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Studies on beta-oxidation in the adult liver fluke Fasciola hepatica
International Journal
for Parasitology.
1976. Yol. 6. pp. 155457.
Pergamon Press. Printed in Great Britain.
STUDIES ON BETA-OXIDATION FLUKE FASCZOLA
IN THE ADULT LIVER
HEPATZCA
J. BARRETTand W. K~~RTING Department
of Zoology, U.C.W., Aberystwyth, and Institute of Zoology and Hydrobiology, of Veterinary Medicine, University of Munich
Faculty
(Received 23 June 1975) i%stmet-BARRETT J. and KtiRnNG W. 1976. Studies on eta-oxidation in the adult liver fluke Fasciola hepaticu. Z~ternat~onai Jourr~uffor Par~~tia~o~y 6: 155-157. All of the enzymes of the g-oxidation sequence have been demonstrated in adult F. hepaticu. However, this parasite does not oxidise exogenous ‘eC-U-palmitate, although lab&led palmitate is readily taken up and incorporated into the neutrai and phospholipid fractions. This indicates, that despite the presence of all the enzymes, the @oxidation pathway is not functional in adult F. hepatiea: possible roles for the h-oxidation enzymes in the metabolism of the parasite are discussed.
INDEX
KEY WORDS:
Fusciulu hepaticu; fi-oxidation;
MATERIALS AND METHODS
INTRODUCTION IN CONTRAST to ~r~hydrate metabolism, lipid catabolism has been little studied in parasitic helminths. A complete sequence of @-oxidation enzymes has been demonstrated in adult Ascaris Zumbrkoides muscle (Ward & Fairbairn, 197Ou)
and a partial series of B-oxidation enzymes has been found in Hymemolepis diminuta, Moniliformis dubius and the parasitic females of S. ratti (Ward & Fairbairn, 1970a; Kiirting & Fairbairn, 1971, 1972). However, none of these parasites were able to oxidize exogenous 14C-U-palmitic acid and there is no good evidence for endogenous lipid utilization in any adult helminth parasite (Fairbairn, 1970). Unlike the parasitic stages, the developing eggs of A. Zumbr~cojdes and the free-living females and infective larvae of 5’. ratti, all of which have a complete sequence of P-oxidation enzymes, readily oxidize r”C-U-palmitic acid indicating the presence of a functional $-oxidation pathway (Ward & Fairbairn, 19706; Korting & Fairbairn, 1971). In adult Fusciola hepaticu lipid constitutes about
12-13 % of the dry weight (Weinland & von Brand, 1926). Nistologically the lipid appears to be associated mainly with the excretory system (Erasmus, 1972) and the excretion of lipid via the protonephridial system has been reported by a number of workers (von Brand, 1928; Stephenson, 1947; Johnson, Ehrlich & Burren, 1967; Burren, Ehrlich & Johnson, 1967; Moss, 1970). In this paper the enzymes of the ~-oxidation sequence have been demonstrated in adult F. heputiccz and the ability of this parasite
to oxidize
fatty acids investigated.
palmitic acid; lipid.
