International Journalfor Printed in Great Britain
Parasirology
Vol. 20, No. 5, pp. 581-585,
1990 0
SPHINGOMYELIN
002Cb7519/90 $3.00 + 0.00 Pergamon Press p/c Socieryfor Pcmsirology
1990 Aurrra/tin
SYNTHESIS IN FASCIOLA HEPATICA I. BANKOV* and J. BARRETT?!
* Central Laboratory of Helminthology, Bulgarian Academy of Sciences, Sofia-I 113, Bulgaria t Department of Biological Sciences, University College of Wales, Aberystwyth, Dyfed SY23 3DA, U.K. (Received 20 September 1989; accepted 12 January 1990)
1990. Sphingomyelin synthesis in Fasciola hepatica. International Whole worms and/or homogenates of F. hepatica incorporate label from cytidine-5-diphospho[methyl-14C]choline, [1-‘“ClpalmitoylCoA, [U-“Clserine, [2-‘4C]methionine, [U-“Clglycine, [U-‘*C]threonine and [U-“Claspartate into the various intermediates of sphingomyelin synthesis (ketosphinganine, sphinganine, sphingosine, ceramide and sphingomyelin). This suggests that sphingomyelin synthesis in F. hepatica occurs by a pathway similar to that found in mammals. However, there is some evidence that in F. hepaticu 3-ketosphinganine may be N-acylated prior to reduction and dehydrogenation.
Abstract-BANKov
I.
and
BARRETTJ.
Journal for Parasitology 20: 581-585.
INDEX KEY WORDS: Fasciolu hepaticu; sphingomyelin; sphingosine.
INTRODUCTION SPHINGOLIPIDS play a vital role in the structure
and function of cell membranes. They are complex lipids which have as their backbone sphingosine or a related base; the most abundant of the sphingolipids is sphingomyelin. The pathway of sphingomyelin synthesis in animals is still uncertain. In mammals there are thought to be five steps involved: (i) the condensation of palmitoylCoA and serine to give 3ketosphinganine (3-dehydrosphinganine); (ii) the reduction of 3-ketosphinganine to sphinganine (dihydrosphingosine); (iii) dehydrogenation of sphinganine to sphingosine; (iv) synthesis of ceramide from sphingosine and fatty acyl-CoA; (v) transfer of choline to the ceramide to yield sphingomyelin. Most of the enzymes involved in sphingolipid synthesis are membrane bound, which makes their purification difficult. In addition many of the substrates and products of the enzymes are insoluble in aqueous
solution making characterization difficult. Oldenborg, van Vugt & van Golde (1975) were unable to detect sphingomyelin in F. hepatica although both sphingomyelin and ceramide were subsequently reported by Hrzenjak & Ehrlich (1975). In this paper the pathway of sphingomyelin synthesis in F. hepatica is demonstrated by following the incorporation of label from radioactive precursors into sphingolipid intermediates. MATERIALS
AND METHODS
Parasite material. Adult F. heputicu were collected from
cattle livers at a local slaughter house and brought to the $ To whom all correspondence should be addressed.
laboratory in warm (37°C) Hedon-Fleig solution (Dawes, 1954). The worms were incubated for approximately 4 h to allow them to void their gut contents, they were then washed in distilled water, blotted dry and homogenized in 5 vol. of extraction medium, in an all-glass homogenizer cooled in ice. The homogenizing medium contained 0.25 M-sucrose, 5 mMtriethanolamine-HCl, 1 mhr-EDTA, pH 7.2. The homogenate was centrifuged at 125 x g for 10 min at 4°C and the supematant fraction used for enzyme assays. In some experiments live flukes were incubated with labelled precursors in Ehrlenmeyer flasks in a shaking water bath, at 37”C, under air. Sphingosine synthesis. The incubation mixture was based on Fujino & Nakano (1971). It contained (in a final volume of 2 ml): 100 mr.r-potassium phosphate buffer, pH 7.6, 0.6 mM-palmitoylCoA, 1.25-48~PM-[D-“C]serine (4.44 GBq mmole-‘), 2.5 mM-ATP, 1 mM-pyridoxal phosphate, 2.5 mMMgCl,, 2.5 mM-dithiothreitol, NADPH generating system (consisting of 3 m&r-NADP, 20 mr+glucose-6-phosphate, 5 pg glucose-6-phosphate dehydrogenase), 0.5 ~1 homogenate. The assay mixture was incubated for 2.5 h in a shaking water bath at 38°C. At the end of the incubation small amounts of sphingosine, sphinganine, 3-ketosphinganine, ceramide and sphingomyelin were added as internal markers. The incubation was terminated by the addition of 4 ml of methanol and the linids extracted and separated by thin layer chromatography (
Parasirology
Vol. 20, No. 5, pp. 581-585,
1990 0
SPHINGOMYELIN
002Cb7519/90 $3.00 + 0.00 Pergamon Press p/c Socieryfor Pcmsirology
1990 Aurrra/tin
SYNTHESIS IN FASCIOLA HEPATICA I. BANKOV* and J. BARRETT?!
