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De novo purine ribonucleotide biosynthesis in adult Angiostrongylus cantonensis (Nematoda: Metastrongyloidea)
Comp. Biochem. Phy.siol., Vol 6211, pp. 129 to 132 © Pergamon Press Ltd 1979. Printed in Great Britain
0305-0491/79/0215-0129S02.00/0
DE NO VO PURINE RIBONUCLEOTIDE BIOSYNTHESIS IN ADULT ANGIOSTRONGYLUS CANTONENSIS (NEMATODA : METASTRONGYLOIDEA) P. C. L. WONG and R. C. Ko Departments of Biochemistry and Zoology, University of Hong Kong, Hong Kong (Received 19 May 1978) A b s t r a c t - - l . Radioactivity was found to be associated with purine ribonucleotides in extracts of gravid
adult A~iostrongylus cantonensis from the lungs of rats when the parasite was incubated in a medium containing [ t'*C]glyeine. 2. The rate of incorporation was at least 0.2 pmole/min per mg of parasite and approximately equal amounts of adenine and guanine ribonucleotides were synthesized. 3. This is the first demonstration of the capacity for a mammalian parasitic helminth to synthesize purines de novo.
INTRODUCTION
Angiostrongylus cantonensis is a member of the metastrongyloid nematode which is a highly specialized group of parasites adapted successfully to live in the pulmonary system, nasal sinuses and other organs of a large varieties of terrestrial and marine mammals. The metastrongyle usually undergoes its different stages of development in specific host's tissues along its migratory route to the final site. Another unique adaptation in this group of parasites is that some members are neurotropic, requiring some unknown factors from the central nervous system of the host for growth and development. The metabolic pathways adopted by these largely tissue dwelling metastrongyles are virtually unknown. A. cantonensis, a normal parasite of rats, is the causal agent of human eosinophilic meningoencephalitis in southeast Asia and south Pacific (Alicata & Jindrak, 1970). The worms develop from the third stage to young adults in the brain of the rodent host and later migrate via the circulatory system to the heart and lungs where they become gravid and oviposition occur. The present report on the biosynthesis of purine ribonucleotides forms part of a programme to survey the nutritional requirements for the growth and development of this parasite in rat tissues. MATERIALS AND METHODS
Chemicals [U-l'*C]Glycine (114 mCi/mmole), [8-14C]adenine (59mCi/mmole), [8-14C]hypoxanthine (60mCi/mmole), [8-14C]guanine (56 mCi/mmole), [8-14C]adenosine (47 mCi/mmole)and [8-14C]inosine (51 mCi/mmole) were obtained from The Radiochemical Centre, Amersham, U.K. Purine ribonucleotides, nucleotides and bases were obtained from Sigma (St. Louis, MI). Other chemicals were of analytical grade from various commercial sources. Source and maintenance of infection The strain of A. cantonensis used in the present study was originally isolated from wild caught Rattus sladeni in Hong Kong in 1974. The worm has since been maintained
in the laboratory in Wistar albino rats and the giant African snail, Achatinafulica, in the following manner: First-stage worms isolated from the lungs and intestine of infected rats using the Baermann's technique were directly injected into the foot or haemocoel of the molluscs. Third-stage worms were recovered by digesting the snails for 3 hr in an artificial gastric juice (0.6~o pepsin and l~o conc HCl in 100 ml distilled water) at 37°C. These worms were then given to anaesthesized rats by stomach intubation. Since many of the established worms will usually die ,due to the intensive host's reactions, a new batch of rats has to be infected every 3-4 months. The adult worms used for the experiments were obtained from the lungs of freshly killed rats. After recovery the worms were examined under a microscope to check for signs of damage. Intact worms could usually liv~in saline for several hours under in vitro conditions. Incubation The worms were rinsed in Krebs-Ringer phosphate solution (pH 7.4). They were blotted lightly by filter paper and their weights recorded. Two to three parasites, weighing approx 20 rag, were transferred into a 25 ml conical flask containing 1 ml of Krebs-Ringer phosphate solution supplemented with 5.5mM glucose. The contents were shaken for l0 rain at 37°C (60 oscillations/min). After that time, various amounts of radioactively labelled precursors were added. An aliquot of 0.01 ml of the medium was removed and the radioactivity associated measured by liquid scintillation counting in 5 ml of Bray's solution (Bray, 1960) with an etficiency of 90~o. The contents were further incubated with shaking at 37°C for up to 120min. At the 60 min mark, a few pg of sodium bicarbonate crystals were added to maintain the pH at 7.4. At the end of the incubation period, an aliquot of 0.01 ml of medium was removed for counting. The difference in radioactivity was taken as the amount of material utilized by the worms. The vitality of the worms during incubation was ascertained by the following parametersi (a) they should maintain normal wriggling motion during the whole period and (b) glucose in the medium is continuously utilized. Acid-soluble and insoluble fractions At the end of the incubation period, the parasites were removed and quickly rinsed in Krebs-Ringer phosphate solution. They were then homogenized in 1 ml of 5~o (w/v) 129
