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Oxidative phosphorylation in Ascaris muscle mitochondria
Comp. Biochera. Physiol., 1974, Vol. 47B, pp. 237 to 242. Pergamon Press. Printed in Great Britain
OXIDATIVE PHOSPHORYLATION IN A S C A R I S MUSCLE MITOCHONDRIA K. S. CHEAH Agricultural Research Council, Meat Research Institute, Langford, Bristol BS18 7DY, U.K. (Received 16 May 1973)
Abstract--1. An improved method of preparing Ascaris muscle mitochondria illustrates for the first time the presence of both respiratory control and oxidative phosphorylation in these mitochondria. 2. Oxidative phosphorylation was demonstrated by the stimulation of oxygen uptake by ADP, the inhibition of the ADP-stimulated respiration by oligomycin and its subsequent relieve of inhibition by the uncoupler, ptrifluoromethoxy-carbonyl-cyanide-phenylhydrazone (FCCP). 3. Ascaris muscle mitochondria contain both coupled Site II and Site III. These were demonstrated by using suecinate or o~-glycerophosphatefor Site II and ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) for Site III. INTRODUCTION THE PRESENCE of a functional cytochrome system in Ascaris lumbrieoides, an intestinal roundworm of pigs, is now established (Kikuchi et al., 1959; Kikuchi & Ban, 1961; Smith, 1969; Cheah & Chance, 1970; Cheah, 1972). Mitochondria isolated from Ascaris muscle contained substrate-reducible cytochromes of types a, b and c (Kikuchi et al., 1959; Kikuchi & Ban, 1961; Cheah & Chance, 1970; Cheah, 1972), with cytochrome as acting as the terminal oxidase (Smith, 1969; Cheah & Chance, 1970). Cytochrome c (Hill et al., 1971 ; Cheah, 1972) and a novel b-type cytochrome, designated Ascaris cytochrome bss0 (Cheah, 1973b), have also been purified from Ascaris muscle and the amino acid composition of both of these cytochromes determined (Hill et al., 1971 ; Cheah, 1973b). Furthermore, enzymes of the tricarboxylic acid cycle were demonstrated to be present in Ascaris muscle (Oya et al., 1971). These findings presented from different groups of workers (Kikuchi et al., 1959; Kikuchi & Ban, 1961 ; Smith, 1969; Cheah & Chance, 1970; Hill et al., 1971; Oya et al., 1971; Cheah, 1972) do not support the view that Ascaris mitochondria function anaerobically (Saz, 1970, 1972), and that Asearis leads a predominantly anaerobic type of metabolism without the participation of cytochromes in the respiratory chain system (Kmetec & Bueding, 1961 ; Saz, 1970, 1972). This paper describes the presence of respiratory control and oxidative phosphorylation in Ascaris muscle mitochondria and also of the existence of coupling Site II and Site III. 237
238
K.S. CsF~,-I MATERIALS AND METHODS
Antimycin A (Type III), oligomycin and the sodium saltsof ADP, A T P (Grade II), malate, pyruvate, succinatc and rotenone were purchased from Sigma Chemical Corp. ; sodium saltsof L-ascorbate, E D T A and T M P D from the British Drug Houses. All other reagents were of analyticalgrade. CrystallineBacillus subtilisproteinasewas obtained from Teikoku Chemical Co., Osaka, and F C C P was kindly supplied by Dr. P. Heytler. Ascaris was obtained from pigs killedat the Meat Research Instituteat Langford. The worms were rinsed in Ringer's solution and were used within 2 hr after removal from the host or after 20 hr storage in Ringer's solution at 2--4°C. The worms were eviscerated, washed in Ringer's solution and then minced. The minced muscle was stirred in the isolationmedium (pH 7"4) containing I00 m M KCI, 50 trim Tris-HCl, I rnM ATP, 5 m M MgCls, I m M E D T A and dissolvedproteinase (3 mg/g wet tissue)for 20 rain at 0°C. The mitochondria were then isolated using the method previously described for isolating
mitochondria from adults of Moniezia expansa (Cheah, 1971a). For electron microscopy, the/lscaris muscle mitochondria were fixed in 2"0% glutaraldehyde in 50 mM cacodylate buffer (pH 7.4) containing 250 mM sucrose, before being embedded in Epon (Cheah & Cheah, 1971). Oxygen uptake was measured with a Clark oxygen electrode [Yellow Spring Biological Oxygen Monitor (Model 53)] at 25°C. The reaction medium (pH 7"2) contained 1"0 mM EDTA, 30"0 mM KCI, 6.0 mM MgCI2, 75"0 mM sucrose and 20 mM KH2PO 4. Other experimental details are given in the legends. Protein was determined by the Folin phenol reagent (Lowry et al., 1951).
