- Email: [email protected]
Amino acid biosynthesis in Haemonchus contortus from C14-labelled precursors, in vitro
Veterinary Parasitology, 15 (1984) 293--299 Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands
293
A M I N O A C I D B I O S Y N T H E S I S I N HA E M O N C H U S C O N T O R T U S F R O M C14-LABELLED PRECURSORS, IN VITRO
JYOTIKA KAPUR and M.L. SOOD Department o f Zoology, Punjab Agricultural University, Ludhiana-141 004 (India)
(Accepted for publication 1 February 1984)
ABSTRACT
Kapur, J. and Sood, M.L., 1984. Amino acid biosynthesis in Haemonchus contortus from C~4-1abelled precursors, in vitro. Vet. Parasitol., 15: 293--299. Adult Haemonchus contortus (Nematoda: Trichostrongylidae) was investigated for its ability to utilize various C'4-1abelled precursors, i.e., glucose, acetate, CO 2 and palmitic acid, for amino acid biosynthesis. H. contortus has been demonstrated to be capable of synthesizing essential as well as non-essential amino acids. Label from all the precursors was detected in aspartic acid, lysine, histidine, cystine, cysteine, glutamic acid, proline, arginine, tyrosine, alanine, glycine, serine, valine, methionine, leucine and isoleucine. Glutamic acid, aspartic acid, alanine, glycine and serine were synthesized to a greater extent relative to the other amino acids, regardless of the precursor employed. However, in case of glucose, there was comparatively less incorporation into glycine and serine. Incorporation of C '4 into various amino acids is evidence for the operation of tricarboxylic acid cycle. Further, the fact that carbon from palmitic acid appears in amino acids, indicates that adult H. contortus is capable of catabolizing long-chain fatty acids. Possible mechanisms for the involvement of various precursors in amino acids biosynthesis are examined here.
INTRODUCTION M a n y n e m a t o d e s have been investigated f o r their a m i n o acid c o m p o s i t i o n (Herlich, 1 9 6 6 ; A b b a s a n d F o o r , 1 9 7 8 ; Nigam, 1 9 7 8 , 1 9 7 9 ; K a p u r and S o o d , 1984). No significant variation in the c o m p o s i t i o n o f a m i n o acids o f n e m a t o d e s i n h a b i t i n g d i f f e r e n t e n v i r o n m e n t s has been f o u n d . This indicates t h a t in n e m a t o d e s the c o m p o s i t i o n o f the a m i n o acid p o o l remains largely u n a f f e c t e d , n o t being d e p e n d e n t on their a b s o r p t i o n f r o m e x t e r n a l environm e n t . Thus, the n e m a t o d e s m u s t be c a p a b l e o f a m i n o acid biosynthesis. This has been d e m o n s t r a t e d in m a n y n e m a t o d e species (Nicholas et al., 1 9 6 0 ; R o t h s t e i n and T o m l i n s o n , 1 9 6 1 , 1 9 6 2 ; R o t h s t e i n and M a y o h , 1 9 6 4 ; P6rez et al., 1 9 6 7 ; S l o n k a et al., 1 9 7 3 ) . In parasitic n e m a t o d e s , w h e r e the m a j o r m e t a b o l i c activities are d i r e c t e d
0304-4017/84/$03.00
© 1984 Elsevier Science Publishers B.V.
294 towards egg production, the emphasis on protein and hence amino acid biosynthesis must be considerable. If we are able to find some differences in the enzymes involved in amino acid biosynthesis by the parasite and the host, we can selectively check the synthesis of amino acids in the parasite. Thus, these would not be available for incorporation into egg proteins and hence the propagation of a species could be checked. Therefore, keeping in view the importance of amino acid biosynthesis in nematodes, adult H. contortus (Rud., 1803) was investigated for determining whether or not it had the potential of synthesizing amino acids and if so, exactly which ones were synthesized. The various precursors employed in the present study include glucose and acetate, known to be incorporated by nematodes into amino acids (Rothstein amd Mayoh, 1964), CO2 known to be incorporated into carbon-chain of TCA cycle related amino acids (Rothstein, 1965) and palmitic acid (to study the relative importance of long chain fatty acids in amino acid biosynthesis). MATERIALS AND METHODS Live adult specimens of H. contortus were collected from the abomasa of infected goats (Capra hircus) from local abattoirs. Worms were washed thoroughly in 0.9% saline and freed from the adhering host material.
