synthesis and interconversion in gravid A. cantonensis. Compounds in parentheses have not been demonstrated but are presumably formed. The rates of conversion of GMP to IMP and AMP to IMP are very small. The conversion of adenosine to adenine is only demonstrable in cell-free extracts. Conversion of adenosine to inosine occurs at a very low rate in the intact worms.

307

Purine nucleotide synthesis in A. cantonensis activity of this enzyme, however, is demonstrable only in ceil-free extracts. In the intact worms, adenine was never observed to be present when they were incubated with adenosine (cf. hypoxanthine was formed when inosine was present). It is not clear at present if this alternative pathway is of any significance in the intact worm. Guanine ribonucleotides can arise directly from guanine by the phosphoribosyltransferase reaction. As in the case for adenine ribonucleotides, the guanosine monophosphate kinase and nucleoside diphosphokinase activities are very high so that GTP is the mostly accumulated nucleotide. A small portion of guanine may be deaminated to xanthine. It is doubtful, however, if xanthine can be used directly for XMP synthesis. The presence of this nucleotide could not be demonstrated in acid-soluble extracts when worms were incubated with guanine. When adult worms were incubated with hypoxanthine, both ATP and GTP were synthesized. The specific enzymes have not been assayed directly and it is only assumed that the pathways of I M P - * s u c cinyl-AMP-~ AMP and of IMP--, XMP ~ GMP exist in these worms. While no attempt was made to measure the synthesis of succinyl-AMP in the whole worm, radioactive XMP was never found to accumulate. This intermediate, if formed, could have been rapidly converted to GMP. Approximately equal amounts of adenine and guanine ribonucleotides are formed in A. cantonensis. In S. mansoni, however, the radioactivity in adenine ribonucleotides synthesized from hypoxanthine was several fold higher than that in the guanine ribonucleotides (Senft et al., 1973a). Similar results have been observed in the mouse brain (Wong & Henderson, 1972) and Ehrlich ascites-tumour cells (Crabtree & Henderson, 1971). Since an appreciable amount of IMP was found to accumulate and reached a steadystate, the pathway from IMP to AMP can be considered as a limiting-step in the synthesis of adenine nucleotides from hypoxanthine. Under the present experimental conditions, a small amount of inosine and hypoxanthine were released into the medium when the worms were incubated with adenosine. They were not formed when adenine was the precursor. This indicates that inosine and hypoxanthine were not catabolites of AMP but were formed directly by adenosine deaminase and inosine phosphorylase. For the latter reaction, the conditions seem to favour the production of hypoxanthine. For this reason, the pathways for the synthesis of nucleotides from inosine was not investigated in the whole worm. Nevertheless, it seems that inosine cannot be directly converted to IMP by the kinase reaction since this enzyme was not detectable in the cell-free extracts. A small but significant amount of radioactive guanine ribonucleotides was formed when the worms were incubated with I-8-14C]adenine. There are two pathways which can account for this observation. Firstly, AMP may be deaminated to IMP by adenylate deaminase. However the activity of this enzyme in cell-free extracts was too low to be detectable by the method used. If this reaction operates in the intact worm, it must be occurring at a very limited rate. Alternatively, AMP may be dephosphorylated by a

nucleotidase to form adenosine. This can then be converted to inosine and hypoxanthine which could give rise to IMP as discussed before. However, none of these intermediary compounds was radioactively labelled when the worms were incubated with radioactive adenine. Therfore, this alternative route for the conversion of AMP to GMP cannot be supported at this moment. When guanine was used as the precursor, a small amount of ATP accumulated in the worms. This is indicative of a pathway leading from GMP to AMP operating at a slow rate. One of the steps in the pathway, the conversion of IMP to AMP, was shown to be rate-limiting as discussed earlier. The other step, the conversion of GMP to IMP, has been shown to be limited by the guanylate reductase (EC 1.6.6.8) activity in Ehrlich ascites-tumour cells (Crabtree & Henderson, 1971). This is probably true also in this worm since neither IMP nor inosine plus hypoxanthine accumulated. Thus, it is seen that A. cantonensis possesses multiple mechanisms for purine ribonucleotides synthesis. Apart from de novo synthesis (Wong & Ko, 1979), the worms are capable of using adenine, adenosine and hypoxanthine for adenine ribonucleotide synthesis and guanine and hypoxanthine for guanine ribonucleotide synthesis. The synthetic activities in these worms are particularly high in comparison with their catabolic activities. This is shown by the observation that in all the studies made, the radioactivity was found to be associated only with nucleotides and unreacted precursors. Under no circumstances did radioactive uric acid or other intermediates along the degradative pathways of nucleotide metabolism accumulate. It is noted, however, that urea is the main end-product of nitrogen metabolism in adult Ascaris lumbricoides (Farland & MacInnis, 1978) as well as in some plant and free-living parasites (Rothstein, 1970). Acknowledgements--This work was supported by a grant from the Wing Lung Bank Medical Research Fund and was performed with the skilful assistance of Mr P. C. Kwan and Miss Janette Chan.

REFERENCES

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purine salvage metabolism in and outside the free malarial parasite. Exp. Parasit. 37, 138-146. MURRAY A. W. (1966) Purine phosphoribosyltransferase

activities in rat and mouse tissues and in Ehrlich ascitestumour cells. Biochem. J. I00, 664-670. MURRAYA. W. (1968) Some properties of adenosine kinase

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Disposition of adenosine by whole worms. Biochem. Pharmac. 22, 437-447. SMILEY K. L., BERRY A. J. & SUELTER C. H. (1967) An improved purification, crystallization, and some properties of rabbit muscle 5'-adenylic acid deaminase. J. biol. Chem. 242, 2502-2506. WONG P. C. L. & HENDERSONJ. F. (1972) Purine ribonucleotide biosynthesis, interconversion and catabolism in mouse brain in vitro. Biochem. J. 129, 1085 1094. WONC P. C. L. & Ko R. C. (1979) De novo purine ribonucleotide biosynthesis in adult Angiostrongylus cantonensis (Nematoda: Metastrongyloidea). Comp. Biochem. Physiol. 62B, 129 132.