Adult F. hepatica were obtained from local slaughterhouses and brought to the laboratory in warm (37°C) Hedon-Fleig’s sofution containing 0.06 g/l benzylpenicillin and 0.1 g/I streptomycin (van Noordwijk & de Wolf, 1963). Enzyme assays. The worms were homogenized in sucrose buffer (0.25 M-sucrose; 0.005 M-tris-Cl, pH 7.5) in a hand driven glass homogenizer. The homogenizer was cooled in ice and the volume of the homogenizing medium was adjusted to give approximately a 1 in 10 homogenate (w/v). The homogenate was centrifuged at 500 g for 10 min at 2°C and the supernatant fraction used for enzyme assays. All the assays were performed at 30% using a Gifford Model 240 recording spectrophotometer, Short chain acyl-CoA synthetases (EC 6.2.1.1 and EC 6.2.1.2) were assayed by following the disappearance of the thiol groups of CoA as described by-Ward & Fairbaim (197Ob) exceut that D.T.N.B. (5.5’-dithiobis-2nitrobenzoic acid) was used to determine the thiol groups instead of the nitroprusside reagent (Hock & Beevers, 1966). Short chain acyl-CoA synthetase (EC 6.2.1.1) was also measured by following the formation of acetyl-CoA (Jones & Lipmann, 1955). In addition, a direct spectrophotometric method was used for short chain acyl-CoA synthetase, the reaction mixture contained (in 1 ml); tris-acetate buffer, pH 7.4, 20 pmol; CoA 2 umol ; ATP, 10 pmol ; MgCI,, 5 pmol. Acetyl-CoA formation was followed by the increase in extinction at 232 nm. Long chain acyl-CoA synthetase (EC 6.2.1.3), acyl-CoA dehydrogenase (EC 1.3.2.2.), enoyl-CoA hydratase (EC 4.2.1.17), 3-hydroxyaeyl-CoA dehydrogena~ (EC 1.1.1.35) and acetyl-CoA acyltransferase (EC 2.3.1.16) were all assayed as described by Ward & Fairbairn (197Ob). The GTP specific acyl-CoA synthetase was assayed as for long chain acyl-CoA
155
156
J,
BARRETT
and W. Kii~rmc;
synthetase except that ATP was replaced by GTP. Protein was determined by the method of Lowry, Rosebrough, Fat-r & Randal (1951); standard errors were calcutated as described by Dean & Dixon (1951). l~o~~pe experime~ifs. Fatty acid oxidation was determined bv measurina the ‘%XL nroduced when adult F. hepatitcrwere incubated with lit?-U-palmitate in HedonFleigs solution plus ~~zyi-penicillin and streptomycin at 37°C. The incubations were carried out and the released 14C0, measured as described by Ward & Fairbairn (1970b). After incubation the worms were washed, dried, weighed and the total lipids extracted by the method of Fetch, Lees & Sloane-Stanley (1957). The lipids were separated by thin-layer chromatography using the double development system of Freeman & West (1966) and the different fractions located under U.V. light after spraying with aqueous rhodamine 6G (0+05% w/v). The lipid bands were scraped off, suspended in 10 ml of toluene-fuor solution containing 0.7% (w/v), 1,3,4-oxadiazole (butyl-PBD), 2.5% (v/v) Beckman Bio-Solve BBS-3 solubilizer and their radioactivity determined by liquid scintillation spectroscopy,
RESULTS AND DISCUSSION
TABLE
I-THE ENZYMES
SPECIFIC ACTKVITY OF BETA-OXIDATION IN EXTRACTS OF Fmc&ka kf&&CQ
Specific activity* Enzyme and substrate
(nmol~min~mg protein)
synthetase Acetate Propionate Butyrate
1.98 + 0.68 0.83 $1 0.28 I.46 & O-12
synthetase Patmitate Stearate Oleate Linoleate
075 0.65 1.54 1.67
Acyl-CoA
Acyl-CoA
Acyl-CoA dehydrogenase Botyryl-CoA Enoyl-CoA hydratase Crotonyl-CoA 3-Hydroxyacyl-CoA dehydrogenase 3-~ydroxybutyry~-boa Acetyl-CoA acyhransferase Acetoacyl-CoA *Mean i
S.E.M. n =
197Oa); although the specific activities of the enzymes in F. Iaeparica are much lower than those in As&at-ismuscle. Wein~and & von Brand (1926} found that there was no appreciable lipid utiIization when adult F. hepatica were incubated under aerobic or anaerobic conditions. On incubating adult F. he~a~~~a with l*C-U-paImitate there was no significant production of labelled carbon dioxide, although the palmitate was readily taken up and incorporated into the neutral and phospholipid fractions (Table 2). The small amount of lvCO, produced by F. ~~~~a~i~a may TABLE 2-INCORFDRATION OF EXOGENOUS "C-u-PALMITATE INTO LIPIDS AND CARBON DIOXfDE BY ADULT F. hepafica
______I__-. Fraction
-
Adult F. hepa~iea were found to possess a complete sequence of &oxidation enzymes (Table l), but no GTP specific acyl-CoA synthetase could be detected, Compared with a tissue such as rat kidney which
i i i rt
0.30 O-12 0.20 0.09
272 -I: 026 76.25 It 14.84 l-04 & 0.17 416 rt: 2.94
10.
