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The fatty acids of each lipid fraction and their use in providing energy source of the plerocercoid of Spirometra erinacei
International
Pergamon 0020-7519(94)EOO58-LJ
Journal/or Parasitology, Vol. 25. No. 1, pp. 15-21. 1995 Copyright 0 1995 Australian Society for Parasitology Ekvier Science Ltd Printed in Great Britain. All rights reserved 002~7519/95 $9.50 + 0.00
The Fatty Acids of each Lipid Fraction and their use in Providing Energy Source of the Plerocercoid of Spirometra erinacei TETSUHITO
FUKUSHIMA,*t AK10 ISOBE,? SHIWAKU,? YOSUKE YAMANEt
NOBUMASA and MOTOMI
HOJO,? KUNINORI TORIIS
fDepartment of Environmental Medicine, Shimane Medical University, Enya-cho 89-1, Izumo 693, Japan .fDepartment of Parasitology, Ehime University School of Medicine, Ehime 791-02, Japan (Received
16 September
1993; accepted
18 March
1994)
Abstract-Fukushima T., Isobe A., Hojo N., Shiwaku K., Yamane Y. and Torii M. 1995. The fatty acids of each lipid fraction and their use in providing energy source of the pierocercoid of Spirometra e&u&. Znteraarionul JouraoZlfor Parasitology 25 1521. The fatty acid concentration of each lipid fraction of plerocercoids of Spirometro erinocei and the host snake serum was investigated. The major fatty acids of phospholipid of the plerocercoids were Cl&l, Cl&O and Cl&O, and those of the host snake serum were Cl&O, ClIkl and Cl&O, in order of amount in both cases The changes of tbe fatty acid composition of phospholipid of the plerocercoids when they were incubated in physiological saline at 18°C and at 37OC for 24 h were investigated in both cases. Polyunsaturated fatty acids increased at WC, and saturated fatty acids increased at 37V. MIchaelis constants (Km) of beta-hydroxyacyl-CoA dehydrogenase (HAD), NADH: ubiquinone oxidoreductase (complex I) (NADH: ferricyanide reaction) and complex I (NADH: ubiquhtone reaction) for NADH were 20.6, 50 and 13.3 FM, respectively. The ATP production in mhochondria of the plerocercoids was accelerated by adding ADP and inhibited by adding such electron transport system inhibitors as rotenone, antimycin A and sodium cyanide. These results suggested that the fatty acids in the plerocercoids played an important role in regulating tbe fluidity of membrane by changing the composition in membrane lipid corresponding with the change of temperature circumstance. The NADH reduced by HAD might be accepted by the complex I in the electron transport system, and thus the parasites were capable of ATP production in a classical pathway of the oxidative phosphorylation system. Key
words:
Spirometra
phosphorylation
erinacei;
plerocercoid; fatty acid; membrane fluidity; ATP production;
oxidative
system.
INTRODUCTION
In the human,
infection with the plerocercoid of Spirometraerinaceicauses “sparganosis”. It migrates in the body of the host, and invades the subcutaneous adipose tissue or deep organs to stay there for a long period. Diphyllobothriid cestodes maintain
*To whom all correspondence should be addressed.
their characteristic fatty acid compositions by absorbing and modifying fatty acids (Nakagawa, Fukushima & Fukumoto, 1987; Fukushima, Abe, Nakagawa & Yamane, 1988). Studies on the fatty acids of parasites have attracted much interest in connection with the physiological functions of parasites, such as growth, regulation of membrane fluidity, energy source and defense mechanism. Among
16
T. Fukushima
them the function related to the defensemechanism of the plerocercoid of S. erinacei against the host immunesystemhasbeendemonstrated(Fukushima, Isobe, Hojo, Shiwaku, Yamane & Torii, 1993).The plerocercoidabsorbedarachidonicacid and released prostaglandinE2 which was known to suppressthe functions of mononuclearcellsof the host. Fatty acids are chief componentsof the cellular and organellemembranesof living things, and their composition affects the fluidity of membrane. Therefore the fatty acid composition of biomembrane should be an important factor affecting the membraneenzyme reaction or the membranereceptor function. The plerocercoidof S. erinacei can live in both cold-blooded reptiles and warm-blooded mammals. In the mammal, beta-oxidation works to break down long-chainfatty acidsand the NADH reduced by beta-hydroxyacyl-CoA dehydrogenase(HAD) is gained by NADH: ubiqinone oxidoreductase (complex I), which is an enzyme complex in the oxidative phosphorylation systemin mitochondria. The activities of beta-oxidation enzymeshave been demonstratedin severalspecies(Ward & Fairbairn, 1970; Barrett & Kiirting, 1977; Korting & Barrett, 1978), but there is no evidencefor an active betaoxidation sequenceforming NADH in any speciesof cestodes.The Diphyllobothrium latum mitochondria oxidize NADH (Salminen, 1973) and succinate (Salminen, 1974) and a part of the cytochrome chain cestodes seem to be of the classical mammalian-type.But there is no evidencefor ATP synthesiscarried out by the oxidative phosphorylation systemin cestodes. In this study, the expectedfunctions of fatty acids of S. erinacei plerocercoid, regulation of membrane fluidity and provision of energysource,were demonstrated, and the question why the plerocercoid preferred adipose tissue as migrating site was discussed. MATERIALS
AND
METHODS
Plerocercoids of S. erinacei were collectedfrom striped snakes(Elaphe quadrivirgata), which were captured in the field during the period from May to August. Fifty of the plerocercoidswere usedfor measuringlipid fractions and the fatty acid concentration in each fraction. The hundred plerocercoids were used for infection and stored in the subcutaneoustissueof a male, IO-week-old golden hamster (Mesocricetus auratus). The rest of the plerocercoids were washed in physiological saline solution and divided into three groups: control group, incubated at 18°C group and incubated at Materials.
et al.