Incubation m e d i u m The incubation medium used in this investigation had the following composition: 10.7 g NaC1; 0.2 g KC1; 0.265 g CaC12 • 2H20; 0.21 g MgC12 • 6H20; 1.0 g NaHCO3; 0.05 g NaH2PO4; 1.0 g glucose; 3.0 g streptopencillin; 0.01 g nystatin, and the volume made up to 1 1 with double distilled water. For each experiment, 500 mg of worm tissue were transferred to flasks containing 10 ml of the above medium. To each flask 12.5 pCi of the labelled precursor was added. The various precursors employed in the present studies included U-C14-D-glucose, sodium-l-C~4-acetate, sodium-C~4-bicarbonate and C14-1-pabmitic acid. Worms were incubated at 39 -+ 1°C for 4 h on a metabolic shaker. After the completion of incubation period, a few drops of chloroform: methanol (2:1 v/v) were added in order to stop the incorporation. Media were poured out of the flasks and worms washed several times with distilled water.
Extraction o f amino acids Worms of each group were homogenised in 10% trichloroacetic acid (w/v) for 10 min in a teflon pestle homogenizer (Remi Udyog, Bombay). These were then sonicated for 5 min using an ultrasonic vibrator (Model VPL-P2, Vibronic Pvt. Ltd., Bombay) and finally centrifuged in a K24 centrifuge, Jenetzki, DDR) at 4°C and 1800 g for 10 min. Precipitate thus obtained was
295 extracted twice with 5% TCA at 0°C. Subsequently, 10% TCA was added to the precipitate and refluxed on a steam bath for 15 min, centrifuged at 5°C and 1800 g for 10 min. Proteins obtained in the above precipitate were hydrolysed in 6 N HC1 by autoclaving at 15 lb for 4 h. Fractionation of amino acids
Protein hydrolysate (50 pl of each sample) was analysed for amino acids by uni-dimensional descending paper chromatography, using Whatman No. I paper. Solvent system used was n-butanol:acetic acid:water (4:1:5 v/v). Duplicate chromatograms were developed for each hydrolysate. After completion of the development, the chromatograms were air dried. One of the duplicates was sprayed with 0.2% ninhydrin in n-butanol to locate the different amino acids. Measurement o f radioactivity
The spots on the above chromatogram were used as template and the corresponding areas of the other chromatogram were cut out and transferred directly to the scintillation vials. Radioactivity was measured using a ~-scintillation spectrometer. For measurement of radioactivity, Bray's (1960) scintillation fluid was used which had the following composition: 4 g PPO; 200 mg POPOP; 60 g naphthalene; 20 ml ethylene glycol; 100 ml methanol, and the volume made to 1 1 with dioxan. 10 ml of this fluid was added to each vial. RESULTS C 14 has been detected in the proteins of H. contortus (Kapur and Sood, unpublished observations), thus indicating its ability to synthesize proteins, and hence the amino acids. Results of the incorporation of carbon from various precursors into different amino acids of adult H. contortus are given in Table I. It is evident from Table I that the order of incorporation of label from glucose into various amino acids was Glu > Asp > Ala > Tyr > Leu+Ile Pro ;, Arg > Gly+Ser > Val+Met > Cys+Cys-Cys > Lys+His. In case of acetate it was Glu > Asp > Gly+Ser > Ala > Pro=Tyr > Arg > Lys+His Val+Met > Leu+Ile > Cys+Cys-Cys. While in the case of CO2 the order was Ala > Glu > Asp > Gly+Ser > Lys+His ~ Tyr > Pro=Arg > Leu+Ile Val+Met > Cys+Cys-Cys. In the case of palmitic acid, the order of incorporation was Asp > Glu > Ala > Gly+Ser > Val+Met > Arg > Tyr > Leu+Ile Cys+Cys-Cys > Pro > Lys+His. Thus, it is seen that Glu, Asp, Gly, Ser, and Ala are the amino acids in which m a x i m u m label is incorporated, regardless of the precursor employed. However, in the case of glucose, incorporation into Gly and Ser is comparatively less.