* Central Laboratory of Helminthology, Bulgarian Academy of Sciences, Sofia-I 113, Bulgaria t Department of Biological Sciences, University College of Wales, Aberystwyth, Dyfed SY23 3DA, U.K. (Received 20 September 1989; accepted 12 January 1990)
1990. Sphingomyelin synthesis in Fasciola hepatica. International Whole worms and/or homogenates of F. hepatica incorporate label from cytidine-5-diphospho[methyl-14C]choline, [1-‘“ClpalmitoylCoA, [U-“Clserine, [2-‘4C]methionine, [U-“Clglycine, [U-‘*C]threonine and [U-“Claspartate into the various intermediates of sphingomyelin synthesis (ketosphinganine, sphinganine, sphingosine, ceramide and sphingomyelin). This suggests that sphingomyelin synthesis in F. hepatica occurs by a pathway similar to that found in mammals. However, there is some evidence that in F. hepaticu 3-ketosphinganine may be N-acylated prior to reduction and dehydrogenation.
Abstract-BANKov
I.
and
BARRETTJ.
Journal for Parasitology 20: 581-585.
INDEX KEY WORDS: Fasciolu hepaticu; sphingomyelin; sphingosine.
INTRODUCTION SPHINGOLIPIDS play a vital role in the structure
and function of cell membranes. They are complex lipids which have as their backbone sphingosine or a related base; the most abundant of the sphingolipids is sphingomyelin. The pathway of sphingomyelin synthesis in animals is still uncertain. In mammals there are thought to be five steps involved: (i) the condensation of palmitoylCoA and serine to give 3ketosphinganine (3-dehydrosphinganine); (ii) the reduction of 3-ketosphinganine to sphinganine (dihydrosphingosine); (iii) dehydrogenation of sphinganine to sphingosine; (iv) synthesis of ceramide from sphingosine and fatty acyl-CoA; (v) transfer of choline to the ceramide to yield sphingomyelin. Most of the enzymes involved in sphingolipid synthesis are membrane bound, which makes their purification difficult. In addition many of the substrates and products of the enzymes are insoluble in aqueous
solution making characterization difficult. Oldenborg, van Vugt & van Golde (1975) were unable to detect sphingomyelin in F. hepatica although both sphingomyelin and ceramide were subsequently reported by Hrzenjak & Ehrlich (1975). In this paper the pathway of sphingomyelin synthesis in F. hepatica is demonstrated by following the incorporation of label from radioactive precursors into sphingolipid intermediates. MATERIALS
AND METHODS
Parasite material. Adult F. heputicu were collected from
cattle livers at a local slaughter house and brought to the $ To whom all correspondence should be addressed.
laboratory in warm (37°C) Hedon-Fleig solution (Dawes, 1954). The worms were incubated for approximately 4 h to allow them to void their gut contents, they were then washed in distilled water, blotted dry and homogenized in 5 vol. of extraction medium, in an all-glass homogenizer cooled in ice. The homogenizing medium contained 0.25 M-sucrose, 5 mMtriethanolamine-HCl, 1 mhr-EDTA, pH 7.2. The homogenate was centrifuged at 125 x g for 10 min at 4°C and the supematant fraction used for enzyme assays. In some experiments live flukes were incubated with labelled precursors in Ehrlenmeyer flasks in a shaking water bath, at 37”C, under air. Sphingosine synthesis. The incubation mixture was based on Fujino & Nakano (1971). It contained (in a final volume of 2 ml): 100 mr.r-potassium phosphate buffer, pH 7.6, 0.6 mM-palmitoylCoA, 1.25-48~PM-[D-“C]serine (4.44 GBq mmole-‘), 2.5 mM-ATP, 1 mM-pyridoxal phosphate, 2.5 mMMgCl,, 2.5 mM-dithiothreitol, NADPH generating system (consisting of 3 m&r-NADP, 20 mr+glucose-6-phosphate, 5 pg glucose-6-phosphate dehydrogenase), 0.5 ~1 homogenate. The assay mixture was incubated for 2.5 h in a shaking water bath at 38°C. At the end of the incubation small amounts of sphingosine, sphinganine, 3-ketosphinganine, ceramide and sphingomyelin were added as internal markers. The incubation was terminated by the addition of 4 ml of methanol and the linids extracted and separated by thin layer chromatography (