RESULTS AND DISCUSSION An improved method of isolating intact Ascaris muscle mitochondria made it possible to demonstrate for the first time the presence of both respiratory control and oxidative phosphorylation in this intestinal parasite. Oxidative phosphorylation was indicated by the stimulation of oxygen uptake by ADP and the inhibition of oxygen uptake by oligomycin. Unfortunately no clear cut State 3 to State 4 transitions (Chance & Williams, 1956) were observed as the mitochondrial preparations were loosely coupled. Thus, the values of the ADP/O ratio could not be estimated by the polarographic method (Chance & Williams, 1956). Electron microscopic examinations (Fig. 1) show that Ascaris mitochondria contained inner and outer membranes, outer compartments and intracristal spaces. The presence of elementary particles attaching to the mitochondrial cristae have previously been demonstrated in negatively stained mitochondrial preparations (Chance & Parsons, 1963). Thus, like the Moniezia muscle mitochondria (Cheah, 1971a), the mitochondria from ~qscaris muscle have the same structural characteristics as the classical mammalian aerobic mitochondria. With mitochondria from both Ascaris and Moniezia muscle, the mitochondrial matrix appears to be more densely stained than mitochondria igolated from mammalian skeletal muscle (Cheah, 1971b; Cheah & Cheah, 1971). Table 1 illustrates the effects of ADP, ATP, oligomycin and FCCP on the mitochondrial oxidation of succinate by Ascaris muscle mitochondria. The data dearly indicate the presence of oxidative phosphorylation as the oxygen uptake due to succinate oxidation was inhibited by oligomycin, a classical inhibitor of A T P
FIG. 1. Thin section of Ascaris muscle mitochondria. The mitochondria were stained with uranyl acetate and lead citrate before examination with an AEI (Model EM6-B) electron microscope. Magnification: x 20,000.
OXIDATIVE PHOSPHORYLATION IN ASCAR1S MUSCLE MITOCHONDRIA
239
TABLE I~RESPIRATORY CONTROL AND OXIDATIVE PHOSPHORYLATION IN AscaFi$ MUSCLE MITOCHONDRIA.* (A) SUCCINOXIDASE SYSTEM
Additions
Respiratory rate (%)
Rotenone + succinate Rotenone + succinate + ADP Rotenone + suecinate + ADP + oligomycin Rotenone + succinate + ADP + oligomycin + FCCP Rotenone + suceinate + ATP Rotenone + succinate + ATP + oligomycin Rotenone + succinate + ATP + oligomycin + FCCP
100 (4) 100 (2) 70 (1) 85 (1) 41 (2) 33 (1) 60 (1)
Respiratory control index (RCI)
1 "43
2"44 3"03
The oxygen uptake of Ascaris muscle mitochondria was estimated polarographicaUy at 25°C using a Clark oxygen electrode. For convenience the respiratory rates were expressed in per cent. Total volume, 2-50 ml. Final pH 7"2. Final concentration of additions: rotenone, 2/zM; succinate, 10 mM; ADP, 300/zM; ATP, 500 ftM; oligomycin, 0.5/zg/mg protein; FCCP, 1"0/~M. The numbers in parentheses refer to the number of estimations. synthesis by mitochondrial respiratory chain system (Slater, 1967). Furthermore, the inhibition of oxygen uptake by oligomycin could be relieved by the uncoupler FCCP. The mitochondrial preparations were loosely coupled since ADP did not increase the rate of oxygen uptake, but the mitochondria still retained the property of control of respiration. That respiratory control is present in Ascaris muscle mitochondria is supported by the inhibition of oxygen uptake by A T P and also by oligomycin. Inhibition of oxygen uptake by ATP and oligomycin could be relieved by FCCP. The values for the