has an active P-oxidation sequence (Ward & Fairbairn, 19706) the activity of 3-hydroxyacyl-CoA dehydrogenase is relatively low in F. hepaticu whilst the levels of acyltransferase are relatively rather high. In general the relative activities of the 8oxidation enzymes in F. ~e~at~cu resemble those found in A. Ii4#z~ricaid~s muscle (Ward & Fairbairn,
I.J.P. VOL.6. 1976
Carbon dioxide Neutral lipids Free-fatty acids Phospholipids
_c.p.m./mg Dry wt/hr ______l-.-~-. 20s 25757 5641 4304
have resulted from the de~r~xyiation of the absorbed palmitate (a-oxidation); if an active poxidation sequence had been present one would have expected a hundred to a thousand times higher counts in the carbon dioxide (Korting & Fairbairn, 1971). Adult F. heparka do not, therefore, appear to have a functional S-oxidation sequence despite the presence of all of the ~-oxidation enzymes. Beta-oxidation is tightIy coupled to the TCA cycle (Green, 1963) and Ward & Fairbairn (1970~) suggested that the absence of a functional Boxidation sequence in adult A. lumbricoides muscle may be due to the absence of a classical TCA cycle in this tissue (Saz, 1971). Adult F. hepurica possess ail of the enzymes of the TCA cycle aIthough the activities of aconitase and &citrate dehydrogenase are very low (Prichard & S~hofieid, 1968; de Zoeten, Posthumo & Tipker, 1969; Sturm, ~ir~hh~user & Zilliken, 1972) and Buist & Schofield (1971) using specifically labelled glucose concluded that the TCA cycle was of minimal importance in the oxidative metabolism of this parasite. The absence of a functional P-oxidation sequence in K hepatica may, therefore, as in A. l~~~~&~~~es, be correlated with the relative unimportance of the classical TCA cycle in these helminths. It is not really possible to assess the signi~~~n~e of low enzyme activities in whole homogenates, since one does not know if this represents a Eow activity throughout the organism or whether the activity is relatively high in some tissues and absent from others. Indeed one cannot even be certain that all of the enzymes are present in the same tissue. There are, however, a number of possible functions for the ~-oxidation enzymes in F. hepnrica. The enzymes might be involved in the metabolism of volatile
I.J.P. VOL.6. 1976
Beta-oxidation
in Fasciola hepatica
157
JONESM. E. & LIPMANNF. 1955. Aceto-CoA-kinase. In Methods in Enzymology (Edited by COLOWICK S. P. & KAPLAN N. O.), Vol. 1, pp. 585-591. Academic Press, New York. K~RTING W. & FAIRBAIRND. 1971. Changes in betaoxidation and related enzymes during thelife cycle of Strongy~oides ratfi (Nematoda). Journal OfParasitoZogy 57: 11.53-1158. KBRTING W. & FAIRBAIRND. 1972. Anaerobic energy metabolism in Moniliformis dubius (Acanthocephala). Journal of Parasitology 58: 45-50. REFERENCES LOWRY 0 H., ROSEBROUGH N. J., FARR A. L. & RANDALL R. J. 1951. Protein measurement with the Folin phenol VON BRAND T. 1928. Be&rag zur Kenntnis der Zusamreagent. Journal of3iological Chemistry 193: 265-275. mensetzung des Fettes von Fasciola hepatica. ZeitsMoss G. D. 1970. The excretory metabolism of the thrift fur Vergleichende Physiotogie 8: 613-624. endoparasitic digenean Fasciola hepntica and its BUISTR. A. & SCHOFIELDP. J. 1971. Some aspects of the relationship to its respiratory metabolism. Parasitology glucose metabolism of F&iota hepatica. International 60: l-19. Journal of Biochemistry 2: 377-383. PRICHARDR. K. & SCHOFIELDP. J. 1968. A comparative BURRENC. H., EHRLICHI. & JOHNSONP. 