130
P . C . L . WONG and R. C. Ko
cold trichloroacetic acid (TCAJ, The homogenate was centrifuged and the precipitate was washed with 1 ml of TCA. The combined supernatant portions were extracted eight times with 2ml of ether saturated with H20 and then freeze-dried. The precipitate obtained after addition of TCA was washed twice with TCA and the lipids extracted by solvents in the following order: 10ml of ethanol, 10ml of ethanol chloroform {3 : 1), 10 ml of ethanol ether (3 : 1) and finally, 10ml of ether.
based scintillation fluid. The efficiencies of counting were corrected for quenching by the channels ratio method. The results represent the radioactivity in each metabolite rather than the total amount of each component. The RNA in the acid-insoluble fraction was hydrolysed by the method of Crandell & Tremblay (1976) and the nucleotides in the hydrolysate were separated as described by Katz & Comb (1963)
Analysis o1' purme contents
The gravid adults of this metastrongyloid were shown to synthesize purine ribonucleotides from various precursors. As illustrated in Table 1. [~4C]glycine was rapidly taken up by both male and female worms. As much as 21% of the total radioac~ tivity taken up was found in fractions containing purine ribonucleotides and a small but significant a m o u n t in fractions containing RNA. Adenine, adenosine, hypoxanthine and inosine were all utilized for nucleotide synthesis. An analysis of the purine contents in the medium after the worms were incubated with inosine for 2 hr revealed that only 32% of the added inosine remained and 68% of the total radioactivity was found to be associated with hypoxanthine. G u a n i n e was not taken up by the worms. The rates of incorporation of radioactivity into purine ribonucleotides and RNA from [J4C]glycine were studied. In the presence of 8.6 # M glycine, a rate of approx 0.2 pmole of nucleotides formed/min per m g of parasite was measured over a 2 hr period (Fig. 1). At the end of this time~ 42°0 of the glycine in the medium was taken up. A small but constant rate of radioactively labelled RNA formation (0.005 pmole/min per m g of parasite) was also measurable. The radioactivities were proved to be associated with purine derivatives by the following lines of evidence. 1. The radioactively labelled c o m p o n e n t s in the acid-soluble fraction were separated on Dowex-1 (formate form) columns. The results are shown in Fig. 2. These c o m p o u n d s were identified by comparison with the elution pattern of authentic compounds. Radioactivity was found in peaks 4, 5, 6, 7, 8 and 9 which corresponded to A M P , G M P + X M P + IMP, ADP. ATP, G D P and G T P , respectively. Peak 1 contained unreacted glycine and related amino acids and peak
The freeze-dried material of the acid-soluble fraction was dissolved in 1),5 ml of H20. The purine nucleotides were separated from the nucleosides, bases and glycine by either onc of the following systems. System 1 : 0.05 ml of the solution was applied on Whatman No. 1 paper and the components were separated by high-voltage electrophoresis (3 kV/60cm, 45 min)in 0.05 M sodium borate buffer, pH 9.5. In this system, glycine and purine nucleosides and bases remained close to the origin whereas the purine nuclcotides covered a distance of 12-f7cm from the origin. System 2:0.01 ml of the solution was applied on to BakerFlex polyethyleneimine (PEI)-cellulose thin layers on Mylar sheets and the components separated by development in 707g propan-l-ol (Wong & Henderson, 1972). Two methods were also employed to quantitate the amount of radioactivity associated with individual purine nucleotides. First. 0.01 ml of the acid-soluble fraction was applied on PEI-cellulose thin layers and the nucleotides separated by the method of Crabtree & Henderson (1971). Second, all of the acid-soluble material were applied on ~o a Dowex-1 (formate form) column and the nucleotides were separated by stepwise elution with increasing concentations of formic acid and formic acid-ammonium formate mixtures (Blair & Rainnie, 1967). In some cases, the components of the "nucleotide spot" in the chromatograms from system 2 above were characterized. The PEl-cellulose resins at and 1 cm from the origin were scraped into a tube containing 1 ml of 1 N HCI. The contents were heated for 90rain in a boiling water-bath with occasional swirling. The resins were removed by centrifugation and the supernatant solution was evaporated to dryness. The residue was dissolved in 0.5 ml of 1 N HC1 and 0.05 ml of this was applied on Whatman No. 1 paper and developed for 16 hr in a mixture of isopropanol, cone. HC1 and H20 (65:16.7:18.3, v/v). The R z values for guanine, adenine and hypoxanthine were 0.23~ 0.33 and 0.7Z respectively. The radioactivities associated with the various areas were determined by direct counting in 10ml of toluene-