296 TABLEI
Incorporation of carbon from C~4-1abelled glucose, acetate, sodium bicarbonate and palmitic acid into amino acids of adult Haemonchus contortus. The results are expressed as the relative percentage of incorporation of total amino acids recovered from protein hydrolysate Amino acid
Cystine (Cys-Cys) + Cysteine (Cys) Aspartic acid (Asp) Lysine (Lys) + Histidine (His) Glutamic acid (Glu) Proline (Pro) Arginine (Arg) Tyrosine (Tyr) Alanine (Ala) Glycine (Gly) + Serine (Set) Valine (Val) + Methionine (Met) Leucine (Leu) + Isoleucine (Ile)
Precursor Glucose
Acetate
Carbon dioxide (HCO;)
Palmitic acid
4.3 15.6 4.1 25.7 6.6 6.5 8.1 11.0 5.8 5.6 6.8
4.4 15.5 5.0 19.2 8.7 7.1 8.7 9.5 12.5 4.8 4.6
4.1 11.1 9.1 16.9 5.2 5.2 7.7 20.3 10.8 4.5 5.0
4.9 20.6 3.7 16.3 4.4 7.8 5.9 12.6 9.9 8.3 5.4
DISCUSSION The ability to synthesize essential amino acids had been a t t r i b u t e d only to plants and microorganisms. However, there are a few reports indicating t hat certain helminth species also have this p r o p e r t y . These include Caenorhabditis briggsae (Rothstein and Tomlinson, 1961, p r o b a b l y t he first r e p o r t o f essential amino acids biosynthesis by a multicellular animal species), Cooperia punctata (Slonka et al., 1973), Clinostomum campanulatum (Thomas and Gallicchio, 1967), Moniliformis dubius (Graff, 1964) and A n c y l o s t o m a caninum (P~rez et al., 1967). The concept is f u r t h e r r e f u t e d by our present studies in which H. contortus has been d e m o n s t r a t e d to be capable o f essential amino acid biosynthesis. The p r o p o s e d scheme for the involvement of different precursors in amino acid biosynthesis is given in Fig. 1. It is evident from t he figure t hat the inc o r p o r a t i o n o f c a r b o n f r om each precursor into amino acids is via the TCA cycle. Also, the low activities of aconitase and isocitrate dehydrogenase in adult H. contortus (Kaur and Sood, 1983) support the idea t hat the TCA cycle has only a m i n o r f u n c t i o n as regards the energy p r o d u c t i o n . It is possibly c o n c e r n e d with the interconversions of carbon-skeletons into amino acids metabolism (Saz and Vidrine, 1959; Oya et al., 1962, 1965). Most of the label is f o u n d in Glu, Asp, Ala, Gly and Ser, regardless of the precursor e m p l o y e d . This has also been d e m o n s t r a t e d in o t h e r helminths (Rothstein, 1963; Slonka et al., 1973). Higher a m o u n t of label in Asp and Glu suggests
297
I GLUCOSEI
I -[P.OSP.A GLUCOS r- ERYTHROSE-4- PHOS. / PHATE I
I TRIOSE PHOSPHATE
SERINE q T RYP'I'OPHAN l
[,'AL..,.cAC,O)
PHOSPHOENOL PYRU VATE I |
k LA~IINE,e~
~,
'~
/I
/I
+
"TRYP~PHAN/)2
PYRUVATE ., LEUCrNE
/I
/
#
TYROSINE
i
~1 '
/
I
j
~ "ACETYLCoA.-~LEUCINE VALINE
ISOLEUCINE ALANINE
LYSINE~
t
I~
ASPARTATE.-,P.THREONINE<-.bGLYCINE
t J,"~SERINE
/OXALOACETATE,~ FUMARATE
ARGIf NINEI
METHIONINE
CITRATE
TCACYCLE
1
~, E CYSI'EIN
SERINE
CYS~rINE
~.- KETOGLUTARATE
~ S U C C I N Y L CoA/ ARGININE4,.~ GLUTAMATE
TRYPTOPHAN ~//~R~!,NE PHENYLALANINE Fig. 1.Proposed scheme for the involvement of C'4-glucose,acetate,carbon dioxide and palmiticacid in amino acid biosynthesisin adultsof Haernonchus contortus.
the presence of TCA cycle intermediates (a-ketoglutaric acid and oxaloacetic acid) which are :important links between amino acid and carbohydrate metabolism. Adult H. contortus, unlike C. punctata (Slonka et al., 1973} is able to synthesize Val and Met from glucose, though only to a small extent. C. briggsae is also capable of synthesizing Val (Nicholas et al., 1960). Ala, Glu and Asp are among the top amino acids labelled on incubation with C14-glucose. This observation is supported by the findings of Slonka et al. (1973) on C. punctata. However, in Moniliformis dubius (Graff, 1964) no label was found in Glu. In C. briggsae, C 14 from glucose was incorporated in the following order: Val > Ala > Lys > Glu > Asp > Ser > Thr > His > Arg > Gly (Nicholas et al., 1960; Rothstein and Tomlinson, 1961). In contrast to these observations, in the present studies, valine is synthesized to a small extent. Also, there was incorporation into Cys-Cys, Cys, Tyr, Leu,
298
Ile, Pro and Met in the present studies which was n o t detected in C. briggsae. As regards the biosynthesis of amino acids from acetate and CO2, H. contortus like C. briggsae (Nicholas et al., 1960; Rothstein, 1965; Rothstein and Mayoh, 1964) is capable of synthesizing amino acids related to TCA cycle. In addition, it is also capable of incorporating into Cys-Cys, Cys, Pro, Tyr, Met. Presence of C '4 in Ala, Glu and Asp in H. contortus is a good evidence that at least part of the TCA cycle operates. These results might be explained by implicating succinate as the primary p r o d u c t of CO: fixation, as also reported in Ascaris (Saz and Vidrine, 1959). So far there is no report regarding the role of long chain fatty acids in amino acid biosynthesis. In the present studies, we have demonstrated that the label from palmitic acid is incorporated in all the 16 amino acids. From these observations, it can be speculated that adult H. contortus is capable of catabolizing palmitic acid to acetyl CoA, which is then incorporated into amino acids via TCA cycle. CONCLUSIONS
Adult H. contortus has the potential to synthesize essential as well as nonessential amino acids in vitro. However, from the present data, it is n o t possible to predict whether or not the synthesis of amino acids is at a level commensurate with reproduction. Therefore, further studies need to be carried out on these lines. Also, it would be interesting to elucidate the pathways of amino acid biosynthesis. These studies can give us a deeper insight into the metabolism of H. contortus. ACKNOWLEDGEMENTS
The authors wish to thank Professor S.S. Guraya, Head of the Department for the laboratory facilities. J. Kaput also wishes to thank the authorities of C.S.I.R., New Delhi for the Junior/Senior Research Fellowship grant.