1967. Excretion study of the tricarboxylic acid cycle enzymes in of lipids by the liver fluke (Fasciola heputica L). Lipids Fasciola hepatica and rat liver. Comparative Bio2: 353-356. chemistry and Physiology 25: 100-1019. DEAN R. B. & DIXON W. J. 1951. Simplified statistics for SAZ H. J. 1971. Facultative anaerobiosis in the invertesmall number of observations. Analytical Chemistry brates: pathways and control systems. American 23: 636-638. Zoologist 11: 125-135. DE ZOETEN L. W., POSTHUMOD. & TIPKER J. 1969. Intermediary metabolism of the liver fluke Faxi& SEUBERTW., LAMBERTSI., KRAMERR. & OHLY B. 1968. On the mechanisms of malonyl-COG-independent hepatica, 1 Biosynthesis of propionic acid. Hoppefatty acid synthesis. I. The mechanism of elongation of Seyler’s Zeitschrift fur Physiologische Chemie 350: long chain acids by acetyl-CoA. Biochimica et Bio683-690. physica Acta 164: 498-517. ERASMUSD. A. 1972. The Biology of Trematodes. Arnold, STEPHENSON W. 1947. Physiologi~l and hist~hemical London. observations on the adult liver fluke, Fasciola hepatica FAIRBAIRND. 1970. Biochemical adaptation and loss of L. IV. The excretory system. Parasitology 38: 140-144, genetic capacity in helminth parasites. Biological Reviews (Cambridge) 45: 29-72. STURM G.. HIRSCHH~USERC. & ZILLIKEN F. 1972. Vergleichende Bestimmung von Enzymaktiviraten in FOLCH J., LEES M. & SLOANE-STANLEY G. H. 1957. A Dicrocoelium dendriticum, Wirtsleber (Rind) und simple method for the isolation and purification of Fascioia hepaf ica. Zeitschrift fir Par~itenk~de 38 : total lipides from animal tissue. JournaE of Bio~ogicaZ 45-47. Chemistry 226: 497-509. FREEMANC. P. & WEST D. 1966. Complete separation of VANNOORDWIJKJ. & DE WOLF J. N. 1963. Effect of pH and Ca*+ concentration on the paralytic activity of lipid classes on a single thin-layer plate. Journal of isopelletierine studied on the liver fluke. Acta PhysioLipid Research 7: 324327. GREEN D. 1963. Fatty acid oxidation. In Progress in the togica et Pharmacologica Neerlandica 12: 30-47. Chemistry of Fats and other Lipids (Edited by HOLMAN WARD C. W. & FAIRBAIRN D. 1970a. Enzymes of betaoxidation and the tricarboxylic acid cycle in adult R. T., LANDBERGW. 0. & MALKIN T.). Vol. 6. no. Hymenoiepis diminuta (Cestoda) and Ascaris lumbri88-115. Pergamon Press, Oxford. ” ’ Ah coides (Nematoda). Journal of Parasitology 56: HOCK B. & BEEVERSH. 1966. Development and decline of the glyoxylate-cycle enzymes in watermelon seedlings 1009-1012. (Citrullus vtdgaris Schrad.). Zeitschrif fur PflunzenWARD C. W. & FAIRBAIRN D. 19706. Enzymes of Boxidation and their function during development of physio/ogie 55 : 405414. JOHNSONP., EHRLICH1. & BURRENC. H. 1967. Excretion Ascaris ~umbr~coideseggs. ~eve[opmenta~ Biology 22: of lipids by the liver fluke (Fascioia hepatica L.) and 366-387. incorporation of labelled precursors into fluke lipids WEINLANDE. & VONBRANDT. 1926. Beobachtungen an in vitro. Federation of European Biochemical Societies Fasciola hepatica (Stoffwechsel und Lebensweise). Zeitschrift fiir vergleichende Physiologie 4: 212-285. Symposium 167, p. 62.
acids, as has been suggested in adult A. lumbr~~o~des by Ward & Fairbairn (1970a) or they may be involved in the malonyl-CoA independent elongation of long chain fatty acids (Seubert, Lamberts, Kramer 8c Ohly, 1968). Alternatively the B-oxidation enzymes might be concentrated in the developing eggs in preparation for the free-Iiving miracidial stage.
for Parasitology.