RESI~LTS
Table 1. Uptake of purine ribonucleotide precursors by adult A. cantonensis [~]'{'LllI'~{)[
['Ol]Ltllt/';lriOn { HI
Ill~ o r
worm
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1,1
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I. 2
11
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120
18
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fi.2
11.0
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120
59
21
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120
22
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l3
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21.5
25
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120
29
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120
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g . (,
25
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f~
~
' ;is !;~?~\
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:1.2
~. 2
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De nova purine synthesis in adult Angiostrongylus cantonensis
4oo!
131
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16
l
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o~ E
soo
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E
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L-~,
12
,
1 8
ao
~ 200
6
~
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2 .~
1013
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.,
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I 4
Radioactivity
f
6
I
a
I
8
I
cpm x 10 - 2
t3.
20
40
60 80 Time, min
I00
120
Fig. 1. Uptake of [14C]glycine and incorporation into purine ribonucleotides and RNA by A. cantonensis. The nucleotides in the acid-soluble fraction were separated by system 1 (see Methods) and that in the RNA hydrolysate by system 2.
Fig. 3. Chromatography of acid-soluble extract on PEIcellulose. The incubation conditions were the same as described in Fig. 2. Sections of the chromatogram were cut out and the associated radioactivity determined. A diagrammatic representation of the positions of internal markers is also shown.
method of Crabtree & Henderson (1971), radioactivity was found to be associated with areas corresponding to various purine ribonucleotides. Figure 3 2 contained the nucleosides. Peak 3 was unidentified. shows a typical example in which the radioactivity The ratio of adenine to guanine nucleotides (plus was mainly found in areas containing ATP and GTP. The ratio of adenine to guanine nucleotides (plus IMP and XMP) was estimated to be 1:1.4. 2. When an aliquot of the acid-soluble extract was IMP and XMP) was found to be 1:1.1. In this case, chromatographed on PEI-cellulose thin layers by the the results are probably affected by incomplete removal of [14C]amino acids, as shown by the high radioactivity which remained at the top of the chromateIO gram. 3. When an acid hydrolysate of the material contained in the area corresponding to nucleotides in the chromatograms prepared by system 2 was analyzed o for purine bases, it was found that over 90~ of o 6 the radioactivity was accounted for in the purines. × The ratio of adenine:guanine:hypoxanthine was u 1:0.95:0.5. 4 4. The alkaline hydrolysate of RNA obtained from # the acid-insoluble fraction was analyzed for AMP and 5 9 GMP. After separation in a Dowex-50 (H + form) 2 column, only 1.6~o of the tota L radioactivity was .o "0 accounted for in the fractions containing AMP and GMP. About 4 ~ was eluted by HC1. The rest ! 0 remained associated with the column. The small I0 2 3 40 50 Fraction number amount of radioactively labelled A M P and GMP was T T t ~ T T ! also detectable if the components of the RNA hydroi 2 3 4 5 6 7 8 9 lysate was separated by high voltage electrophoresis Fig. 2. Chromatography of acid-soluble extract on (system 1). Dowex-I (formate form) column. Female parasites (70 mg) were incubated for 2 hr in 2 ml of Krebs-Ringer phosphate DISCUSSION supplemented with 5.5 mM glucose and 11 #M [14C]glycine. Each fraction contained 10ml of eluant and 0.5 ml The results show conclusively that adult A. canwas used for measurement of radioactivity. The eluants were added to the column in succeeding order as indicated tonensis obtained from lungs of rats possess the capaby the arrows. The concentrations of formic acid (FA) and city of utilizing the carbon atoms of glycine for purine ammonium formate (AF) and volumes (in parenthesis) ribonucleotide synthesis. The rate of synthesis were: 1. HzO (40ml); 2. 0.05 M FA (50ml); 3. 0.1 M FA measured in the present study represents the mini(60ml); 4. 0.4M FA (50mi); 5. 2M FA (70ml); 6. 3M mum capacity since the intracellular pool size of glyFA (60ml); 7. 4M FA+0.15M AF (60ml); 8, 4M cine in this parasite is not determined. Approximately FA + 0.3M AF (60mi); 9. 4M FA + 0.6M AF (120ml). equal amounts of radioactivity were found in adenine