37°C group (each group having 25 plerocercoids). Incubation was done in 2 ml of sterilized physiological saline at the two different temperatures.Five plerocercoids were taken from each group for measurementof fatty acids. The hamster was fed with solid feed (Oriental Kobo Ltd, Japan), given water ad Iibitum in an animal experiment station at 24°C for 6 months.It waskilled by deepether anaesthesia, and plerocercoidswere collected from the subcutaneousadiposetissue.They were washedfirst in ice-cold physiological salinesolution and then in distilled water. Measurement of lipid fractions, lipid separation of each fraction and extraction of fatty acids. The
amount of phospholipid, cholesterol and triglyceridesin plerocercoidsfrom snakesand the host serum were measuredby enzymatic determination and the free fatty acid was measuredwith NEFATest. All the reagentswere products of Wako Pure ChemicalIndustries(Tokyo, Japan). Total lipids of plerocercoidsand the host serumwere extracted by the method of Folch, Lees & Stanley (1957). The extracts were spotted on thin layer plates of silica gel, and chromatographed with a solvent system containing petroleum ether-diethyl ether-aceticacid (80:20:1). Pieces of silica gel, each of which contained a separatedlipid fraction, were scraped from the plate, and the fatty acidswere esterifiedby the method of Metcalfe & Schmitz (1961) and Wijngaarden (1967).C 17:O(margaric acid) standard was added to each samplefor quantitative analysis, and the extracted fatty acidswereanalyzed usinggas chromatogram. Gas chromatography. A Hitachi Model 163 GC equippedwith a flame ionization detector wasused. The gaschromatography wascarried out on 50 m x 0.24 mm (insidediameter)ULBON HR SS-10capillary column (Chromato Packing Center, Kyoto, Japan). The separationwas achievedon column at the range 170-220°C(2”Cmin). The split ratio was 10:1, and nitrogen was used as carrier gas with a flow rate of 18 ml/min. Peaks were identified with standard fatty acids, and were confirmed by a JMS-D 300 massspectrometer (JEOL Company, Tokyo, Japan). Subcellular fractionation. Fifty plerocercoidsfrom the hamster were gently homogenizedin 0.25 Msucrose/l0 mM-potassiumphosphatedibasic. 2 MTris base was added to it to adjust pH 7.0. The homogenatewas centrifuged at 1600g for 12 min. The supematant was filtered through 4 layers of cheeseclothand added buffer containing 0.25 Msucrose, 10 mM-Trischloride buffer (Tris-Cl), 1 mM-EDTA, pH 7.8 (0.25 volumesin the filtrate) and
Fatty acids and ATP production in S. erinacei centrifuged at 27300 g for 30 min. The precipitate was washed twice with buffer containing 0.25-M sucrose, 10 mM-Tris-Cl, pH 7.8, and a crushed mitochondriai fraction was obtained by rough homogenizing, without foaming, with a small amount of the same buffer. The protein determination was done by the biuret assay. The protein concentration of the mitochondrial fraction was 29.7 mgml. Measurement of enzyme activities. For measurement of all the enzyme activities we determined the micromole values of the products formed per min at 37°C. Solutions used for the assay of each enzyme were as follows: for HAD assay, 0.1 M-potassium phosphate buffer, pH 7.2, 100 PM-s-acetoacetyl CoA, 110 ~.LM-NADH and 148.5 p,g mitochondria protein; for NADH: ferricyanide reaction (NFR) assay, 33 mM-Tris-Cl, pH 7.5, 1.25 mM-potassium ferricyanide, 100 ELM-NADH and 148.5 pg mitochondria protein; for NADH: ubiquinone reaction (NQR) assay, 20 mM-potassium phosphate buffer, pH 8.0, 300 FM-coenzyme Qo (CoQo), 100 ELMNADH and 148.5 kg mitochondria protein. The NADH oxidation and the ferricyanide reduction were monitored at 340 nm and 410 nm, respectively, with a HITACHI U-300 spectrophotometer. Measurement of ATP. ATP was measured at room temperature with 17 mM-Tris-Cl buffer, pH 7.5 containing 0.7 mM-EDTA, ImM ADP, 170 FMNADH, 1.5 mg mitochondria, ATP releasing reagent and luciferin luciferase mixture. Ten PM-rotenone, 100 PM-antimycin A and 100 FM-sodium cyanide were used as electron transport system inhibitors. A Luminocounter ATP-237 (Toyo Kagaku Sangyo, LTD., Japan) was used for measurement. Reagents. Methanol, chloroform, hexane were purchased from Wako Chemical Industries (Tokyo, Japan), and were purified by distillation. BF3methanol was also from Wako Chemical Industries. Standard fatty acids were purchased from GL Sciences (Tokyo, Japan). We purchased CoQo, NADH, s-acetoacetyl CoA, antimycin A and Tris base from Sigma Chemical Co. (St Louis, MO, U.S.A.), rotenone, sodium cyanide, EDTA, potassium phosphate dibasic, potassium ferricyanide and BSA from Nacalai Tesque (Kyoto, Japan), and ATP releasing reagent and luciferin luciferase mixture from Laboscience (Tokyo, Japan). RESULTS
Table 1 shows the lipid fractions of S. erinacei plerocercoids and the host snake serum. Phospholipid was the chief lipid of the plerocercoids and the host snake serum, and triglyceride of the
Table l-Lipid
17
fractions of S. erinacei plerocercoids and the host snake serum Plerocercoid
Phospholipid 144.8 + 28.8 mg/100g B.W. Cholesterol 95.5 * 23.5 mg/lO@g B.W. Triglyceride 111.0 * 18.9 mg/lOCJgB.W. Free fatty acid 0.39 k 0.14 mEq/kg B.W.
Snake sernm 466.8 mg/dl 432.9 mg/dl 132.2 mg/dl 0.33 mEq/l
Each value of plerocercoid is the mean t SD. of 5 measurements. B.W.: body weight; mEq: milliequivalent. plerocercoids and cholesterol of the host snake serum were relatively important lipids. Comparing the fatty acid concentrations of each fraction of the plerocercoids with those of the host snake serum, we find the parasite maintained its characteristic fatty acid concentration (Tables 2 and 3). The major fatty acids of the phospholipid of the plerocercoids were C18:1, C18:O and Cl&O, and those of the host snake serum were C 16:0, C 18: 1 and C18:0, in order of amount in both cases. The major fatty acids of the cholesterol ester of the plerocercoids were C18:1, C16:O and C18:0, and those of the host serum were C18:2, C18:l and C20:4, in order of amount. The major fatty acids of the triglyceride of the plerocercoids were C18:O and C16:O which are saturated fatty acids, and those of the host serum were C18:2, C18:l and C20:4 which are unsaturated fatty acids. Table 4 shows the change of fatty acid composition of phospholipid of the plerocercoids before and after incubation in physiological saline at 18 and at 37°C for 24 h. Monounsaturated fatty acids decreased at both temperatures. Polyunsaturated fatty acids increased at 18°C (lower temperature), and saturated fatty acids increased at 37°C (higher temperature). Though polyunsaturated fatty acids also increased at 37°C (higher temperature), it should have been a result affected by a sharp decrease of monounsaturated fatty acids. Table 5 shows values of Michaelis constant (Km) and maximal velocity (V,,,) for NADH of HAD, an enzyme in beta-oxidation of fatty acids and a donor of NADH to electron transport system, and of complex I, an enzyme complex in the electron transport system and an acceptor of NADH from dehydrogenases such as HAD in the plerocercoids of S. erinacei. All the activities of the enzymes, HAD and complex I (NFR) and complex I (NQR) were detectable and their Km and V,,,, values were calculated. The values of Km for NADH of the enzymes are as follows; HAD, 20.6 (*M; complex I (NFR), 50.0 FM; complex I (NQR), 13.3 FM. The ATP synthesis in mitochondria of the plero-
T. Fukushima et al.
18 Table 2-Relative
fatty acid concentrations of phospholipids, free fatty acids, triglycerides and cholesterol esters of S. erinacei plerocercoids from snake
Phospholipids 124.0 2.9 149.6 197.7 10.0 1.3 11.2 17.7 1.5 2.0 8.9 2.4 2.6 2.1
C 160 %I
Go %I CE.2 C 183 C 20.0 Go:, Go:, cm3
C,,:, GO5 C 22.5 C*,:,
Cholesterol esters
? 12.4 k 0.9 f 4.0 f 20.9 f 1.8 * 0.8 AL 1.7 f 3.3 Ii 1.0 k 1.7 k 2.9 f 1.0 f 1.0 + 1.3
Triglycerides
16.3 f 2.6 1.0 f 0.4 13.4 * 2.2 51.9 f 12.8 6.0 f 0.9 0.1 f 0.1 0.4 * 0.3 2.9 f 0.7 0.7 f 0.1 0.1 ?I 0.1 2.0 f 0.6 0.6 + 0.1 1.3 f 0.3 3.1 + 0.8
5.4 0.9 8.4 1.4
f 0.4 2 0.3 f 1.4 f 0.2 N.D. N.D. N.D. 1.7 t 0.3 N.D. N.D. N.D. N.D. N.D. 0.8 k 0.2
Free fatty acids 2.5 0.5 6.7 0.8
+ 0.4 f 0.2 f 1.4 f 0.2 N.D. N.D. N.D. 1.6 f 0.6 N.D. N.D. N.D. N.D. N.D. N.D.