1976. Yol. 6. pp. 155457.
Pergamon Press. Printed in Great Britain.
STUDIES ON BETA-OXIDATION FLUKE FASCZOLA
IN THE ADULT LIVER
HEPATZCA
J. BARRETTand W. K~~RTING Department
of Zoology, U.C.W., Aberystwyth, and Institute of Zoology and Hydrobiology, of Veterinary Medicine, University of Munich
Faculty
(Received 23 June 1975) i%stmet-BARRETT J. and KtiRnNG W. 1976. Studies on eta-oxidation in the adult liver fluke Fasciola hepaticu. Z~ternat~onai Jourr~uffor Par~~tia~o~y 6: 155-157. All of the enzymes of the g-oxidation sequence have been demonstrated in adult F. hepaticu. However, this parasite does not oxidise exogenous ‘eC-U-palmitate, although lab&led palmitate is readily taken up and incorporated into the neutrai and phospholipid fractions. This indicates, that despite the presence of all the enzymes, the @oxidation pathway is not functional in adult F. hepatiea: possible roles for the h-oxidation enzymes in the metabolism of the parasite are discussed.
INDEX
KEY WORDS:
Fusciulu hepaticu; fi-oxidation;
MATERIALS AND METHODS
INTRODUCTION IN CONTRAST to ~r~hydrate metabolism, lipid catabolism has been little studied in parasitic helminths. A complete sequence of @-oxidation enzymes has been demonstrated in adult Ascaris Zumbrkoides muscle (Ward & Fairbairn, 197Ou)
and a partial series of B-oxidation enzymes has been found in Hymemolepis diminuta, Moniliformis dubius and the parasitic females of S. ratti (Ward & Fairbairn, 1970a; Kiirting & Fairbairn, 1971, 1972). However, none of these parasites were able to oxidize exogenous 14C-U-palmitic acid and there is no good evidence for endogenous lipid utilization in any adult helminth parasite (Fairbairn, 1970). Unlike the parasitic stages, the developing eggs of A. Zumbr~cojdes and the free-living females and infective larvae of 5’. ratti, all of which have a complete sequence of P-oxidation enzymes, readily oxidize r”C-U-palmitic acid indicating the presence of a functional $-oxidation pathway (Ward & Fairbairn, 19706; Korting & Fairbairn, 1971). In adult Fusciola hepaticu lipid constitutes about
12-13 % of the dry weight (Weinland & von Brand, 1926). Nistologically the lipid appears to be associated mainly with the excretory system (Erasmus, 1972) and the excretion of lipid via the protonephridial system has been reported by a number of workers (von Brand, 1928; Stephenson, 1947; Johnson, Ehrlich & Burren, 1967; Burren, Ehrlich & Johnson, 1967; Moss, 1970). In this paper the enzymes of the ~-oxidation sequence have been demonstrated in adult F. heputiccz and the ability of this parasite
to oxidize
fatty acids investigated.
palmitic acid; lipid.