TT
132
P . C . L . WONG and R. C. Ko
and guanine nucleotides. If the generally accepted pathways of interconversion of purine ribonucleotides can be assumed for this parasite, it seems that the rates of synthesis of these nucleotides are not restricted by the enzymes responsible to divert IMP, the common intermediate, into the two pathways. Both adenine and hypoxanthine were actively utilized for nucleotide synthesis. The relative rates of accumulation of adenine and guanine ribonucleotides may therefore be measured more accurately by using radioactively labelled hypoxanthine as the precursor. Studies on the malarial organisms Plasmodium herghei and Plasmodium lophurae have shown the lack of de novo purine biosynthesis (Van Dyke et al., 1968; Walsh & Shearman, 19681. Among the trematodes, the blood fluke Schistosoma mansoni also failed to utilize glycine or glucose for purine synthesis (Senti et al., 1972). Heath & Hart (1970a) observed that in the tapeworm Mesocetoides tetrathyridia, de novo purine synthesis did not occur if preformed bases were present in the synthetic medium. However, if the sucker-bearing fragments of the tapeworm were allowed to regenerate, growth could occur without the addition of purines in the medium, thus indicating that de novo purine synthesis must have taken place (Heath & Hart, 1970b). Nevertheless, these workers could not demonstrate the incorporation of [~4C]glycine into nucleic acids. It is tempting, therefore, to generalize that utilization of preformed purines produced by the hosts is a highly specialized parasitic adaptation resulting in the formation of a host-dependent system. The present study reveals an exceptional case in A. cantonensis in which the parasite is shown to be quite capable of synthesizing its own purines from simple nutrients. However, it is still too early to speculate on the significance of this unique capability before more is known about similar synthesis in other metastrongyloids and various groups of nematodes. Because of the differences in habitats, methods and requirements of nutrient uptake, some nematodes may have selectively adopted a system unlike that of the haematozoans, blood flukes or intestinal cestodes.
Acknowled.qements--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.
REFERENCES
ALICATA J. E. & JINDRAK K. (19701 Angiostrongylosis m the Pacifi c and Southeast Asia. C. C. Thomas, Springfield, IU BLAIR D. G. R. & RAINNIED. J. 11967) Acid-soluble metabolites of 2,6-diaminopurine in L strain mouse cells. Can. J. Biochem. 45, 347 350. BRAY G. A. (1960) A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillation counter. Analyt. Biochem. 1, 279-285. CRABTREE G. W. & HENDERSON J. F. (1971) Purine ribonucleotide interconversions in Ehrlich ascites tumor cells in vitro: rate limiting steps. Cancer Res. 31,985 991. CRANDELL D. E. & TREMBLAYG. C. (19761 Pyrimidine biosynthesis and its regulation in embryos of the sea urchin, Arbacia punctulata. Comp. Biochem. Physiol. 55B, 571 581. HEATH R. L. & HART J. L. 0970a) Biosynthesis de novo of purines and pyrimidines in Mesocestoides (Cestodal I. J. Parasit. 56, 98-102. HEATH R. L. & HART J. L. (1970b) Biosynthesis de novo of purines and pyrimidines in Mesocestoides (Cestoda) II. J. Parasit. 56, 340-345. KATZ S. & COMB D. G. (19631 A new method for the determination of the base composition of ribonucleic acid. J. biol. Chem. 238, 3065-3067. SENFT A. W., MIECH R. P., BROWN P. R. & SENFT D. G. (1972) Purine metabolism in Schistosoma mansoni. Int. J. Parasit. 2, 249-260. VAN DYKE K., TREMBLAYG., SZUSTKJEWICZC. & SAXE L. H. (19681 Preliminary studies of purine and pyrimidine incorporation and biosynthesis into nucleic acids of rat blood parasitized by Plasmodium herclhei. J. Protozool. Suppl. 15, 23. WALSH C. & SHERMAN I. W. 11968) Purine and pyrimidine synthesis by the avian malaria parasite, Plasmodium lophurae. J. Protozool. 15. 763 770. WONG P. C. L. & HENDERSONJ. F. (19721 Purine ribonucleotide biosynthesis, interconversion and catabolism in mouse brain in vitro. Biochem. J. 129, 1085- 1094.