Each value is the mean + SD. (mg/lOO g B.W.) of 5 measurements. N.D.: not detected. Table 3-Relative
fatty acid concentrations
Phospholipids
Cl,:, Cl,:, c,s:o C 18I C 182 cm3
cm0 CZO,, Go:2 Go:, Go:.4 GO3 C225 C22 6
48.7 f 1.5 3.1 2 0.04 11.8 kO.1 28.4 k 0.4 9.8 + 0.8 0.5 * 0.1 0.6 f 0.1 1.2 * 0.5 0.4 k 0.04 0.3 f 0.1 4.4 f 0.6 0.6 + 0.2 0.4 f 0.1 0.5 + 0.1
of phospholipids, free fatty acids, triglycerides and cholesterol esters of host snake serum
Cholesterol esters
Triglycerides
2.3 f 0.6 1.7 f 0.6 0.7 f 0.3 6.1 + 1.3 15.4 * 5.0 0.9 + 0.4 0.1 f 0.02 0.2 f 0.1 0.4 2 0.04 0.2 I! 0.1 3.9 t 2.0 0.7 * 0.3 N.D. 0.3 * 0.2
11.7 f 0.6 4.5 f 0.2 1.9 * 0.7 24.6 f 1.7 33.2 2 6.8 3.1 50.6 0.2 f 0.1 0.6 f 0.2 0.5 f 0.1 0.4 * 0.1 17.4 * 3.4 3.3 * 0.8 1.9 + 0.6 2.1 f 0.6
Free fatty acids 1.9 f 0.04 0.3 f 0.1 1.4 + 0.5 2.3 + 0.4 1.5 + 0.2 0.1 fO.l 0.1 * 0.03 0.03 It 0.01 0.1 f 0.02 N.D. 0.3 f 0.2 0.1 f0.1 N.D. 0.1 + 0.1
Each value is the mean AZS.D. (mg/dl) of 4 measurements. N.D.: not detected. cercoid S. erinacei was demonstrated (Fig. 1). The concentration of ATP in the mitochondria was 0.7 nmoVmg protein. When 170 ~J.M-NADH was added to the mixture containing 1.5 mg mitochondria protein, ATP increased to 1.0 nmoYmg protein. The ATP production in the mitochondria was accelerated by adding 1 mM-ADP and the concentration of ATP was 23.8 nmol/mg protein. But the ATP concentration was decreased to 18.7 nmol/mg protein by adding 170 FM-NADH to the mixture containing 1.5 mg mitochondria protein and 1 mMADP. Inhibition of the ATP synthesis in the mitochondria of plerocercoid S. erinacei using electron transport system inhibitors was demonstrated in order to investigate the plerocercoids’ ability to produce ATP by the oxidative phosphorylation system (Table 6).
The ATP production in the mitochondria was started by adding 1 mM-ADP and the concentration of ATP was 23.8 nmol/mg protein. The ATP synthesis was inhibited to 48.6% (11.6 nmol/mg protein) by 10 FM-rotenone, to 13.9% (3.3 nmol/mg protein) by 100 l.r,M-antimycin A and to 37.5% (8.9 nmol/mg protein) by 100 PM-sodium cyanide. DISCUSSION
The fatty acid composition of parasites is generally different from that of host serum or of the parasitic tissue. The present study suggested that plerocercoids of S. erinacei maintained its characteristic fatty acid concentration in each lipid fraction by absorbing and modifying lipids. Beach, Holz, Semprevivo & Honigberg (1982) certified by culture study that C18, C20 and C22
Fatty acids and ATP production in S. erinacei Table
4-Fatty acid compositions of phospholipid of plerocercoids from snake b&fore and after incubation in physiological saline at 18°C or at 37°C for 24 h S. erinacei
Before incubation C,,:, C 16I c,tw CM:, c,,:, Gl:, GO3 GO3 C*o:4 C,,:, C 22.5 cm SFA MUFA PUFA M/S PJS
23.4 + 0.6 0.5 t 0.1 28.1 f 1.6 37.1 f 1.6 1.8 * 0.3 2.1 k 0.2 3.3 f 0.4 0.3 t 0.1 1.6 f 0.6 0.3 f 0.1 0.5 * 0.2 0.4 f 0.2 53.6 + 1.1 40.8 * 1.5 5.6 f 0.5 0.76 I!C0.04 0.10 f 0.01
30 25 I
24.2 If:2.0 0.5 f 0.2 29.4 f 1.6 28.1 f 2.5 *** 4.2 + 1.0 *** 1.6 + 0.3 * 2.8 i 0.3 0.5 k 0.3 4.2 f 1.0 *** 0.5 * 0.2 2.1 f0.3 **** 1.4 f 0.2 **** 55.2 f 1.6 31.4 f 2.3 **** 13.4 + 2.2 **** 0.57 f 0.05 *** 0.24 i 0.04 ****
20 -
Each value is the mean + S.D. (weight %) of 5 measurements. SFA: Saturated fatty acid; MUFA: monounsaturated fatty acid; PUFA: polyunsaturated fatty acid; M/S: monounsaturatedJsaturated ratio; P/S: polyunsaturated/ saturated ratio. * P
nmo I /mg
After incubation at 18°C at 37°C 26.6 i 2.2 * 0.4 t 0.2 32.7 i 2.5 + 25.3 k 1.5 **** 3.3 f 0.5 *** 1.9 f 0.4 3.5 k 0.4 0.2 2 0.1 3.6 ?I 1.6 * 0.7 + 0.4 1.0 f 0.6 0.7 * 0.4 61.2 f 2.2 **** 29.2 f 1.7 **** 9.7 It 2.2 ** 0.48 f 0.04 **** 0.16 k 0.04 *
dehydrogenase
50.0 13.3 20.6
V,,, WJw) 0.357 0.067 0.006
19
15 lo50
rY?vn A
m B
C
D
Fig. 1. ATP synthesis in plerocercoid of S. erinacei mitochondria. A, ATP amount in mitochondria. Measurements were made at room temperature with 17 mM-Tris-Cl buffer, pH 7.5 containing 0.7 mM-EDTA, 1.5 mg mitochondria, ATP releasing reagent and luciferin luciferase mixture. B, ATP amount in mitochondria after adding 170 FM-NADH. Measurements were made at room temperature with 17 mM-Tris-Cl buffer, pH 7.5 containing 0.7 mM-EDTA, 1.5 mg mitochondria, 170 FM-NADH, ATP releasing reagent and luciferin luciferase mixture. C, ATP amount in mitochondria after adding lmM-ADP. Measurements were made at room temperature with 17 mM-Tris-Cl buffer, pH 7.5 containing 0.7 mM-EDTA, 1.5 mg mitochondria, 1 mM-ADP, ATP releasing reagent and luciferin luciferase mixture. D, ATP amount in mitochondria after adding 1 mM ADP and 170 )LM-NADH. Measurements were made at room temperature with 17 mM-Tri-Cl buffer, pH 7.5 containing 0.7 mM-EDTA, 1.5 mg mitochondria, 1 mM-ADP, 170 FM-NADH, ATP releasing reagent and luciferin luciferase mixture.
Measureme& were made at 37°C. Solutions were 33 mM-Tris-Cl, pH 7.5, 1.25 mM-potassium ferricyanide and 148.5 pg mitochondria (NFR assay), 20 mM-potassium phosphate buffer, pH 8.0, 300 FM-CoQo and 148.5 Fg mitochondria (NQR assay), 0.1 M potassium phosphate buffer pH 7.2, 100 FM-s-acetoacetyl CoA and 148.5 wg mitochondria (P-hydroxyacyl-CoA dehydrogenase assay). NADH oxidation and ferricyanide reduction were monitored at 340 nm and 410 nm, respectively.
Table 6-Inhibition of ATP synthesis in the plerocercoid S. erinacei mitochondria with electron transport system inhibitors
polyunsaturated fatty acids in membrane phospholipids of Leishmuniu donovani decreased with the ascent of temperature. It is generally known that the shorter the chain of fatty acids and the higher the degree of desaturation, the lower the melting points. In our incubation study, polyunsaturated fatty acids increased at 18°C (lower temperature), and saturated fatty acids increased at 37°C (higher temperature). The melting points of fatty acids in membrane lipid played an important role in regulating the fluidity of
Measurements were made at room temperature with 17 mM-Tris4 buffer, pH 7.5, containing 0.7 mM-EDTA, lmM-ADP, 170 FM-NADH, each inhibitor (10 FMrotenone, 100 PM-antimycin A and 100 PM-sodium cyanide), 1.5 mg mitochondria, ATP releasing reagent and luciferin luciferase mixture.