REFERENCES Abbas, M. and Foor, W.E., 1978. Ascaris s u u m : Free amino acids and proteins in the pseudocoelom, seminal vesicle, and glandular vas deferens. Exp. Parasitol., 45: 263-273. Bray, G.A., 1960. A simple efficient scintillator for counting aqueous solutions in a liquid scintillation counter. An. Biochem., 1 : 279--285. Graff, D.J., 1964. Metabolism of C'4-glucose by Moniliforrnis dubius (Acanthocephala). J. Parasitol., 50: 230--234. Herlich, H., 1966. Amino acid composition of some strongyle parasites of cattle. Proc. Helminthol. Soc. Wash., 33: 103--105. Kapur, J. and Sood, M.L., 1984. Amino acid composition of the adults of H a e m o n c h u s c o n t o r t u s (Nematoda: Trichostrongylidae). Helminthologia, 21: in press.
299
Kaur, R. and Sood, M.L., 1983. The effects o f dl-tetramisole and rafoxanide on tricarboxylic acid cycle enzymes of Haemonchus contortus, in vitro. Vet. Parasitol., 13: 333--340. Nicholas, W.L., Dougherty, E.C. and Hansen, E.L., 1960. The incorporation of 14C from sodium acetate-2-14C into the amino acids of the soil-inhabiting nematode, Caenorhabditis briggsae. J. Exp. Biol., 37: 435--443. Nigam, S.C., 1978. Free amino acids from Ascaridia galli. Indian J. Parasitol., 2: 157-158. Nigam, S.C., 1979. Amino acid composition of nematode parasites. Indian J. Helminthol., 31: 69--71. Oya, H., Kikuchi, G., Hayashi, H. and Bando, T., 1962. The occurrence of tricarboxylic acid cycle in the muscle of Ascaris lurnbricoides var. suis. Jpn. J. Parasitol., 11 : 291. Oya, H., Kikuchi, G., Bando, T. and Hayashi, H., 1965. Muscle tricarboxylic acid cycle in Ascaris lumbricoides vat. suis. Exp. Parasitol., 17: 229--240. P~rez Gim~nez, M.E., Gim~nez, A. and Gaede, K., 1967. Metabolic transformation of 1tiC-glucose into tissue proteins of Ancylostoma caninum. Exp. Parasitol., 21: 215-233. Rothstein, M., 1963. Nematode biochemistry--III. Excretion products. Comp. Biochem. Physiol., 9 : 51--59. Rothstein, M., 1965. Nematode biochemistry--V. Intermediary metabolism and amino acid interconversions in Caenorhabditis briggsae. Comp. Biochem. Physiol., 14: 541-552. Rothstein, M. a r d Mayoh, H., 1964. Nematode biochemistry IV. On isocitrate lyase in Caenorhabditis briggsae. Arch. Biochem. Biophys., 108: 134--142. Rothstein, M. and Tomlinson, G., 1961. Biosynthesis of amino acids by the nematode Caenorhabditis briggsae. Biochem. Biophys. Acta, 49: 625--627. Rothstein, M. and Tomlinson, G., 1962. Nematode biochemistry. II. Biosynthesis of amino acids. Biochem. Biophys. Acta, 63: 471--480. Saz, H.J. and Vidrine, A., Jr., 1959. The mechanism of formation of succinate and propionate by Ascaris lumbricoides muscle. J. Biol. Chem., 234: 2001--2005. Slonka, G.F., Ridley, R.K. and Leland, S.E., Jr., 1973. The use of in vitro grown Cooperia punctata (Nematoda: Triehostrongylidae) to study incorporation of carbon from D-glucose-U-~4C into major chemical fractions. J. Parasitol., 59: 282--288. Thomas, R.E. and Galliechio, V., 1967. Metabolism of 14C-glucose by metacercariae of Clinostomum carnpanulaturn (Trematoda). J. Parasitol., 53: 981--984.