0305-0491/79/0215-0129S02.00/0
DE NO VO PURINE RIBONUCLEOTIDE BIOSYNTHESIS IN ADULT ANGIOSTRONGYLUS CANTONENSIS (NEMATODA : METASTRONGYLOIDEA) P. C. L. WONG and R. C. Ko Departments of Biochemistry and Zoology, University of Hong Kong, Hong Kong (Received 19 May 1978) A b s t r a c t - - l . Radioactivity was found to be associated with purine ribonucleotides in extracts of gravid
adult A~iostrongylus cantonensis from the lungs of rats when the parasite was incubated in a medium containing [ t'*C]glyeine. 2. The rate of incorporation was at least 0.2 pmole/min per mg of parasite and approximately equal amounts of adenine and guanine ribonucleotides were synthesized. 3. This is the first demonstration of the capacity for a mammalian parasitic helminth to synthesize purines de novo.
INTRODUCTION
Angiostrongylus cantonensis is a member of the metastrongyloid nematode which is a highly specialized group of parasites adapted successfully to live in the pulmonary system, nasal sinuses and other organs of a large varieties of terrestrial and marine mammals. The metastrongyle usually undergoes its different stages of development in specific host's tissues along its migratory route to the final site. Another unique adaptation in this group of parasites is that some members are neurotropic, requiring some unknown factors from the central nervous system of the host for growth and development. The metabolic pathways adopted by these largely tissue dwelling metastrongyles are virtually unknown. A. cantonensis, a normal parasite of rats, is the causal agent of human eosinophilic meningoencephalitis in southeast Asia and south Pacific (Alicata & Jindrak, 1970). The worms develop from the third stage to young adults in the brain of the rodent host and later migrate via the circulatory system to the heart and lungs where they become gravid and oviposition occur. The present report on the biosynthesis of purine ribonucleotides forms part of a programme to survey the nutritional requirements for the growth and development of this parasite in rat tissues. MATERIALS AND METHODS
Chemicals [U-l'*C]Glycine (114 mCi/mmole), [8-14C]adenine (59mCi/mmole), [8-14C]hypoxanthine (60mCi/mmole), [8-14C]guanine (56 mCi/mmole), [8-14C]adenosine (47 mCi/mmole)and [8-14C]inosine (51 mCi/mmole) were obtained from The Radiochemical Centre, Amersham, U.K. Purine ribonucleotides, nucleotides and bases were obtained from Sigma (St. Louis, MI). Other chemicals were of analytical grade from various commercial sources. Source and maintenance of infection The strain of A. cantonensis used in the present study was originally isolated from wild caught Rattus sladeni in Hong Kong in 1974. The worm has since been maintained
in the laboratory in Wistar albino rats and the giant African snail, Achatinafulica, in the following manner: First-stage worms isolated from the lungs and intestine of infected rats using the Baermann's technique were directly injected into the foot or haemocoel of the molluscs. Third-stage worms were recovered by digesting the snails for 3 hr in an artificial gastric juice (0.6~o pepsin and l~o conc HCl in 100 ml distilled water) at 37°C. These worms were then given to anaesthesized rats by stomach intubation. Since many of the established worms will usually die ,due to the intensive host's reactions, a new batch of rats has to be infected every 3-4 months. The adult worms used for the experiments were obtained from the lungs of freshly killed rats. After recovery the worms were examined under a microscope to check for signs of damage. Intact worms could usually liv~in saline for several hours under in vitro conditions. Incubation The worms were rinsed in Krebs-Ringer phosphate solution (pH 7.4). They were blotted lightly by filter paper and their weights recorded. Two to three parasites, weighing approx 20 rag, were transferred into a 25 ml conical