respiratory control index (RCI) vary from 1.43 to 3.03; the RCI was based on the rate of oxygen uptake due to succinate oxidation divided by the rate in the presence of either ATP or A T P plus oligomycin. Analogous to the oxidation of succinate, the oxidation of ~-glycerophosphate was not stimulated by the addition of ADP (Table 2). This was expected since the mitochondrial preparations were loosely coupled. The subsequent addition of oligomycin inhibited the rate of oxygen uptake and this inhibition could again be relieved by FCCP. These observations again support the presence of oxidative phosphorylation and respiratory control in Ascaris muscle mitochondria. The oxidation of a-glycerophosphate linking to oxidative phosphorylation and the previous reports on the oxidation of ~-glyeerophosphate by Ascaris muscle mitochondria (Cheah & Chance, 1970) are contrary to a recent claim that this particular system is absent in Ascaris muscle (Barrett & Beis, 1973). The cytochrome oxidase (E.C. 1.9.31) activity of Ascaris muscle mitochondria was assayed using ascorbate plus T M P D . Unlike the succinoxidase and the ~glycerophosphate oxidase systems, the addition of ADP to mitochondria oxidizing ascorbate plus T M P D resulted in an increase in the rate of oxygen uptake. Unfortunately, no clear transition of the State 3 to State 4 rate was observed. Oxidative
240
K . S . CH~H
TABLE 2--RRSPIRATORY CONTROL AND OXIDATIVE PHOSPHORYLATION IN Ascaris MUSCLE MITOCHONDRIA: (B) ¢X-GLYCRROPHOSPHATE OXIDASE SYSTEM
Additions
Respiratory rate (%)
~-Glycerophosphate ~-Glycerophosphate + ADP ~-Glycerophosphate + ADP + oligomycin ~-Glycerophosphate + ADP + oligomycin + FCCP
100 100 72 91
Respiratory control index (RCI)
1"39
Experimental details as described in the legends to Table 1. Final concentration of additions: ~-glycerophosphate, 10 mM; ADP, 300/~M; oligomycin, 0-5/~g/mg protein; FCCP, 1"0/~M. phosphorylation was taking place as oligomycin inhibited the A D P - i n d u c e d rate (State 3) and this inhibited rate could be relieved by FCCP. Crude A T P , containing 6 per cent ADP, also stimulated respiration. T h e respiratory rate stimulated by crude A T P appears to be greater than that stimulated by ADP. In one particular experiment, not included in Table 3, a fivefold increase in rate was observed. At this stage, it is difficult to explain this phenomenon. T h e respiratory control index TABLE 3--RESPIRATORY CONTROL AND OXIDATIVE PHOSPHORYLATION IN Ascaris MUSCLE MITOCHONDRIA: ( C ) ASCORBATE-TMPD OXIDASE SYSTEM
Additions Ascorbate + TMPD Ascorbate + TMPD + ADP Ascorbate + TMPD + ATP (crude) Ascorbate + T M P D + ATP (crude) + oligomycin Ascorbate + TMPD + ADP + ATP (crude) Ascorbate + T M P D + ADP + ATP (crude) + oligomycin Ascorbate + TMPD + ADP + ATP (crude) + oligomycin + FCCP
Respiratory rate (%) 100 (7) 162 (5) 169 (3) 127 (1) 192 (4) 138 (3)
Respiratory control index (RCI)
1"33 1"39
198 (3)
Experimental details as described in the legends to Table 1 except that antimycin A (0"5/~g/mg protein) was added prior to the addition of ascorbate plus TMPD. Final concentration of additions: ascorbate, 4 mM; TMPD, 0.4 mM; ADP, 300/~M; crude ATP, 5000/~M containing 300/~M ADP. based on either A D P or the crude A T P - i n d u c e d oxygen uptake rate divided by the rate of oxygen uptake in the presence of oligomycin was about 1.3. T h e data in Table 3 thus favour the presence of a loosely coupled Site I I I in Ascaris muscle mitochondria. T h e average State 3 rate based on seven estimations from four different mitochondrial preparations was 66 natoms O/rain per mg protein at 25°C (pH = 7.2), and this rate was comparable with the State 3 rate of 52 natoms