293
A M I N O A C I D B I O S Y N T H E S I S I N HA E M O N C H U S C O N T O R T U S F R O M C14-LABELLED PRECURSORS, IN VITRO
JYOTIKA KAPUR and M.L. SOOD Department o f Zoology, Punjab Agricultural University, Ludhiana-141 004 (India)
(Accepted for publication 1 February 1984)
ABSTRACT
Kapur, J. and Sood, M.L., 1984. Amino acid biosynthesis in Haemonchus contortus from C~4-1abelled precursors, in vitro. Vet. Parasitol., 15: 293--299. Adult Haemonchus contortus (Nematoda: Trichostrongylidae) was investigated for its ability to utilize various C'4-1abelled precursors, i.e., glucose, acetate, CO 2 and palmitic acid, for amino acid biosynthesis. H. contortus has been demonstrated to be capable of synthesizing essential as well as non-essential amino acids. Label from all the precursors was detected in aspartic acid, lysine, histidine, cystine, cysteine, glutamic acid, proline, arginine, tyrosine, alanine, glycine, serine, valine, methionine, leucine and isoleucine. Glutamic acid, aspartic acid, alanine, glycine and serine were synthesized to a greater extent relative to the other amino acids, regardless of the precursor employed. However, in case of glucose, there was comparatively less incorporation into glycine and serine. Incorporation of C '4 into various amino acids is evidence for the operation of tricarboxylic acid cycle. Further, the fact that carbon from palmitic acid appears in amino acids, indicates that adult H. contortus is capable of catabolizing long-chain fatty acids. Possible mechanisms for the involvement of various precursors in amino acids biosynthesis are examined here.
INTRODUCTION M a n y n e m a t o d e s have been investigated f o r their a m i n o acid c o m p o s i t i o n (Herlich, 1 9 6 6 ; A b b a s a n d F o o r , 1 9 7 8 ; Nigam, 1 9 7 8 , 1 9 7 9 ; K a p u r and S o o d , 1984). No significant variation in the c o m p o s i t i o n o f a m i n o acids o f n e m a t o d e s i n h a b i t i n g d i f f e r e n t e n v i r o n m e n t s has been f o u n d . This indicates t h a t in n e m a t o d e s the c o m p o s i t i o n o f the a m i n o acid p o o l remains largely u n a f f e c t e d , n o t being d e p e n d e n t on their a b s o r p t i o n f r o m e x t e r n a l environm e n t . Thus, the n e m a t o d e s m u s t be c a p a b l e o f a m i n o acid biosynthesis. This has been d e m o n s t r a t e d in m a n y n e m a t o d e species (Nicholas et al., 1 9 6 0 ; R o t h s t e i n and T o m l i n s o n , 1 9 6 1 , 1 9 6 2 ; R o t h s t e i n and M a y o h , 1 9 6 4 ; P6rez et al., 1 9 6 7 ; S l o n k a et al., 1 9 7 3 ) . In parasitic n e m a t o d e s , w h e r e the m a j o r m e t a b o l i c activities are d i r e c t e d
0304-4017/84/$03.00
© 1984 Elsevier Science Publishers B.V.
294 towards egg production, the emphasis on protein and hence amino acid biosynthesis must be considerable. If we are able to find some differences in the enzymes involved in amino acid biosynthesis by the parasite and the host, we can selectively check the synthesis of amino acids in the parasite. Thus, these would not be available for incorporation into egg proteins and hence the propagation of a species could be checked. Therefore, keeping in view the importance of amino acid biosynthesis in nematodes, adult H. contortus (Rud., 1803) was investigated for determining whether or not it had the potential of synthesizing amino acids and if so, exactly which ones were synthesized. The various precursors employed in the present study include glucose and acetate, known to be incorporated by nematodes into amino acids (Rothstein amd Mayoh, 1964), CO2 known to be incorporated into carbon-chain of TCA cycle related amino acids (Rothstein, 1965) and palmitic acid (to study the relative importance of long chain fatty acids in amino acid biosynthesis). MATERIALS AND METHODS Live adult specimens of H. contortus were collected from the abomasa of infected goats (Capra hircus) from local abattoirs. Worms were washed thoroughly in 0.9% saline and freed from the adhering host material.