flask containing 1 ml of Krebs-Ringer phosphate solution supplemented with 5.5mM glucose. The contents were shaken for l0 rain at 37°C (60 oscillations/min). After that time, various amounts of radioactively labelled precursors were added. An aliquot of 0.01 ml of the medium was removed and the radioactivity associated measured by liquid scintillation counting in 5 ml of Bray's solution (Bray, 1960) with an etficiency of 90~o. The contents were further incubated with shaking at 37°C for up to 120min. At the 60 min mark, a few pg of sodium bicarbonate crystals were added to maintain the pH at 7.4. At the end of the incubation period, an aliquot of 0.01 ml of medium was removed for counting. The difference in radioactivity was taken as the amount of material utilized by the worms. The vitality of the worms during incubation was ascertained by the following parametersi (a) they should maintain normal wriggling motion during the whole period and (b) glucose in the medium is continuously utilized. Acid-soluble and insoluble fractions At the end of the incubation period, the parasites were removed and quickly rinsed in Krebs-Ringer phosphate solution. They were then homogenized in 1 ml of 5~o (w/v) 129
130
P . C . L . WONG and R. C. Ko
cold trichloroacetic acid (TCAJ, The homogenate was centrifuged and the precipitate was washed with 1 ml of TCA. The combined supernatant portions were extracted eight times with 2ml of ether saturated with H20 and then freeze-dried. The precipitate obtained after addition of TCA was washed twice with TCA and the lipids extracted by solvents in the following order: 10ml of ethanol, 10ml of ethanol chloroform {3 : 1), 10 ml of ethanol ether (3 : 1) and finally, 10ml of ether.
based scintillation fluid. The efficiencies of counting were corrected for quenching by the channels ratio method. The results represent the radioactivity in each metabolite rather than the total amount of each component. The RNA in the acid-insoluble fraction was hydrolysed by the method of Crandell & Tremblay (1976) and the nucleotides in the hydrolysate were separated as described by Katz & Comb (1963)
Analysis o1' purme contents
The gravid adults of this metastrongyloid were shown to synthesize purine ribonucleotides from various precursors. As illustrated in Table 1. [~4C]glycine was rapidly taken up by both male and female worms. As much as 21% of the total radioac~ tivity taken up was found in fractions containing purine ribonucleotides and a small but significant a m o u n t in fractions containing RNA. Adenine, adenosine, hypoxanthine and inosine were all utilized for nucleotide synthesis. An analysis of the purine contents in the medium after the worms were incubated with inosine for 2 hr revealed that only 32% of the added inosine remained and 68% of the total radioactivity was found to be associated with hypoxanthine. G u a n i n e was not taken up by the worms. The rates of incorporation of radioactivity into purine ribonucleotides and RNA from [J4C]glycine were studied. In the presence of 8.6 # M glycine, a rate of approx 0.2 pmole of nucleotides formed/min per m g of parasite was measured over a 2 hr period (Fig. 1). At the end of this time~ 42°0 of the glycine in the medium was taken up. A small but constant rate of radioactively labelled RNA formation (0.005 pmole/min per m g of parasite) was also measurable. The radioactivities were proved to be associated with purine derivatives by the following lines of evidence. 1. The radioactively labelled c o m p o n e n t s in the acid-soluble fraction were separated on Dowex-1 (formate form) columns. The results are shown in Fig. 2. These c o m p o u n d s were identified by comparison with the elution pattern of authentic compounds. Radioactivity was found in peaks 4, 5, 6, 7, 8 and 9 which corresponded to A M P , G M P + X M P + IMP, ADP. ATP, G D P and G T P , respectively. Peak 1 contained unreacted glycine and related amino acids and peak