OXIDATIVE P H O S P H O R Y L A T I O N I N .~,.qCAIUS MUSCLE M I T O C H O N D R I A
241
O/min per mg protein for Moniezia muscle mitoehondria (Cheah, 1971a). It thus appears that the State 3 rate for a coupled functional Site I I I in large intestinal parasites like .dscaris and Moniezia is about 25-30 per cent of the State 3 rate for Site I I I in mitochondria isolated from aerobic skeletal muscle (Cheah, 1973a). T h e present data on oxidative phosphorylation and respiratory control in Ascaris do not support claims (Kmetec & Bueding, 1961; Saz, 1970, 1972) that .dscaris possesses a predominantly anaerobic type of metabolism. Our present findings together with previous work on other large intestinal parasites strongly suggest that mitochondria from these parasites (Cheah, 1967; Cheah & Chance, 1970; Cheah, 1971a) have functional cytochromes in their respiratory chain systems, and also that oxidation of the various substrates is linked to oxidative phosphorylation (Cheah, 1971a). Acknowledgements--The author would like to thank Mrs. A. M. Cheah for technical assistance.
REFERENCES BARRETTJ. & BEIS I. (1973) Studies on the glycolysis in the muscle tissue of Ascaris lumbricoides (Nematoda). Comp. Biochem. Physiol. 44B, 751-761. CHANCE B. & PARSONSD. P. (1963) Cytochrome function in relation to inner membrane structure of mitoehondria. Science 142, 1176-1180. CHANCEB. & WI~-LXAMSG. R. (1956) The respiratory chain and oxidative phosphorylafion. In Advances in Enzymology (Edited by NO~D F. F.), Vol. 17, pp. 65-134. Interseienee, New York. CHWH K. S. (1967) Spectrophotometric studies on the succinate oxidase system of Taenia hydatigena. Comp. Biochem. Physiol. 20, 867-875. CrlEAH K. S. (1971a) Oxidative phosphorylation in Moniezia-muscle mitochondria. Biochim. biophys..4cta 253, 1-11. CH~H K. S. (1971b) Effect of loss of cytochrome c following storage of mitochondria in situ. F E B S Lett. 19, 105-108. CrmAH K. S. (1972) Cytochromes in Ascaris and Moniezia. In Comparative Biochemistry of Parasites (Edited by VAN DEN Bosscrm H.), pp. 417-432. Academic Press, New York. CHEAH K. S. (1973a) Comparative studies of the mitochondrial properties of Longissimus dorsi muscle of Pietrain and Large White pigs. ft. Sci. Fd .4gric. 24, 51-61. CHEAH K. S. (1973b) Purification and properties of Ascaris cytochrome bseo. ft. biol. Chem. 248, 4101-4105. C~AH K. S. & CHANC~ B. (1970) The oxidase systems of .4scaris-muscle mitoehondria. Biochim. biophys. Aeta 223, 55-60. CHEArI K. S. & CHWH A. M. (1971) Post-mortem changes in structure and function of ox muscle mitoehondria--1. Electron microscopic and polarographic investigations. J. Bioenerg. 2, 85-92. HXLL G. C., P~.RKOWSKXC. A. & MAXHEWSONN. W. (1971) Purification and properties of cytochrome c55o from Ascaris lumbricoides var. suum. Biochim. biophys. Acta 236, 242245. KIRUCrlI G. & BAN S. (1961) Cytochrome in the particulate preparation of the Ascaris lumbricoides muscle. Biochim. biophys. Acta 51, 387-389. KXRUCm G., RAMIm~XJ. & BAa~aONE. S. G. (1959) Electron transport system in Ascaris lumbricoides. Biochim. biophys. Acta 36, 335-342. KMEX~C E. & BtmDXNO E. (1961) Succinic and reduced diphosphopyridine nueleotide oxidase system of Ascaris muscle. J. biol. Chem. 236, 584--591.