Adult F. hepatica were obtained from local slaughterhouses and brought to the laboratory in warm (37°C) Hedon-Fleig’s sofution containing 0.06 g/l benzylpenicillin and 0.1 g/I streptomycin (van Noordwijk & de Wolf, 1963). Enzyme assays. The worms were homogenized in sucrose buffer (0.25 M-sucrose; 0.005 M-tris-Cl, pH 7.5) in a hand driven glass homogenizer. The homogenizer was cooled in ice and the volume of the homogenizing medium was adjusted to give approximately a 1 in 10 homogenate (w/v). The homogenate was centrifuged at 500 g for 10 min at 2°C and the supernatant fraction used for enzyme assays. All the assays were performed at 30% using a Gifford Model 240 recording spectrophotometer, Short chain acyl-CoA synthetases (EC 6.2.1.1 and EC 6.2.1.2) were assayed by following the disappearance of the thiol groups of CoA as described by-Ward & Fairbaim (197Ob) exceut that D.T.N.B. (5.5’-dithiobis-2nitrobenzoic acid) was used to determine the thiol groups instead of the nitroprusside reagent (Hock & Beevers, 1966). Short chain acyl-CoA synthetase (EC 6.2.1.1) was also measured by following the formation of acetyl-CoA (Jones & Lipmann, 1955). In addition, a direct spectrophotometric method was used for short chain acyl-CoA synthetase, the reaction mixture contained (in 1 ml); tris-acetate buffer, pH 7.4, 20 pmol; CoA 2 umol ; ATP, 10 pmol ; MgCI,, 5 pmol. Acetyl-CoA formation was followed by the increase in extinction at 232 nm. Long chain acyl-CoA synthetase (EC 6.2.1.3), acyl-CoA dehydrogenase (EC 1.3.2.2.), enoyl-CoA hydratase (EC 4.2.1.17), 3-hydroxyaeyl-CoA dehydrogena~ (EC 1.1.1.35) and acetyl-CoA acyltransferase (EC 2.3.1.16) were all assayed as described by Ward & Fairbairn (197Ob). The GTP specific acyl-CoA synthetase was assayed as for long chain acyl-CoA
155
156
J,
BARRETT
and W. Kii~rmc;
synthetase except that ATP was replaced by GTP. Protein was determined by the method of Lowry, Rosebrough, Fat-r & Randal (1951); standard errors were calcutated as described by Dean & Dixon (1951). l~o~~pe experime~ifs. Fatty acid oxidation was determined bv measurina the ‘%XL nroduced when adult F. hepatitcrwere incubated with lit?-U-palmitate in HedonFleigs solution plus ~~zyi-penicillin and streptomycin at 37°C. The incubations were carried out and the released 14C0, measured as described by Ward & Fairbairn (1970b). After incubation the worms were washed, dried, weighed and the total lipids extracted by the method of Fetch, Lees & Sloane-Stanley (1957). The lipids were separated by thin-layer chromatography using the double development system of Freeman & West (1966) and the different fractions located under U.V. light after spraying with aqueous rhodamine 6G (0+05% w/v). The lipid bands were scraped off, suspended in 10 ml of toluene-fuor solution containing 0.7% (w/v), 1,3,4-oxadiazole (butyl-PBD), 2.5% (v/v) Beckman Bio-Solve BBS-3 solubilizer and their radioactivity determined by liquid scintillation spectroscopy,
RESULTS AND DISCUSSION
TABLE
I-THE ENZYMES
SPECIFIC ACTKVITY OF BETA-OXIDATION IN EXTRACTS OF Fmc&ka kf&&CQ
Specific activity* Enzyme and substrate
(nmol~min~mg protein)
synthetase Acetate Propionate Butyrate
1.98 + 0.68 0.83 $1 0.28 I.46 & O-12
synthetase Patmitate Stearate Oleate Linoleate
075 0.65 1.54 1.67
Acyl-CoA
Acyl-CoA
Acyl-CoA dehydrogenase Botyryl-CoA Enoyl-CoA hydratase Crotonyl-CoA 3-Hydroxyacyl-CoA dehydrogenase 3-~ydroxybutyry~-boa Acetyl-CoA acyhransferase Acetoacyl-CoA *Mean i
S.E.M. n =
197Oa); although the specific activities of the enzymes in F. Iaeparica are much lower than those in As&at-ismuscle. Wein~and & von Brand (1926} found that there was no appreciable lipid utiIization when adult F. hepatica were incubated under aerobic or anaerobic conditions. On incubating adult F. he~a~~~a with l*C-U-paImitate there was no significant production of labelled carbon dioxide, although the palmitate was readily taken up and incorporated into the neutral and phospholipid fractions (Table 2). The small amount of lvCO, produced by F. ~~~~a~i~a may TABLE 2-INCORFDRATION OF EXOGENOUS "C-u-PALMITATE INTO LIPIDS AND CARBON DIOXfDE BY ADULT F. hepafica
______I__-. Fraction
-
Adult F. hepa~iea were found to possess a complete sequence of &oxidation enzymes (Table l), but no GTP specific acyl-CoA synthetase could be detected, Compared with a tissue such as rat kidney which
i i i rt
0.30 O-12 0.20 0.09
272 -I: 026 76.25 It 14.84 l-04 & 0.17 416 rt: 2.94
10.