The freeze-dried material of the acid-soluble fraction was dissolved in 1),5 ml of H20. The purine nucleotides were separated from the nucleosides, bases and glycine by either onc of the following systems. System 1 : 0.05 ml of the solution was applied on Whatman No. 1 paper and the components were separated by high-voltage electrophoresis (3 kV/60cm, 45 min)in 0.05 M sodium borate buffer, pH 9.5. In this system, glycine and purine nucleosides and bases remained close to the origin whereas the purine nuclcotides covered a distance of 12-f7cm from the origin. System 2:0.01 ml of the solution was applied on to BakerFlex polyethyleneimine (PEI)-cellulose thin layers on Mylar sheets and the components separated by development in 707g propan-l-ol (Wong & Henderson, 1972). Two methods were also employed to quantitate the amount of radioactivity associated with individual purine nucleotides. First. 0.01 ml of the acid-soluble fraction was applied on PEI-cellulose thin layers and the nucleotides separated by the method of Crabtree & Henderson (1971). Second, all of the acid-soluble material were applied on ~o a Dowex-1 (formate form) column and the nucleotides were separated by stepwise elution with increasing concentations of formic acid and formic acid-ammonium formate mixtures (Blair & Rainnie, 1967). In some cases, the components of the "nucleotide spot" in the chromatograms from system 2 above were characterized. The PEl-cellulose resins at and 1 cm from the origin were scraped into a tube containing 1 ml of 1 N HCI. The contents were heated for 90rain in a boiling water-bath with occasional swirling. The resins were removed by centrifugation and the supernatant solution was evaporated to dryness. The residue was dissolved in 0.5 ml of 1 N HC1 and 0.05 ml of this was applied on Whatman No. 1 paper and developed for 16 hr in a mixture of isopropanol, cone. HC1 and H20 (65:16.7:18.3, v/v). The R z values for guanine, adenine and hypoxanthine were 0.23~ 0.33 and 0.7Z respectively. The radioactivities associated with the various areas were determined by direct counting in 10ml of toluene-
RESI~LTS
Table 1. Uptake of purine ribonucleotide precursors by adult A. cantonensis [~]'{'LllI'~{)[
['Ol]Ltllt/';lriOn { HI
Ill~ o r
worm
~.'x
Firw
i)f
it~cllbl~tioll fmin]
'i, taken
tip
: ;i-. !lut I(.>tides
(1,1
.
(1
Hale
12n
1,1
i . ~,
I. 2
11
Hale
120
18
5.{)
fi.2
11.0
7()
rema 1 e
120
59
21
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It,.9
1S
Female
120
22
2B
L2
]I)-p o x,'~l/t h ] TIC
I O. !)
1 6 . F,
Fema 1 e
I 2(l
l3
25
~ . (]
~dcnos i ne
21.5
25
Fema l o
120
29
.84
i . {I
In,,~ine
to. 0
IS
Female
120
q
39
2. 1
8.9
9
Fema 1 e
12O
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g . (,
25
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f~c)
f~
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~. 2
0
De nova purine synthesis in adult Angiostrongylus cantonensis
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l
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6
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1013
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.,
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I 4
Radioactivity
f
6
I
a
I
8
I
cpm x 10 - 2
t3.
20
40
60 80 Time, min
I00
120
Fig. 1. Uptake of [14C]glycine and incorporation into purine ribonucleotides and RNA by A. cantonensis. The nucleotides in the acid-soluble fraction were separated by system 1 (see Methods) and that in the RNA hydrolysate by system 2.
Fig. 3. Chromatography of acid-soluble extract on PEIcellulose. The incubation conditions were the same as described in Fig. 2. Sections of the chromatogram were cut out and the associated radioactivity determined. A diagrammatic representation of the positions of internal markers is also shown.