ATP (nmolJmg) Per cent Mitochondria Mitochondria Mitochondria Mitochondria
+ + + +
ADP Rotenone + ADP Antimycin A + ADP Cyanide + ADP
23.8 11.6 3.3 8.9
100 48.6 13.9 37.5
membrane (Singer & Nicolson, 1972; Martin, Jr, 1985). The plerocercoid should regulate the fluidity of membrane to maintain the function of membrane proteins, and for that purpose it changes the fatty
20
T. Fukushima et al.
acid composition in membrane lipid as the host temperature circumstance changes. In the mammal, HAD functions as a beta-oxidation enzyme, giving NADH to the oxidative phosphorylation system and inciting ATP synthesis. But there is no evidence for an active beta-oxidation sequence to form NADH and make ATP synthesize in the oxidative phosphorylation system of any species of cestodes. The beta-oxidation enzymes have been assumed to be used for the formation of branched C5 and C6 acids (Ward & Fairbairn, 1970; Kijrting & Barrett, 1978) or the elongation of longchain fatty acids (Seubert, Lamberts, Kramer & Ohly, 1968). The chief source of energy might be glycolysis, so it is difficult to explain the source of the large energy that enables active mobility and rapid growth of the plerocercoids of S. erinacei. Mitochondrial functions, especially its oxidative phosphorylation system have been studied energetically with protozoa (Bienen, Webster & Fish, 1991; Gozar, O’Sullivan & Bagnara, 1992) and nematoda (Wang, Takamiya, Kita, Oya & Aoki, 1992; Kita, 1992). In cestodes, isolated Hymenolepis diminuta and Moniezia expansa mitochondria are capable of oxidative phosphorylation and respiratory control (Cheah, 1971; Yorke & Turton, 1974) and the D. datum mitochondria oxidizes NADH (Salminen, 1973) and succinate (Salminen, 1974), and probably a part of the cytochrome chain in cestodes is of the classical mammalian-type. Allen (1973) reported that the presence of rhodoquinone was demonstrated, but no ubiquinone was found in an adult M. expansa. On the contrary, Dhandayuthapani, Nellaiappan & Ramalingam (1983) reported that ubiquinone and vitamin K were detected in adult Penetrocephalus ganapatii. There is no evidence, however, for the ATP synthesis carried out by the oxidative phosphorylation system in cestodes. Activities of the beta-oxidation enzyme (HAD) and the electron transport system enzyme complex (complex I) in the plerocercoids of S. erinacei were detected and kinetic characteristics of these enzymes were presented. And then the ATP synthesis and the inhibition of ATP synthesis by electron transport system inhibitors in the mitochondria of plerocercoid S. erinacei were demonstrated. Our results suggested that the plerocercoids of S. erinacei could make ATP synthesize by the oxidative phosphorylation system. Ubiquinone might exist in the electron transport system of the plerocercoids of S. erinacei, because the ATP synthesis was inhibited by rotenone, an inhibitor of the reduction of ubiquinone. Considering that the ATP synthesis was inhibited by other inhibitors of the cytochrome
chain, a part of the cytochrome chain in the plerocercoids of S. erinacei might be of the classical mammalian type. The plerocercoid migrates in the host adipose tissue. Probably the tissue is a more suitable site for escaping from the host immune system than the abdominal cavity. Our results tell that the plerocercoids use fatty acids in adipose tissue positively for its physiological functions. Their preference of the adipose tissue as migration site could have a deep connection with their existence. Acknowledgements-We thank Masamitsu Fukushima and Toshimi Yoneyama, the technical officials, for the technical assistance. REFERENCES
Allen P. C. 1973. Helminths: comparison of their rhodoquionone. Experimental Parasitology 34: 211-219. Barrett J. & Kdrting W. 1977. Lipid catabolism in the plerocercoids of Schistocephalus solidus (Cestoda: Pseudophyllidea). International Journal for Parasitology 7: 419422. Beach D. H., Holz G. G., Semprevivo L. H. & Honigberg B. M. 1982. Temperature-dependent fatty acyl group changes in phospholipids of 37”C-adapted Leishmania donovani promastigotes. Journal of Parasitology 68: 1004-1009. Bienen E. J., Webster P. & Fish W. R. 1991. Trypanosoma (Nannomonas) congolense: changes in respiratory metabolism during the life cycle. Experimental Parasitology 73: 403412. Cheah K. S. 1971. Oxidative phosphorylation in Moniezia muscle mitochondria. Biochemica et Biophysics Acta 253: l-11. Dhandayuthapani S., Nellaiappan K. & Ramalingam K. 1983. Quinones in Penetrocephalus ganapatii (Cestoda:Pseudophyllidea). Journal of Parasitology 69: 996-998.
Folch J., Lees M. & Stanley G. H. 1957. A simple method for the isolation and purification of total lipids from animal tissue. Journal of Biological Chemistry 226: 497-509.
Fukushima T., Abe K., Nakagawa A. & Yamane Y. 1988. Fatty acid composition of plerocercoid and adult of Spirometra erinacei and the host-parasite relationship. International
Journal
for Parasitology
18: 27-31.
Fukushima T., Isobe A., Hojo N., Shiwaku K., Yamane Y. & Torii M. 1993. The metabolism of arachidonic acid to PGE2 in plerocercoid of Spirometra erinacei. Parasitology
Research
79: 634-638.
Gozar M. M. G., O’Sullivan W. J. & Bagnara A.S. 1992. Mitochondrial function in Babesia bovis. International Journal for Parasitology 22: 165-17 1. Kita K. 1992. Electron-transfer complexes of mitochondria in Ascaris suum. Parasitology Today 8: 155-l 59. Kiirting W. & Barrett J. 1978. Studies on beta-oxidation in the plerocercoids of Ligula intestinalis (Cestoda:
Fatty
acids and ATP
Pseudophyllidea). Zeitschrift fiir Parasitenkunde 57: 243-246. Martin Jr. D. W. 1985. The fluid mosaic model of membrane structure. In: Harper’s Review of Biochemistry (Edited by Martin D. W. Jr., Mayes P. A., Rodwell V. W. & Granner D. K.), pp. 454456. Maruzen Co. Ltd, Tokyo. Metcalfe L. D. & Schmitz A. A. 1961. The rapid preparation of fatty acid esters for gas chromatographic analysis. Analytical Chemistry 33: 363-364. Nakagawa A., Fukushima T. & Fukumoto S. 1987. Fatty acid composition of diphyllobothriid cestodes with reference to their hosts. Yonago Acta Medica 30: 65-80. Salminen K. 1973. The oxidation of external NADH adult and plerocercoid Diphytlobothrium by latum. Comparative Biochemistry and Physiology 44B: 283-289. Salminen K. 1974. Succinate dehydrogenase and cytochrome oxidase in adult and plerocercoid Diphyllobothrium latum. Comparative Biochemistry and Physiology 49B: 87-92. Seubert W., Lamberts I., Kramer R. & Ohly B. 1968. On
production
in S. erinacei
21
the mechanism of malonyl-CoA-independent fatty acid synthesis. I. The mechanism of elongation of long-chain fatty acids by acetyl-CoA. Biochimica et Biophysics Acta 164: 498-517. Singer S. J. & Nicolson G. L. 1972. The fluid mosaic model of the structure of cell membranes. Science 175: 720. Wang H., Takamiya S., Kita K., Oya H. & Aoki T. 1992. Comparative study on the primary structure of the ironsulfur subunit of complex II in Ascaris suum mitochondria. Japanese Journal of Parasitology 1: 122-131. Ward C. W. & Fairbaim D. 1970. Enzymes of beta-oxidation and the tricarboxylic acid cycle in adult Hymenolepis diminuta (Cestoda) and Ascaris lumbricoides (Nematoda). Journal of Parasitology 56: 1009-1012. Wijngaarden D. V. 1967. Modified rapid preparation of fatty acid esters from lipids for gas chromatographic analysis. Analytical Chemistry 39: 848-849. Yorke R. E. & Turton J. A. 1974. Effects of fasciolicidal and anti-cestode agents on the respiration of isolated Hymenolepis diminuta mitochondria. Zeitschrift fir Parasitenkunde 45: l-10.