Incubation m e d i u m The incubation medium used in this investigation had the following composition: 10.7 g NaC1; 0.2 g KC1; 0.265 g CaC12 • 2H20; 0.21 g MgC12 • 6H20; 1.0 g NaHCO3; 0.05 g NaH2PO4; 1.0 g glucose; 3.0 g streptopencillin; 0.01 g nystatin, and the volume made up to 1 1 with double distilled water. For each experiment, 500 mg of worm tissue were transferred to flasks containing 10 ml of the above medium. To each flask 12.5 pCi of the labelled precursor was added. The various precursors employed in the present studies included U-C14-D-glucose, sodium-l-C~4-acetate, sodium-C~4-bicarbonate and C14-1-pabmitic acid. Worms were incubated at 39 -+ 1°C for 4 h on a metabolic shaker. After the completion of incubation period, a few drops of chloroform: methanol (2:1 v/v) were added in order to stop the incorporation. Media were poured out of the flasks and worms washed several times with distilled water.
Extraction o f amino acids Worms of each group were homogenised in 10% trichloroacetic acid (w/v) for 10 min in a teflon pestle homogenizer (Remi Udyog, Bombay). These were then sonicated for 5 min using an ultrasonic vibrator (Model VPL-P2, Vibronic Pvt. Ltd., Bombay) and finally centrifuged in a K24 centrifuge, Jenetzki, DDR) at 4°C and 1800 g for 10 min. Precipitate thus obtained was
295 extracted twice with 5% TCA at 0°C. Subsequently, 10% TCA was added to the precipitate and refluxed on a steam bath for 15 min, centrifuged at 5°C and 1800 g for 10 min. Proteins obtained in the above precipitate were hydrolysed in 6 N HC1 by autoclaving at 15 lb for 4 h. Fractionation of amino acids
Protein hydrolysate (50 pl of each sample) was analysed for amino acids by uni-dimensional descending paper chromatography, using Whatman No. I paper. Solvent system used was n-butanol:acetic acid:water (4:1:5 v/v). Duplicate chromatograms were developed for each hydrolysate. After completion of the development, the chromatograms were air dried. One of the duplicates was sprayed with 0.2% ninhydrin in n-butanol to locate the different amino acids. Measurement o f radioactivity
The spots on the above chromatogram were used as template and the corresponding areas of the other chromatogram were cut out and transferred directly to the scintillation vials. Radioactivity was measured using a ~-scintillation spectrometer. For measurement of radioactivity, Bray's (1960) scintillation fluid was used which had the following composition: 4 g PPO; 200 mg POPOP; 60 g naphthalene; 20 ml ethylene glycol; 100 ml methanol, and the volume made to 1 1 with dioxan. 10 ml of this fluid was added to each vial. RESULTS C 14 has been detected in the proteins of H. contortus (Kapur and Sood, unpublished observations), thus indicating its ability to synthesize proteins, and hence the amino acids. Results of the incorporation of carbon from various precursors into different amino acids of adult H. contortus are given in Table I. It is evident from Table I that the order of incorporation of label from glucose into various amino acids was Glu > Asp > Ala > Tyr > Leu+Ile Pro ;, Arg > Gly+Ser > Val+Met > Cys+Cys-Cys > Lys+His. In case of acetate it was Glu > Asp > Gly+Ser > Ala > Pro=Tyr > Arg > Lys+His Val+Met > Leu+Ile > Cys+Cys-Cys. While in the case of CO2 the order was Ala > Glu > Asp > Gly+Ser > Lys+His ~ Tyr > Pro=Arg > Leu+Ile Val+Met > Cys+Cys-Cys. In the case of palmitic acid, the order of incorporation was Asp > Glu > Ala > Gly+Ser > Val+Met > Arg > Tyr > Leu+Ile Cys+Cys-Cys > Pro > Lys+His. Thus, it is seen that Glu, Asp, Gly, Ser, and Ala are the amino acids in which m a x i m u m label is incorporated, regardless of the precursor employed. However, in the case of glucose, incorporation into Gly and Ser is comparatively less.