has an active P-oxidation sequence (Ward & Fairbairn, 19706) the activity of 3-hydroxyacyl-CoA dehydrogenase is relatively low in F. hepaticu whilst the levels of acyltransferase are relatively rather high. In general the relative activities of the 8oxidation enzymes in F. ~e~at~cu resemble those found in A. Ii4#z~ricaid~s muscle (Ward & Fairbairn,
I.J.P. VOL.6. 1976
Carbon dioxide Neutral lipids Free-fatty acids Phospholipids
_c.p.m./mg Dry wt/hr ______l-.-~-. 20s 25757 5641 4304
have resulted from the de~r~xyiation of the absorbed palmitate (a-oxidation); if an active poxidation sequence had been present one would have expected a hundred to a thousand times higher counts in the carbon dioxide (Korting & Fairbairn, 1971). Adult F. heparka do not, therefore, appear to have a functional S-oxidation sequence despite the presence of all of the ~-oxidation enzymes. Beta-oxidation is tightIy coupled to the TCA cycle (Green, 1963) and Ward & Fairbairn (1970~) suggested that the absence of a functional Boxidation sequence in adult A. lumbricoides muscle may be due to the absence of a classical TCA cycle in this tissue (Saz, 1971). Adult F. hepurica possess ail of the enzymes of the TCA cycle aIthough the activities of aconitase and &citrate dehydrogenase are very low (Prichard & S~hofieid, 1968; de Zoeten, Posthumo & Tipker, 1969; Sturm, ~ir~hh~user & Zilliken, 1972) and Buist & Schofield (1971) using specifically labelled glucose concluded that the TCA cycle was of minimal importance in the oxidative metabolism of this parasite. The absence of a functional P-oxidation sequence in K hepatica may, therefore, as in A. l~~~~&~~~es, be correlated with the relative unimportance of the classical TCA cycle in these helminths. It is not really possible to assess the signi~~~n~e of low enzyme activities in whole homogenates, since one does not know if this represents a Eow activity throughout the organism or whether the activity is relatively high in some tissues and absent from others. Indeed one cannot even be certain that all of the enzymes are present in the same tissue. There are, however, a number of possible functions for the ~-oxidation enzymes in F. hepnrica. The enzymes might be involved in the metabolism of volatile
I.J.P. VOL.6. 1976
Beta-oxidation
in Fasciola hepatica
157
JONESM. E. & LIPMANNF. 1955. Aceto-CoA-kinase. In Methods in Enzymology (Edited by COLOWICK S. P. & KAPLAN N. O.), Vol. 1, pp. 585-591. Academic Press, New York. K~RTING W. & FAIRBAIRND. 1971. Changes in betaoxidation and related enzymes during thelife cycle of Strongy~oides ratfi (Nematoda). Journal OfParasitoZogy 57: 11.53-1158. KBRTING W. & FAIRBAIRND. 1972. Anaerobic energy metabolism in Moniliformis dubius (Acanthocephala). Journal of Parasitology 58: 45-50. REFERENCES LOWRY 0 H., ROSEBROUGH N. J., FARR A. L. & RANDALL R. J. 1951. Protein measurement with the Folin phenol VON BRAND T. 1928. Be&rag zur Kenntnis der Zusamreagent. Journal of3iological Chemistry 193: 265-275. mensetzung des Fettes von Fasciola hepatica. ZeitsMoss G. D. 1970. The excretory metabolism of the thrift fur Vergleichende Physiotogie 8: 613-624. endoparasitic digenean Fasciola hepntica and its BUISTR. A. & SCHOFIELDP. J. 1971. Some aspects of the relationship to its respiratory metabolism. Parasitology glucose metabolism of F&iota hepatica. International 60: l-19. Journal of Biochemistry 2: 377-383. PRICHARDR. K. & SCHOFIELDP. J. 1968. A comparative BURRENC. H., EHRLICHI. & JOHNSONP. 