Pergamon 0020-7519(94)EOO58-LJ
Journal/or Parasitology, Vol. 25. No. 1, pp. 15-21. 1995 Copyright 0 1995 Australian Society for Parasitology Ekvier Science Ltd Printed in Great Britain. All rights reserved 002~7519/95 $9.50 + 0.00
The Fatty Acids of each Lipid Fraction and their use in Providing Energy Source of the Plerocercoid of Spirometra erinacei TETSUHITO
FUKUSHIMA,*t AK10 ISOBE,? SHIWAKU,? YOSUKE YAMANEt
NOBUMASA and MOTOMI
HOJO,? KUNINORI TORIIS
fDepartment of Environmental Medicine, Shimane Medical University, Enya-cho 89-1, Izumo 693, Japan .fDepartment of Parasitology, Ehime University School of Medicine, Ehime 791-02, Japan (Received
16 September
1993; accepted
18 March
1994)
Abstract-Fukushima T., Isobe A., Hojo N., Shiwaku K., Yamane Y. and Torii M. 1995. The fatty acids of each lipid fraction and their use in providing energy source of the pierocercoid of Spirometra e&u&. Znteraarionul JouraoZlfor Parasitology 25 1521. The fatty acid concentration of each lipid fraction of plerocercoids of Spirometro erinocei and the host snake serum was investigated. The major fatty acids of phospholipid of the plerocercoids were Cl&l, Cl&O and Cl&O, and those of the host snake serum were Cl&O, ClIkl and Cl&O, in order of amount in both cases The changes of tbe fatty acid composition of phospholipid of the plerocercoids when they were incubated in physiological saline at 18°C and at 37OC for 24 h were investigated in both cases. Polyunsaturated fatty acids increased at WC, and saturated fatty acids increased at 37V. MIchaelis constants (Km) of beta-hydroxyacyl-CoA dehydrogenase (HAD), NADH: ubiquinone oxidoreductase (complex I) (NADH: ferricyanide reaction) and complex I (NADH: ubiquhtone reaction) for NADH were 20.6, 50 and 13.3 FM, respectively. The ATP production in mhochondria of the plerocercoids was accelerated by adding ADP and inhibited by adding such electron transport system inhibitors as rotenone, antimycin A and sodium cyanide. These results suggested that the fatty acids in the plerocercoids played an important role in regulating tbe fluidity of membrane by changing the composition in membrane lipid corresponding with the change of temperature circumstance. The NADH reduced by HAD might be accepted by the complex I in the electron transport system, and thus the parasites were capable of ATP production in a classical pathway of the oxidative phosphorylation system. Key
words:
Spirometra
phosphorylation
erinacei;
plerocercoid; fatty acid; membrane fluidity; ATP production;
oxidative
system.
INTRODUCTION
In the human,
infection with the plerocercoid of Spirometraerinaceicauses “sparganosis”. It migrates in the body of the host, and invades the subcutaneous adipose tissue or deep organs to stay there for a long period. Diphyllobothriid cestodes maintain
*To whom all correspondence should be addressed.
their characteristic fatty acid compositions by absorbing and modifying fatty acids (Nakagawa, Fukushima & Fukumoto, 1987; Fukushima, Abe, Nakagawa & Yamane, 1988). Studies on the fatty acids of parasites have attracted much interest in connection with the physiological functions of parasites, such as growth, regulation of membrane fluidity, energy source and defense mechanism. Among
16
T. Fukushima
them the function related to the defensemechanism of the plerocercoid of S. erinacei against the host immunesystemhasbeendemonstrated(Fukushima, Isobe, Hojo, Shiwaku, Yamane & Torii, 1993).The plerocercoidabsorbedarachidonicacid and released prostaglandinE2 which was known to suppressthe functions of mononuclearcellsof the host. Fatty acids are chief componentsof the cellular and organellemembranesof living things, and their composition affects the fluidity of membrane. Therefore the fatty acid composition of biomembrane should be an important factor affecting the membraneenzyme reaction or the membranereceptor function. The plerocercoidof S. erinacei can live in both cold-blooded reptiles and warm-blooded mammals. In the mammal, beta-oxidation works to break down long-chainfatty acidsand the NADH reduced by beta-hydroxyacyl-CoA dehydrogenase(HAD) is gained by NADH: ubiqinone oxidoreductase (complex I), which is an enzyme complex in the oxidative phosphorylation systemin mitochondria. The activities of beta-oxidation enzymeshave been demonstratedin severalspecies(Ward & Fairbairn, 1970; Barrett & Kiirting, 1977; Korting & Barrett, 1978), but there is no evidencefor an active betaoxidation sequenceforming NADH in any speciesof cestodes.The Diphyllobothrium latum mitochondria oxidize NADH (Salminen, 1973) and succinate (Salminen, 1974) and a part of the cytochrome chain cestodes seem to be of the classical mammalian-type.But there is no evidencefor ATP synthesiscarried out by the oxidative phosphorylation systemin cestodes. In this study, the expectedfunctions of fatty acids of S. erinacei plerocercoid, regulation of membrane fluidity and provision of energysource,were demonstrated, and the question why the plerocercoid preferred adipose tissue as migrating site was discussed. MATERIALS
AND
METHODS
Plerocercoids of S. erinacei were collectedfrom striped snakes(Elaphe quadrivirgata), which were captured in the field during the period from May to August. Fifty of the plerocercoidswere usedfor measuringlipid fractions and the fatty acid concentration in each fraction. The hundred plerocercoids were used for infection and stored in the subcutaneoustissueof a male, IO-week-old golden hamster (Mesocricetus auratus). The rest of the plerocercoids were washed in physiological saline solution and divided into three groups: control group, incubated at 18°C group and incubated at Materials.
et al.