242
K.S. CH~
Loway O. H., ROSF.BllOUGHN. J., FARR A. L. & RANDALLR. J. (1951) Protein measurement with the Folin phenol reagent, j~. biol. Chem. 193, 265-275. OYA H., KXKUCm G., BANDO T. & HAYASm H. (1965) Muscle tricarboxylic acid cycle in .4scaris lumbricoides vat. suis. Expl Parasit. 17, 229-240. SAZH. J. (1970) Comparative energy metabolisms of some parasitic helminths..7. Parasit. 56, 634-642. S~z H. J. (1972) Comparative biochemistry of carbohydrates in nematodes and cestodes. In Comparative Biochemistry of Parasites (Edited by VAN DZN BOSSCHZ H.), pp. 33-47. Academic Press, New York. SLAT~ E. C. (1967) Application of inhibitors and uncouplers for a study of oxidative phosphorylation. In Methods of Enzymology (Edited by ESTABROOKR. W. & PULLMAN M. E.), Vol. X, pp. 48-57. Academic Press, New York. SMXTHM. H. (1969) Do intestinal parasites require oxygen ? Nature, Lond. 223, 11291132.
Key Word Index---.dscaris lumbricoides muscle mitocbondria; oxidative phosphorylation; respiratory control; succinoxidase; cytochrome oxidase (as).
OXIDATIVE PHOSPHORYLATION IN A S C A R I S MUSCLE MITOCHONDRIA K. S. CHEAH Agricultural Research Council, Meat Research Institute, Langford, Bristol BS18 7DY, U.K. (Received 16 May 1973)
Abstract--1. An improved method of preparing Ascaris muscle mitochondria illustrates for the first time the presence of both respiratory control and oxidative phosphorylation in these mitochondria. 2. Oxidative phosphorylation was demonstrated by the stimulation of oxygen uptake by ADP, the inhibition of the ADP-stimulated respiration by oligomycin and its subsequent relieve of inhibition by the uncoupler, ptrifluoromethoxy-carbonyl-cyanide-phenylhydrazone (FCCP). 3. Ascaris muscle mitochondria contain both coupled Site II and Site III. These were demonstrated by using suecinate or o~-glycerophosphatefor Site II and ascorbate plus N,N,N',N'-tetramethyl-p-phenylenediamine (TMPD) for Site III. INTRODUCTION THE PRESENCE of a functional cytochrome system in Ascaris lumbrieoides, an intestinal roundworm of pigs, is now established (Kikuchi et al., 1959; Kikuchi & Ban, 1961; Smith, 1969; Cheah & Chance, 1970; Cheah, 1972). Mitochondria isolated from Ascaris muscle contained substrate-reducible cytochromes of types a, b and c (Kikuchi et al., 1959; Kikuchi & Ban, 1961; Cheah & Chance, 1970; Cheah, 1972), with cytochrome as acting as the terminal oxidase (Smith, 1969; Cheah & Chance, 1970). Cytochrome c (Hill et al., 1971 ; Cheah, 1972) and a novel b-type cytochrome, designated Ascaris cytochrome bss0 (Cheah, 1973b), have also been purified from Ascaris muscle and the amino acid composition of both of these cytochromes determined (Hill et al., 1971 ; Cheah, 1973b). Furthermore, enzymes of the tricarboxylic acid cycle were demonstrated to be present in Ascaris muscle (Oya et al., 1971). These findings presented from different groups of workers (Kikuchi et al., 1959; Kikuchi & Ban, 1961 ; Smith, 1969; Cheah & Chance, 1970; Hill et al., 1971; Oya et al., 1971; Cheah, 1972) do not support the view that Ascaris mitochondria function anaerobically (Saz, 1970, 1972), and that Asearis leads a predominantly anaerobic type of metabolism without the participation of cytochromes in the respiratory chain system (Kmetec & Bueding, 1961 ; Saz, 1970, 1972). This paper describes the presence of respiratory control and oxidative phosphorylation in Ascaris muscle mitochondria and also of the existence of coupling Site II and Site III. 237
238
K.S. CsF~,-I MATERIALS AND METHODS