296 TABLEI
Incorporation of carbon from C~4-1abelled glucose, acetate, sodium bicarbonate and palmitic acid into amino acids of adult Haemonchus contortus. The results are expressed as the relative percentage of incorporation of total amino acids recovered from protein hydrolysate Amino acid
Cystine (Cys-Cys) + Cysteine (Cys) Aspartic acid (Asp) Lysine (Lys) + Histidine (His) Glutamic acid (Glu) Proline (Pro) Arginine (Arg) Tyrosine (Tyr) Alanine (Ala) Glycine (Gly) + Serine (Set) Valine (Val) + Methionine (Met) Leucine (Leu) + Isoleucine (Ile)
Precursor Glucose
Acetate
Carbon dioxide (HCO;)
Palmitic acid
4.3 15.6 4.1 25.7 6.6 6.5 8.1 11.0 5.8 5.6 6.8
4.4 15.5 5.0 19.2 8.7 7.1 8.7 9.5 12.5 4.8 4.6
4.1 11.1 9.1 16.9 5.2 5.2 7.7 20.3 10.8 4.5 5.0
4.9 20.6 3.7 16.3 4.4 7.8 5.9 12.6 9.9 8.3 5.4
DISCUSSION The ability to synthesize essential amino acids had been a t t r i b u t e d only to plants and microorganisms. However, there are a few reports indicating t hat certain helminth species also have this p r o p e r t y . These include Caenorhabditis briggsae (Rothstein and Tomlinson, 1961, p r o b a b l y t he first r e p o r t o f essential amino acids biosynthesis by a multicellular animal species), Cooperia punctata (Slonka et al., 1973), Clinostomum campanulatum (Thomas and Gallicchio, 1967), Moniliformis dubius (Graff, 1964) and A n c y l o s t o m a caninum (P~rez et al., 1967). The concept is f u r t h e r r e f u t e d by our present studies in which H. contortus has been d e m o n s t r a t e d to be capable o f essential amino acid biosynthesis. The p r o p o s e d scheme for the involvement of different precursors in amino acid biosynthesis is given in Fig. 1. It is evident from t he figure t hat the inc o r p o r a t i o n o f c a r b o n f r om each precursor into amino acids is via the TCA cycle. Also, the low activities of aconitase and isocitrate dehydrogenase in adult H. contortus (Kaur and Sood, 1983) support the idea t hat the TCA cycle has only a m i n o r f u n c t i o n as regards the energy p r o d u c t i o n . It is possibly c o n c e r n e d with the interconversions of carbon-skeletons into amino acids metabolism (Saz and Vidrine, 1959; Oya et al., 1962, 1965). Most of the label is f o u n d in Glu, Asp, Ala, Gly and Ser, regardless of the precursor e m p l o y e d . This has also been d e m o n s t r a t e d in o t h e r helminths (Rothstein, 1963; Slonka et al., 1973). Higher a m o u n t of label in Asp and Glu suggests
297
I GLUCOSEI
I -[P.OSP.A GLUCOS r- ERYTHROSE-4- PHOS. / PHATE I
I TRIOSE PHOSPHATE
SERINE q T RYP'I'OPHAN l
[,'AL..,.cAC,O)
PHOSPHOENOL PYRU VATE I |
k LA~IINE,e~
~,
'~
/I
/I
+
"TRYP~PHAN/)2
PYRUVATE ., LEUCrNE
/I
/
#
TYROSINE
i
~1 '
/
I
j
~ "ACETYLCoA.-~LEUCINE VALINE
ISOLEUCINE ALANINE
LYSINE~
t
I~
ASPARTATE.-,P.THREONINE<-.bGLYCINE
t J,"~SERINE
/OXALOACETATE,~ FUMARATE
ARGIf NINEI
METHIONINE
CITRATE
TCACYCLE
1
~, E CYSI'EIN
SERINE
CYS~rINE
~.- KETOGLUTARATE
~ S U C C I N Y L CoA/ ARGININE4,.~ GLUTAMATE
TRYPTOPHAN ~//~R~!,NE PHENYLALANINE Fig. 1.Proposed scheme for the involvement of C'4-glucose,acetate,carbon dioxide and palmiticacid in amino acid biosynthesisin adultsof Haernonchus contortus.
the presence of TCA cycle intermediates (a-ketoglutaric acid and oxaloacetic acid) which are :important links between amino acid and carbohydrate metabolism. Adult H. contortus, unlike C. punctata (Slonka et al., 1973} is able to synthesize Val and Met from glucose, though only to a small extent. C. briggsae is also capable of synthesizing Val (Nicholas et al., 1960). Ala, Glu and Asp are among the top amino acids labelled on incubation with C14-glucose. This observation is supported by the findings of Slonka et al. (1973) on C. punctata. However, in Moniliformis dubius (Graff, 1964) no label was found in Glu. In C. briggsae, C 14 from glucose was incorporated in the following order: Val > Ala > Lys > Glu > Asp > Ser > Thr > His > Arg > Gly (Nicholas et al., 1960; Rothstein and Tomlinson, 1961). In contrast to these observations, in the present studies, valine is synthesized to a small extent. Also, there was incorporation into Cys-Cys, Cys, Tyr, Leu,
298
Ile, Pro and Met in the present studies which was n o t detected in C. briggsae. As regards the biosynthesis of amino acids from acetate and CO2, H. contortus like C. briggsae (Nicholas et al., 1960; Rothstein, 1965; Rothstein and Mayoh, 1964) is capable of synthesizing amino acids related to TCA cycle. In addition, it is also capable of incorporating into Cys-Cys, Cys, Pro, Tyr, Met. Presence of C '4 in Ala, Glu and Asp in H. contortus is a good evidence that at least part of the TCA cycle operates. These results might be explained by implicating succinate as the primary p r o d u c t of CO: fixation, as also reported in Ascaris (Saz and Vidrine, 1959). So far there is no report regarding the role of long chain fatty acids in amino acid biosynthesis. In the present studies, we have demonstrated that the label from palmitic acid is incorporated in all the 16 amino acids. From these observations, it can be speculated that adult H. contortus is capable of catabolizing palmitic acid to acetyl CoA, which is then incorporated into amino acids via TCA cycle. CONCLUSIONS
Adult H. contortus has the potential to synthesize essential as well as nonessential amino acids in vitro. However, from the present data, it is n o t possible to predict whether or not the synthesis of amino acids is at a level commensurate with reproduction. Therefore, further studies need to be carried out on these lines. Also, it would be interesting to elucidate the pathways of amino acid biosynthesis. These studies can give us a deeper insight into the metabolism of H. contortus. ACKNOWLEDGEMENTS
The authors wish to thank Professor S.S. Guraya, Head of the Department for the laboratory facilities. J. Kaput also wishes to thank the authorities of C.S.I.R., New Delhi for the Junior/Senior Research Fellowship grant.