37°C group (each group having 25 plerocercoids). Incubation was done in 2 ml of sterilized physiological saline at the two different temperatures.Five plerocercoids were taken from each group for measurementof fatty acids. The hamster was fed with solid feed (Oriental Kobo Ltd, Japan), given water ad Iibitum in an animal experiment station at 24°C for 6 months.It waskilled by deepether anaesthesia, and plerocercoidswere collected from the subcutaneousadiposetissue.They were washedfirst in ice-cold physiological salinesolution and then in distilled water. Measurement of lipid fractions, lipid separation of each fraction and extraction of fatty acids. The
amount of phospholipid, cholesterol and triglyceridesin plerocercoidsfrom snakesand the host serum were measuredby enzymatic determination and the free fatty acid was measuredwith NEFATest. All the reagentswere products of Wako Pure ChemicalIndustries(Tokyo, Japan). Total lipids of plerocercoidsand the host serumwere extracted by the method of Folch, Lees & Stanley (1957). The extracts were spotted on thin layer plates of silica gel, and chromatographed with a solvent system containing petroleum ether-diethyl ether-aceticacid (80:20:1). Pieces of silica gel, each of which contained a separatedlipid fraction, were scraped from the plate, and the fatty acidswere esterifiedby the method of Metcalfe & Schmitz (1961) and Wijngaarden (1967).C 17:O(margaric acid) standard was added to each samplefor quantitative analysis, and the extracted fatty acidswereanalyzed usinggas chromatogram. Gas chromatography. A Hitachi Model 163 GC equippedwith a flame ionization detector wasused. The gaschromatography wascarried out on 50 m x 0.24 mm (insidediameter)ULBON HR SS-10capillary column (Chromato Packing Center, Kyoto, Japan). The separationwas achievedon column at the range 170-220°C(2”Cmin). The split ratio was 10:1, and nitrogen was used as carrier gas with a flow rate of 18 ml/min. Peaks were identified with standard fatty acids, and were confirmed by a JMS-D 300 massspectrometer (JEOL Company, Tokyo, Japan). Subcellular fractionation. Fifty plerocercoidsfrom the hamster were gently homogenizedin 0.25 Msucrose/l0 mM-potassiumphosphatedibasic. 2 MTris base was added to it to adjust pH 7.0. The homogenatewas centrifuged at 1600g for 12 min. The supematant was filtered through 4 layers of cheeseclothand added buffer containing 0.25 Msucrose, 10 mM-Trischloride buffer (Tris-Cl), 1 mM-EDTA, pH 7.8 (0.25 volumesin the filtrate) and
Fatty acids and ATP production in S. erinacei centrifuged at 27300 g for 30 min. The precipitate was washed twice with buffer containing 0.25-M sucrose, 10 mM-Tris-Cl, pH 7.8, and a crushed mitochondriai fraction was obtained by rough homogenizing, without foaming, with a small amount of the same buffer. The protein determination was done by the biuret assay. The protein concentration of the mitochondrial fraction was 29.7 mgml. Measurement of enzyme activities. For measurement of all the enzyme activities we determined the micromole values of the products formed per min at 37°C. Solutions used for the assay of each enzyme were as follows: for HAD assay, 0.1 M-potassium phosphate buffer, pH 7.2, 100 PM-s-acetoacetyl CoA, 110 ~.LM-NADH and 148.5 p,g mitochondria protein; for NADH: ferricyanide reaction (NFR) assay, 33 mM-Tris-Cl, pH 7.5, 1.25 mM-potassium ferricyanide, 100 ELM-NADH and 148.5 pg mitochondria protein; for NADH: ubiquinone reaction (NQR) assay, 20 mM-potassium phosphate buffer, pH 8.0, 300 FM-coenzyme Qo (CoQo), 100 ELMNADH and 148.5 kg mitochondria protein. The NADH oxidation and the ferricyanide reduction were monitored at 340 nm and 410 nm, respectively, with a HITACHI U-300 spectrophotometer. Measurement of ATP. ATP was measured at room temperature with 17 mM-Tris-Cl buffer, pH 7.5 containing 0.7 mM-EDTA, ImM ADP, 170 FMNADH, 1.5 mg mitochondria, ATP releasing reagent and luciferin luciferase mixture. Ten PM-rotenone, 100 PM-antimycin A and 100 FM-sodium cyanide were used as electron transport system inhibitors. A Luminocounter ATP-237 (Toyo Kagaku Sangyo, LTD., Japan) was used for measurement. Reagents. Methanol, chloroform, hexane were purchased from Wako Chemical Industries (Tokyo, Japan), and were purified by distillation. BF3methanol was also from Wako Chemical Industries. Standard fatty acids were purchased from GL Sciences (Tokyo, Japan). We purchased CoQo, NADH, s-acetoacetyl CoA, antimycin A and Tris base from Sigma Chemical Co. (St Louis, MO, U.S.A.), rotenone, sodium cyanide, EDTA, potassium phosphate dibasic, potassium ferricyanide and BSA from Nacalai Tesque (Kyoto, Japan), and ATP releasing reagent and luciferin luciferase mixture from Laboscience (Tokyo, Japan). RESULTS
Table 1 shows the lipid fractions of S. erinacei plerocercoids and the host snake serum. Phospholipid was the chief lipid of the plerocercoids and the host snake serum, and triglyceride of the
Table l-Lipid
17
fractions of S. erinacei plerocercoids and the host snake serum Plerocercoid
Phospholipid 144.8 + 28.8 mg/100g B.W. Cholesterol 95.5 * 23.5 mg/lO@g B.W. Triglyceride 111.0 * 18.9 mg/lOCJgB.W. Free fatty acid 0.39 k 0.14 mEq/kg B.W.
Snake sernm 466.8 mg/dl 432.9 mg/dl 132.2 mg/dl 0.33 mEq/l
Each value of plerocercoid is the mean t SD. of 5 measurements. B.W.: body weight; mEq: milliequivalent. plerocercoids and cholesterol of the host snake serum were relatively important lipids. Comparing the fatty acid concentrations of each fraction of the plerocercoids with those of the host snake serum, we find the parasite maintained its characteristic fatty acid concentration (Tables 2 and 3). The major fatty acids of the phospholipid of the plerocercoids were C18:1, C18:O and Cl&O, and those of the host snake serum were C 16:0, C 18: 1 and C18:0, in order of amount in both cases. The major fatty acids of the cholesterol ester of the plerocercoids were C18:1, C16:O and C18:0, and those of the host serum were C18:2, C18:l and C20:4, in order of amount. The major fatty acids of the triglyceride of the plerocercoids were C18:O and C16:O which are saturated fatty acids, and those of the host serum were C18:2, C18:l and C20:4 which are unsaturated fatty acids. Table 4 shows the change of fatty acid composition of phospholipid of the plerocercoids before and after incubation in physiological saline at 18 and at 37°C for 24 h. Monounsaturated fatty acids decreased at both temperatures. Polyunsaturated fatty acids increased at 18°C (lower temperature), and saturated fatty acids increased at 37°C (higher temperature). Though polyunsaturated fatty acids also increased at 37°C (higher temperature), it should have been a result affected by a sharp decrease of monounsaturated fatty acids. Table 5 shows values of Michaelis constant (Km) and maximal velocity (V,,,) for NADH of HAD, an enzyme in beta-oxidation of fatty acids and a donor of NADH to electron transport system, and of complex I, an enzyme complex in the electron transport system and an acceptor of NADH from dehydrogenases such as HAD in the plerocercoids of S. erinacei. All the activities of the enzymes, HAD and complex I (NFR) and complex I (NQR) were detectable and their Km and V,,,, values were calculated. The values of Km for NADH of the enzymes are as follows; HAD, 20.6 (*M; complex I (NFR), 50.0 FM; complex I (NQR), 13.3 FM. The ATP synthesis in mitochondria of the plero-
T. Fukushima et al.
18 Table 2-Relative
fatty acid concentrations of phospholipids, free fatty acids, triglycerides and cholesterol esters of S. erinacei plerocercoids from snake
Phospholipids 124.0 2.9 149.6 197.7 10.0 1.3 11.2 17.7 1.5 2.0 8.9 2.4 2.6 2.1
C 160 %I
Go %I CE.2 C 183 C 20.0 Go:, Go:, cm3
C,,:, GO5 C 22.5 C*,:,
Cholesterol esters
? 12.4 k 0.9 f 4.0 f 20.9 f 1.8 * 0.8 AL 1.7 f 3.3 Ii 1.0 k 1.7 k 2.9 f 1.0 f 1.0 + 1.3
Triglycerides
16.3 f 2.6 1.0 f 0.4 13.4 * 2.2 51.9 f 12.8 6.0 f 0.9 0.1 f 0.1 0.4 * 0.3 2.9 f 0.7 0.7 f 0.1 0.1 ?I 0.1 2.0 f 0.6 0.6 + 0.1 1.3 f 0.3 3.1 + 0.8
5.4 0.9 8.4 1.4
f 0.4 2 0.3 f 1.4 f 0.2 N.D. N.D. N.D. 1.7 t 0.3 N.D. N.D. N.D. N.D. N.D. 0.8 k 0.2
Free fatty acids 2.5 0.5 6.7 0.8
+ 0.4 f 0.2 f 1.4 f 0.2 N.D. N.D. N.D. 1.6 f 0.6 N.D. N.D. N.D. N.D. N.D. N.D.