1967. Excretion study of the tricarboxylic acid cycle enzymes in of lipids by the liver fluke (Fasciola heputica L). Lipids Fasciola hepatica and rat liver. Comparative Bio2: 353-356. chemistry and Physiology 25: 100-1019. DEAN R. B. & DIXON W. J. 1951. Simplified statistics for SAZ H. J. 1971. Facultative anaerobiosis in the invertesmall number of observations. Analytical Chemistry brates: pathways and control systems. American 23: 636-638. Zoologist 11: 125-135. DE ZOETEN L. W., POSTHUMOD. & TIPKER J. 1969. Intermediary metabolism of the liver fluke Faxi& SEUBERTW., LAMBERTSI., KRAMERR. & OHLY B. 1968. On the mechanisms of malonyl-COG-independent hepatica, 1 Biosynthesis of propionic acid. Hoppefatty acid synthesis. I. The mechanism of elongation of Seyler’s Zeitschrift fur Physiologische Chemie 350: long chain acids by acetyl-CoA. Biochimica et Bio683-690. physica Acta 164: 498-517. ERASMUSD. A. 1972. The Biology of Trematodes. Arnold, STEPHENSON W. 1947. Physiologi~l and hist~hemical London. observations on the adult liver fluke, Fasciola hepatica FAIRBAIRND. 1970. Biochemical adaptation and loss of L. IV. The excretory system. Parasitology 38: 140-144, genetic capacity in helminth parasites. Biological Reviews (Cambridge) 45: 29-72. STURM G.. HIRSCHH~USERC. & ZILLIKEN F. 1972. Vergleichende Bestimmung von Enzymaktiviraten in FOLCH J., LEES M. & SLOANE-STANLEY G. H. 1957. A Dicrocoelium dendriticum, Wirtsleber (Rind) und simple method for the isolation and purification of Fascioia hepaf ica. Zeitschrift fir Par~itenk~de 38 : total lipides from animal tissue. JournaE of Bio~ogicaZ 45-47. Chemistry 226: 497-509. FREEMANC. P. & WEST D. 1966. Complete separation of VANNOORDWIJKJ. & DE WOLF J. N. 1963. Effect of pH and Ca*+ concentration on the paralytic activity of lipid classes on a single thin-layer plate. Journal of isopelletierine studied on the liver fluke. Acta PhysioLipid Research 7: 324327. GREEN D. 1963. Fatty acid oxidation. In Progress in the togica et Pharmacologica Neerlandica 12: 30-47. Chemistry of Fats and other Lipids (Edited by HOLMAN WARD C. W. & FAIRBAIRN D. 1970a. Enzymes of betaoxidation and the tricarboxylic acid cycle in adult R. T., LANDBERGW. 0. & MALKIN T.). Vol. 6. no. Hymenoiepis diminuta (Cestoda) and Ascaris lumbri88-115. Pergamon Press, Oxford. ” ’ Ah coides (Nematoda). Journal of Parasitology 56: HOCK B. & BEEVERSH. 1966. Development and decline of the glyoxylate-cycle enzymes in watermelon seedlings 1009-1012. (Citrullus vtdgaris Schrad.). Zeitschrif fur PflunzenWARD C. W. & FAIRBAIRN D. 19706. Enzymes of Boxidation and their function during development of physio/ogie 55 : 405414. JOHNSONP., EHRLICH1. & BURRENC. H. 1967. Excretion Ascaris ~umbr~coideseggs. ~eve[opmenta~ Biology 22: of lipids by the liver fluke (Fascioia hepatica L.) and 366-387. incorporation of labelled precursors into fluke lipids WEINLANDE. & VONBRANDT. 1926. Beobachtungen an in vitro. Federation of European Biochemical Societies Fasciola hepatica (Stoffwechsel und Lebensweise). Zeitschrift fiir vergleichende Physiologie 4: 212-285. Symposium 167, p. 62.
acids, as has been suggested in adult A. lumbr~~o~des by Ward & Fairbairn (1970a) or they may be involved in the malonyl-CoA independent elongation of long chain fatty acids (Seubert, Lamberts, Kramer 8c Ohly, 1968). Alternatively the B-oxidation enzymes might be concentrated in the developing eggs in preparation for the free-Iiving miracidial stage.