REFERENCES Abbas, M. and Foor, W.E., 1978. Ascaris s u u m : Free amino acids and proteins in the pseudocoelom, seminal vesicle, and glandular vas deferens. Exp. Parasitol., 45: 263-273. Bray, G.A., 1960. A simple efficient scintillator for counting aqueous solutions in a liquid scintillation counter. An. Biochem., 1 : 279--285. Graff, D.J., 1964. Metabolism of C'4-glucose by Moniliforrnis dubius (Acanthocephala). J. Parasitol., 50: 230--234. Herlich, H., 1966. Amino acid composition of some strongyle parasites of cattle. Proc. Helminthol. Soc. Wash., 33: 103--105. Kapur, J. and Sood, M.L., 1984. Amino acid composition of the adults of H a e m o n c h u s c o n t o r t u s (Nematoda: Trichostrongylidae). Helminthologia, 21: in press.
299
Kaur, R. and Sood, M.L., 1983. The effects o f dl-tetramisole and rafoxanide on tricarboxylic acid cycle enzymes of Haemonchus contortus, in vitro. Vet. Parasitol., 13: 333--340. Nicholas, W.L., Dougherty, E.C. and Hansen, E.L., 1960. The incorporation of 14C from sodium acetate-2-14C into the amino acids of the soil-inhabiting nematode, Caenorhabditis briggsae. J. Exp. Biol., 37: 435--443. Nigam, S.C., 1978. Free amino acids from Ascaridia galli. Indian J. Parasitol., 2: 157-158. Nigam, S.C., 1979. Amino acid composition of nematode parasites. Indian J. Helminthol., 31: 69--71. Oya, H., Kikuchi, G., Hayashi, H. and Bando, T., 1962. The occurrence of tricarboxylic acid cycle in the muscle of Ascaris lurnbricoides var. suis. Jpn. J. Parasitol., 11 : 291. Oya, H., Kikuchi, G., Bando, T. and Hayashi, H., 1965. Muscle tricarboxylic acid cycle in Ascaris lumbricoides vat. suis. Exp. Parasitol., 17: 229--240. P~rez Gim~nez, M.E., Gim~nez, A. and Gaede, K., 1967. Metabolic transformation of 1tiC-glucose into tissue proteins of Ancylostoma caninum. Exp. Parasitol., 21: 215-233. Rothstein, M., 1963. Nematode biochemistry--III. Excretion products. Comp. Biochem. Physiol., 9 : 51--59. Rothstein, M., 1965. Nematode biochemistry--V. Intermediary metabolism and amino acid interconversions in Caenorhabditis briggsae. Comp. Biochem. Physiol., 14: 541-552. Rothstein, M. a r d Mayoh, H., 1964. Nematode biochemistry IV. On isocitrate lyase in Caenorhabditis briggsae. Arch. Biochem. Biophys., 108: 134--142. Rothstein, M. and Tomlinson, G., 1961. Biosynthesis of amino acids by the nematode Caenorhabditis briggsae. Biochem. Biophys. Acta, 49: 625--627. Rothstein, M. and Tomlinson, G., 1962. Nematode biochemistry. II. Biosynthesis of amino acids. Biochem. Biophys. Acta, 63: 471--480. Saz, H.J. and Vidrine, A., Jr., 1959. The mechanism of formation of succinate and propionate by Ascaris lumbricoides muscle. J. Biol. Chem., 234: 2001--2005. Slonka, G.F., Ridley, R.K. and Leland, S.E., Jr., 1973. The use of in vitro grown Cooperia punctata (Nematoda: Triehostrongylidae) to study incorporation of carbon from D-glucose-U-~4C into major chemical fractions. J. Parasitol., 59: 282--288. Thomas, R.E. and Galliechio, V., 1967. Metabolism of 14C-glucose by metacercariae of Clinostomum carnpanulaturn (Trematoda). J. Parasitol., 53: 981--984.