Each value is the mean + SD. (mg/lOO g B.W.) of 5 measurements. N.D.: not detected. Table 3-Relative
fatty acid concentrations
Phospholipids
Cl,:, Cl,:, c,s:o C 18I C 182 cm3
cm0 CZO,, Go:2 Go:, Go:.4 GO3 C225 C22 6
48.7 f 1.5 3.1 2 0.04 11.8 kO.1 28.4 k 0.4 9.8 + 0.8 0.5 * 0.1 0.6 f 0.1 1.2 * 0.5 0.4 k 0.04 0.3 f 0.1 4.4 f 0.6 0.6 + 0.2 0.4 f 0.1 0.5 + 0.1
of phospholipids, free fatty acids, triglycerides and cholesterol esters of host snake serum
Cholesterol esters
Triglycerides
2.3 f 0.6 1.7 f 0.6 0.7 f 0.3 6.1 + 1.3 15.4 * 5.0 0.9 + 0.4 0.1 f 0.02 0.2 f 0.1 0.4 2 0.04 0.2 I! 0.1 3.9 t 2.0 0.7 * 0.3 N.D. 0.3 * 0.2
11.7 f 0.6 4.5 f 0.2 1.9 * 0.7 24.6 f 1.7 33.2 2 6.8 3.1 50.6 0.2 f 0.1 0.6 f 0.2 0.5 f 0.1 0.4 * 0.1 17.4 * 3.4 3.3 * 0.8 1.9 + 0.6 2.1 f 0.6
Free fatty acids 1.9 f 0.04 0.3 f 0.1 1.4 + 0.5 2.3 + 0.4 1.5 + 0.2 0.1 fO.l 0.1 * 0.03 0.03 It 0.01 0.1 f 0.02 N.D. 0.3 f 0.2 0.1 f0.1 N.D. 0.1 + 0.1
Each value is the mean AZS.D. (mg/dl) of 4 measurements. N.D.: not detected. cercoid S. erinacei was demonstrated (Fig. 1). The concentration of ATP in the mitochondria was 0.7 nmoVmg protein. When 170 ~J.M-NADH was added to the mixture containing 1.5 mg mitochondria protein, ATP increased to 1.0 nmoYmg protein. The ATP production in the mitochondria was accelerated by adding 1 mM-ADP and the concentration of ATP was 23.8 nmol/mg protein. But the ATP concentration was decreased to 18.7 nmol/mg protein by adding 170 FM-NADH to the mixture containing 1.5 mg mitochondria protein and 1 mMADP. Inhibition of the ATP synthesis in the mitochondria of plerocercoid S. erinacei using electron transport system inhibitors was demonstrated in order to investigate the plerocercoids’ ability to produce ATP by the oxidative phosphorylation system (Table 6).
The ATP production in the mitochondria was started by adding 1 mM-ADP and the concentration of ATP was 23.8 nmol/mg protein. The ATP synthesis was inhibited to 48.6% (11.6 nmol/mg protein) by 10 FM-rotenone, to 13.9% (3.3 nmol/mg protein) by 100 l.r,M-antimycin A and to 37.5% (8.9 nmol/mg protein) by 100 PM-sodium cyanide. DISCUSSION
The fatty acid composition of parasites is generally different from that of host serum or of the parasitic tissue. The present study suggested that plerocercoids of S. erinacei maintained its characteristic fatty acid concentration in each lipid fraction by absorbing and modifying lipids. Beach, Holz, Semprevivo & Honigberg (1982) certified by culture study that C18, C20 and C22
Fatty acids and ATP production in S. erinacei Table
4-Fatty acid compositions of phospholipid of plerocercoids from snake b&fore and after incubation in physiological saline at 18°C or at 37°C for 24 h S. erinacei
Before incubation C,,:, C 16I c,tw CM:, c,,:, Gl:, GO3 GO3 C*o:4 C,,:, C 22.5 cm SFA MUFA PUFA M/S PJS
23.4 + 0.6 0.5 t 0.1 28.1 f 1.6 37.1 f 1.6 1.8 * 0.3 2.1 k 0.2 3.3 f 0.4 0.3 t 0.1 1.6 f 0.6 0.3 f 0.1 0.5 * 0.2 0.4 f 0.2 53.6 + 1.1 40.8 * 1.5 5.6 f 0.5 0.76 I!C0.04 0.10 f 0.01
30 25 I
24.2 If:2.0 0.5 f 0.2 29.4 f 1.6 28.1 f 2.5 *** 4.2 + 1.0 *** 1.6 + 0.3 * 2.8 i 0.3 0.5 k 0.3 4.2 f 1.0 *** 0.5 * 0.2 2.1 f0.3 **** 1.4 f 0.2 **** 55.2 f 1.6 31.4 f 2.3 **** 13.4 + 2.2 **** 0.57 f 0.05 *** 0.24 i 0.04 ****
20 -
Each value is the mean + S.D. (weight %) of 5 measurements. SFA: Saturated fatty acid; MUFA: monounsaturated fatty acid; PUFA: polyunsaturated fatty acid; M/S: monounsaturatedJsaturated ratio; P/S: polyunsaturated/ saturated ratio. * P
nmo I /mg
After incubation at 18°C at 37°C 26.6 i 2.2 * 0.4 t 0.2 32.7 i 2.5 + 25.3 k 1.5 **** 3.3 f 0.5 *** 1.9 f 0.4 3.5 k 0.4 0.2 2 0.1 3.6 ?I 1.6 * 0.7 + 0.4 1.0 f 0.6 0.7 * 0.4 61.2 f 2.2 **** 29.2 f 1.7 **** 9.7 It 2.2 ** 0.48 f 0.04 **** 0.16 k 0.04 *
dehydrogenase
50.0 13.3 20.6
V,,, WJw) 0.357 0.067 0.006
19
15 lo50
rY?vn A
m B
C
D
Fig. 1. ATP synthesis in plerocercoid of S. erinacei mitochondria. A, ATP amount in mitochondria. Measurements were made at room temperature with 17 mM-Tris-Cl buffer, pH 7.5 containing 0.7 mM-EDTA, 1.5 mg mitochondria, ATP releasing reagent and luciferin luciferase mixture. B, ATP amount in mitochondria after adding 170 FM-NADH. Measurements were made at room temperature with 17 mM-Tris-Cl buffer, pH 7.5 containing 0.7 mM-EDTA, 1.5 mg mitochondria, 170 FM-NADH, ATP releasing reagent and luciferin luciferase mixture. C, ATP amount in mitochondria after adding lmM-ADP. Measurements were made at room temperature with 17 mM-Tris-Cl buffer, pH 7.5 containing 0.7 mM-EDTA, 1.5 mg mitochondria, 1 mM-ADP, ATP releasing reagent and luciferin luciferase mixture. D, ATP amount in mitochondria after adding 1 mM ADP and 170 )LM-NADH. Measurements were made at room temperature with 17 mM-Tri-Cl buffer, pH 7.5 containing 0.7 mM-EDTA, 1.5 mg mitochondria, 1 mM-ADP, 170 FM-NADH, ATP releasing reagent and luciferin luciferase mixture.
Measureme& were made at 37°C. Solutions were 33 mM-Tris-Cl, pH 7.5, 1.25 mM-potassium ferricyanide and 148.5 pg mitochondria (NFR assay), 20 mM-potassium phosphate buffer, pH 8.0, 300 FM-CoQo and 148.5 Fg mitochondria (NQR assay), 0.1 M potassium phosphate buffer pH 7.2, 100 FM-s-acetoacetyl CoA and 148.5 wg mitochondria (P-hydroxyacyl-CoA dehydrogenase assay). NADH oxidation and ferricyanide reduction were monitored at 340 nm and 410 nm, respectively.
Table 6-Inhibition of ATP synthesis in the plerocercoid S. erinacei mitochondria with electron transport system inhibitors
polyunsaturated fatty acids in membrane phospholipids of Leishmuniu donovani decreased with the ascent of temperature. It is generally known that the shorter the chain of fatty acids and the higher the degree of desaturation, the lower the melting points. In our incubation study, polyunsaturated fatty acids increased at 18°C (lower temperature), and saturated fatty acids increased at 37°C (higher temperature). The melting points of fatty acids in membrane lipid played an important role in regulating the fluidity of
Measurements were made at room temperature with 17 mM-Tris4 buffer, pH 7.5, containing 0.7 mM-EDTA, lmM-ADP, 170 FM-NADH, each inhibitor (10 FMrotenone, 100 PM-antimycin A and 100 PM-sodium cyanide), 1.5 mg mitochondria, ATP releasing reagent and luciferin luciferase mixture.