method of Crabtree & Henderson (1971), radioactivity was found to be associated with areas corresponding to various purine ribonucleotides. Figure 3 2 contained the nucleosides. Peak 3 was unidentified. shows a typical example in which the radioactivity The ratio of adenine to guanine nucleotides (plus was mainly found in areas containing ATP and GTP. The ratio of adenine to guanine nucleotides (plus IMP and XMP) was estimated to be 1:1.4. 2. When an aliquot of the acid-soluble extract was IMP and XMP) was found to be 1:1.1. In this case, chromatographed on PEI-cellulose thin layers by the the results are probably affected by incomplete removal of [14C]amino acids, as shown by the high radioactivity which remained at the top of the chromateIO gram. 3. When an acid hydrolysate of the material contained in the area corresponding to nucleotides in the chromatograms prepared by system 2 was analyzed o for purine bases, it was found that over 90~ of o 6 the radioactivity was accounted for in the purines. × The ratio of adenine:guanine:hypoxanthine was u 1:0.95:0.5. 4 4. The alkaline hydrolysate of RNA obtained from # the acid-insoluble fraction was analyzed for AMP and 5 9 GMP. After separation in a Dowex-50 (H + form) 2 column, only 1.6~o of the tota L radioactivity was .o "0 accounted for in the fractions containing AMP and GMP. About 4 ~ was eluted by HC1. The rest ! 0 remained associated with the column. The small I0 2 3 40 50 Fraction number amount of radioactively labelled A M P and GMP was T T t ~ T T ! also detectable if the components of the RNA hydroi 2 3 4 5 6 7 8 9 lysate was separated by high voltage electrophoresis Fig. 2. Chromatography of acid-soluble extract on (system 1). Dowex-I (formate form) column. Female parasites (70 mg) were incubated for 2 hr in 2 ml of Krebs-Ringer phosphate DISCUSSION supplemented with 5.5 mM glucose and 11 #M [14C]glycine. Each fraction contained 10ml of eluant and 0.5 ml The results show conclusively that adult A. canwas used for measurement of radioactivity. The eluants were added to the column in succeeding order as indicated tonensis obtained from lungs of rats possess the capaby the arrows. The concentrations of formic acid (FA) and city of utilizing the carbon atoms of glycine for purine ammonium formate (AF) and volumes (in parenthesis) ribonucleotide synthesis. The rate of synthesis were: 1. HzO (40ml); 2. 0.05 M FA (50ml); 3. 0.1 M FA measured in the present study represents the mini(60ml); 4. 0.4M FA (50mi); 5. 2M FA (70ml); 6. 3M mum capacity since the intracellular pool size of glyFA (60ml); 7. 4M FA+0.15M AF (60ml); 8, 4M cine in this parasite is not determined. Approximately FA + 0.3M AF (60mi); 9. 4M FA + 0.6M AF (120ml). equal amounts of radioactivity were found in adenine
TT
132
P . C . L . WONG and R. C. Ko
and guanine nucleotides. If the generally accepted pathways of interconversion of purine ribonucleotides can be assumed for this parasite, it seems that the rates of synthesis of these nucleotides are not restricted by the enzymes responsible to divert IMP, the common intermediate, into the two pathways. Both adenine and hypoxanthine were actively utilized for nucleotide synthesis. The relative rates of accumulation of adenine and guanine ribonucleotides may therefore be measured more accurately by using radioactively labelled hypoxanthine as the precursor. Studies on the malarial organisms Plasmodium herghei and Plasmodium lophurae have shown the lack of de novo purine biosynthesis (Van Dyke et al., 1968; Walsh & Shearman, 19681. Among the trematodes, the blood fluke Schistosoma mansoni also failed to utilize glycine or glucose for purine synthesis (Senti et al., 1972). Heath & Hart (1970a) observed that in the tapeworm Mesocetoides tetrathyridia, de novo purine synthesis did not occur if preformed bases were present in the synthetic medium. However, if the sucker-bearing fragments of the tapeworm were allowed to regenerate, growth could occur without the addition of purines in the medium, thus indicating that de novo purine synthesis must have taken place (Heath & Hart, 1970b). Nevertheless, these workers could not demonstrate the incorporation of [~4C]glycine into nucleic acids. It is tempting, therefore, to generalize that utilization of preformed purines produced by the hosts is a highly specialized parasitic adaptation resulting in the formation of a host-dependent system. The present study reveals an exceptional case in A. cantonensis in which the parasite is shown to be quite capable of synthesizing its own purines from simple nutrients. However, it is still too early to speculate on the significance of this unique capability before more is known about similar synthesis in other metastrongyloids and various groups of nematodes. Because of the differences in habitats, methods and requirements of nutrient uptake, some nematodes may have selectively adopted a system unlike that of the haematozoans, blood flukes or intestinal cestodes.
Acknowled.qements--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.
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