Antimycin A (Type III), oligomycin and the sodium saltsof ADP, A T P (Grade II), malate, pyruvate, succinatc and rotenone were purchased from Sigma Chemical Corp. ; sodium saltsof L-ascorbate, E D T A and T M P D from the British Drug Houses. All other reagents were of analyticalgrade. CrystallineBacillus subtilisproteinasewas obtained from Teikoku Chemical Co., Osaka, and F C C P was kindly supplied by Dr. P. Heytler. Ascaris was obtained from pigs killedat the Meat Research Instituteat Langford. The worms were rinsed in Ringer's solution and were used within 2 hr after removal from the host or after 20 hr storage in Ringer's solution at 2--4°C. The worms were eviscerated, washed in Ringer's solution and then minced. The minced muscle was stirred in the isolationmedium (pH 7"4) containing I00 m M KCI, 50 trim Tris-HCl, I rnM ATP, 5 m M MgCls, I m M E D T A and dissolvedproteinase (3 mg/g wet tissue)for 20 rain at 0°C. The mitochondria were then isolated using the method previously described for isolating
mitochondria from adults of Moniezia expansa (Cheah, 1971a). For electron microscopy, the/lscaris muscle mitochondria were fixed in 2"0% glutaraldehyde in 50 mM cacodylate buffer (pH 7.4) containing 250 mM sucrose, before being embedded in Epon (Cheah & Cheah, 1971). Oxygen uptake was measured with a Clark oxygen electrode [Yellow Spring Biological Oxygen Monitor (Model 53)] at 25°C. The reaction medium (pH 7"2) contained 1"0 mM EDTA, 30"0 mM KCI, 6.0 mM MgCI2, 75"0 mM sucrose and 20 mM KH2PO 4. Other experimental details are given in the legends. Protein was determined by the Folin phenol reagent (Lowry et al., 1951).
RESULTS AND DISCUSSION An improved method of isolating intact Ascaris muscle mitochondria made it possible to demonstrate for the first time the presence of both respiratory control and oxidative phosphorylation in this intestinal parasite. Oxidative phosphorylation was indicated by the stimulation of oxygen uptake by ADP and the inhibition of oxygen uptake by oligomycin. Unfortunately no clear cut State 3 to State 4 transitions (Chance & Williams, 1956) were observed as the mitochondrial preparations were loosely coupled. Thus, the values of the ADP/O ratio could not be estimated by the polarographic method (Chance & Williams, 1956). Electron microscopic examinations (Fig. 1) show that Ascaris mitochondria contained inner and outer membranes, outer compartments and intracristal spaces. The presence of elementary particles attaching to the mitochondrial cristae have previously been demonstrated in negatively stained mitochondrial preparations (Chance & Parsons, 1963). Thus, like the Moniezia muscle mitochondria (Cheah, 1971a), the mitochondria from ~qscaris muscle have the same structural characteristics as the classical mammalian aerobic mitochondria. With mitochondria from both Ascaris and Moniezia muscle, the mitochondrial matrix appears to be more densely stained than mitochondria igolated from mammalian skeletal muscle (Cheah, 1971b; Cheah & Cheah, 1971). Table 1 illustrates the effects of ADP, ATP, oligomycin and FCCP on the mitochondrial oxidation of succinate by Ascaris muscle mitochondria. The data dearly indicate the presence of oxidative phosphorylation as the oxygen uptake due to succinate oxidation was inhibited by oligomycin, a classical inhibitor of A T P
FIG. 1. Thin section of Ascaris muscle mitochondria. The mitochondria were stained with uranyl acetate and lead citrate before examination with an AEI (Model EM6-B) electron microscope. Magnification: x 20,000.