ATP (nmolJmg) Per cent Mitochondria Mitochondria Mitochondria Mitochondria
+ + + +
ADP Rotenone + ADP Antimycin A + ADP Cyanide + ADP
23.8 11.6 3.3 8.9
100 48.6 13.9 37.5
membrane (Singer & Nicolson, 1972; Martin, Jr, 1985). The plerocercoid should regulate the fluidity of membrane to maintain the function of membrane proteins, and for that purpose it changes the fatty
20
T. Fukushima et al.
acid composition in membrane lipid as the host temperature circumstance changes. In the mammal, HAD functions as a beta-oxidation enzyme, giving NADH to the oxidative phosphorylation system and inciting ATP synthesis. But there is no evidence for an active beta-oxidation sequence to form NADH and make ATP synthesize in the oxidative phosphorylation system of any species of cestodes. The beta-oxidation enzymes have been assumed to be used for the formation of branched C5 and C6 acids (Ward & Fairbairn, 1970; Kijrting & Barrett, 1978) or the elongation of longchain fatty acids (Seubert, Lamberts, Kramer & Ohly, 1968). The chief source of energy might be glycolysis, so it is difficult to explain the source of the large energy that enables active mobility and rapid growth of the plerocercoids of S. erinacei. Mitochondrial functions, especially its oxidative phosphorylation system have been studied energetically with protozoa (Bienen, Webster & Fish, 1991; Gozar, O’Sullivan & Bagnara, 1992) and nematoda (Wang, Takamiya, Kita, Oya & Aoki, 1992; Kita, 1992). In cestodes, isolated Hymenolepis diminuta and Moniezia expansa mitochondria are capable of oxidative phosphorylation and respiratory control (Cheah, 1971; Yorke & Turton, 1974) and the D. datum mitochondria oxidizes NADH (Salminen, 1973) and succinate (Salminen, 1974), and probably a part of the cytochrome chain in cestodes is of the classical mammalian-type. Allen (1973) reported that the presence of rhodoquinone was demonstrated, but no ubiquinone was found in an adult M. expansa. On the contrary, Dhandayuthapani, Nellaiappan & Ramalingam (1983) reported that ubiquinone and vitamin K were detected in adult Penetrocephalus ganapatii. There is no evidence, however, for the ATP synthesis carried out by the oxidative phosphorylation system in cestodes. Activities of the beta-oxidation enzyme (HAD) and the electron transport system enzyme complex (complex I) in the plerocercoids of S. erinacei were detected and kinetic characteristics of these enzymes were presented. And then the ATP synthesis and the inhibition of ATP synthesis by electron transport system inhibitors in the mitochondria of plerocercoid S. erinacei were demonstrated. Our results suggested that the plerocercoids of S. erinacei could make ATP synthesize by the oxidative phosphorylation system. Ubiquinone might exist in the electron transport system of the plerocercoids of S. erinacei, because the ATP synthesis was inhibited by rotenone, an inhibitor of the reduction of ubiquinone. Considering that the ATP synthesis was inhibited by other inhibitors of the cytochrome
chain, a part of the cytochrome chain in the plerocercoids of S. erinacei might be of the classical mammalian type. The plerocercoid migrates in the host adipose tissue. Probably the tissue is a more suitable site for escaping from the host immune system than the abdominal cavity. Our results tell that the plerocercoids use fatty acids in adipose tissue positively for its physiological functions. Their preference of the adipose tissue as migration site could have a deep connection with their existence. Acknowledgements-We thank Masamitsu Fukushima and Toshimi Yoneyama, the technical officials, for the technical assistance. REFERENCES
Allen P. C. 1973. Helminths: comparison of their rhodoquionone. Experimental Parasitology 34: 211-219. Barrett J. & Kdrting W. 1977. Lipid catabolism in the plerocercoids of Schistocephalus solidus (Cestoda: Pseudophyllidea). International Journal for Parasitology 7: 419422. Beach D. H., Holz G. G., Semprevivo L. H. & Honigberg B. M. 1982. Temperature-dependent fatty acyl group changes in phospholipids of 37”C-adapted Leishmania donovani promastigotes. Journal of Parasitology 68: 1004-1009. Bienen E. J., Webster P. & Fish W. R. 1991. Trypanosoma (Nannomonas) congolense: changes in respiratory metabolism during the life cycle. Experimental Parasitology 73: 403412. Cheah K. S. 1971. Oxidative phosphorylation in Moniezia muscle mitochondria. Biochemica et Biophysics Acta 253: l-11. Dhandayuthapani S., Nellaiappan K. & Ramalingam K. 1983. Quinones in Penetrocephalus ganapatii (Cestoda:Pseudophyllidea). Journal of Parasitology 69: 996-998.
Folch J., Lees M. & Stanley G. H. 1957. A simple method for the isolation and purification of total lipids from animal tissue. Journal of Biological Chemistry 226: 497-509.
Fukushima T., Abe K., Nakagawa A. & Yamane Y. 1988. Fatty acid composition of plerocercoid and adult of Spirometra erinacei and the host-parasite relationship. International
Journal
for Parasitology
18: 27-31.
Fukushima T., Isobe A., Hojo N., Shiwaku K., Yamane Y. & Torii M. 1993. The metabolism of arachidonic acid to PGE2 in plerocercoid of Spirometra erinacei. Parasitology
Research
79: 634-638.
Gozar M. M. G., O’Sullivan W. J. & Bagnara A.S. 1992. Mitochondrial function in Babesia bovis. International Journal for Parasitology 22: 165-17 1. Kita K. 1992. Electron-transfer complexes of mitochondria in Ascaris suum. Parasitology Today 8: 155-l 59. Kiirting W. & Barrett J. 1978. Studies on beta-oxidation in the plerocercoids of Ligula intestinalis (Cestoda:
Fatty
acids and ATP
Pseudophyllidea). Zeitschrift fiir Parasitenkunde 57: 243-246. Martin Jr. D. W. 1985. The fluid mosaic model of membrane structure. In: Harper’s Review of Biochemistry (Edited by Martin D. W. Jr., Mayes P. A., Rodwell V. W. & Granner D. K.), pp. 454456. Maruzen Co. Ltd, Tokyo. Metcalfe L. D. & Schmitz A. A. 1961. The rapid preparation of fatty acid esters for gas chromatographic analysis. Analytical Chemistry 33: 363-364. Nakagawa A., Fukushima T. & Fukumoto S. 1987. Fatty acid composition of diphyllobothriid cestodes with reference to their hosts. Yonago Acta Medica 30: 65-80. Salminen K. 1973. The oxidation of external NADH adult and plerocercoid Diphytlobothrium by latum. Comparative Biochemistry and Physiology 44B: 283-289. Salminen K. 1974. Succinate dehydrogenase and cytochrome oxidase in adult and plerocercoid Diphyllobothrium latum. Comparative Biochemistry and Physiology 49B: 87-92. Seubert W., Lamberts I., Kramer R. & Ohly B. 1968. On
production
in S. erinacei
21
the mechanism of malonyl-CoA-independent fatty acid synthesis. I. The mechanism of elongation of long-chain fatty acids by acetyl-CoA. Biochimica et Biophysics Acta 164: 498-517. Singer S. J. & Nicolson G. L. 1972. The fluid mosaic model of the structure of cell membranes. Science 175: 720. Wang H., Takamiya S., Kita K., Oya H. & Aoki T. 1992. Comparative study on the primary structure of the ironsulfur subunit of complex II in Ascaris suum mitochondria. Japanese Journal of Parasitology 1: 122-131. Ward C. W. & Fairbaim D. 1970. Enzymes of beta-oxidation and the tricarboxylic acid cycle in adult Hymenolepis diminuta (Cestoda) and Ascaris lumbricoides (Nematoda). Journal of Parasitology 56: 1009-1012. Wijngaarden D. V. 1967. Modified rapid preparation of fatty acid esters from lipids for gas chromatographic analysis. Analytical Chemistry 39: 848-849. Yorke R. E. & Turton J. A. 1974. Effects of fasciolicidal and anti-cestode agents on the respiration of isolated Hymenolepis diminuta mitochondria. Zeitschrift fir Parasitenkunde 45: l-10.