OXIDATIVE PHOSPHORYLATION IN ASCAR1S MUSCLE MITOCHONDRIA
239
TABLE I~RESPIRATORY CONTROL AND OXIDATIVE PHOSPHORYLATION IN AscaFi$ MUSCLE MITOCHONDRIA.* (A) SUCCINOXIDASE SYSTEM
Additions
Respiratory rate (%)
Rotenone + succinate Rotenone + succinate + ADP Rotenone + suecinate + ADP + oligomycin Rotenone + succinate + ADP + oligomycin + FCCP Rotenone + suceinate + ATP Rotenone + succinate + ATP + oligomycin Rotenone + succinate + ATP + oligomycin + FCCP
100 (4) 100 (2) 70 (1) 85 (1) 41 (2) 33 (1) 60 (1)
Respiratory control index (RCI)
1 "43
2"44 3"03
The oxygen uptake of Ascaris muscle mitochondria was estimated polarographicaUy at 25°C using a Clark oxygen electrode. For convenience the respiratory rates were expressed in per cent. Total volume, 2-50 ml. Final pH 7"2. Final concentration of additions: rotenone, 2/zM; succinate, 10 mM; ADP, 300/zM; ATP, 500 ftM; oligomycin, 0.5/zg/mg protein; FCCP, 1"0/~M. The numbers in parentheses refer to the number of estimations. synthesis by mitochondrial respiratory chain system (Slater, 1967). Furthermore, the inhibition of oxygen uptake by oligomycin could be relieved by the uncoupler FCCP. The mitochondrial preparations were loosely coupled since ADP did not increase the rate of oxygen uptake, but the mitochondria still retained the property of control of respiration. That respiratory control is present in Ascaris muscle mitochondria is supported by the inhibition of oxygen uptake by A T P and also by oligomycin. Inhibition of oxygen uptake by ATP and oligomycin could be relieved by FCCP. The values for the respiratory control index (RCI) vary from 1.43 to 3.03; the RCI was based on the rate of oxygen uptake due to succinate oxidation divided by the rate in the presence of either ATP or A T P plus oligomycin. Analogous to the oxidation of succinate, the oxidation of ~-glycerophosphate was not stimulated by the addition of ADP (Table 2). This was expected since the mitochondrial preparations were loosely coupled. The subsequent addition of oligomycin inhibited the rate of oxygen uptake and this inhibition could again be relieved by FCCP. These observations again support the presence of oxidative phosphorylation and respiratory control in Ascaris muscle mitochondria. The oxidation of a-glycerophosphate linking to oxidative phosphorylation and the previous reports on the oxidation of ~-glyeerophosphate by Ascaris muscle mitochondria (Cheah & Chance, 1970) are contrary to a recent claim that this particular system is absent in Ascaris muscle (Barrett & Beis, 1973). The cytochrome oxidase (E.C. 1.9.31) activity of Ascaris muscle mitochondria was assayed using ascorbate plus T M P D . Unlike the succinoxidase and the ~glycerophosphate oxidase systems, the addition of ADP to mitochondria oxidizing ascorbate plus T M P D resulted in an increase in the rate of oxygen uptake. Unfortunately, no clear transition of the State 3 to State 4 rate was observed. Oxidative
240
K . S . CH~H
TABLE 2--RRSPIRATORY CONTROL AND OXIDATIVE PHOSPHORYLATION IN Ascaris MUSCLE MITOCHONDRIA: (B) ¢X-GLYCRROPHOSPHATE OXIDASE SYSTEM
Additions
Respiratory rate (%)
~-Glycerophosphate ~-Glycerophosphate + ADP ~-Glycerophosphate + ADP + oligomycin ~-Glycerophosphate + ADP + oligomycin + FCCP
100 100 72 91
Respiratory control index (RCI)
1"39
Experimental details as described in the legends to Table 1. Final concentration of additions: ~-glycerophosphate, 10 mM; ADP, 300/~M; oligomycin, 0-5/~g/mg protein; FCCP, 1"0/~M. phosphorylation was taking place as oligomycin inhibited the A D P - i n d u c e d rate (State 3) and this inhibited rate could be relieved by FCCP. Crude A T P , containing 6 per cent ADP, also stimulated respiration. T h e respiratory rate stimulated by crude A T P appears to be greater than that stimulated by ADP. In one particular experiment, not included in Table 3, a fivefold increase in rate was observed. At this stage, it is difficult to explain this phenomenon. T h e respiratory control index TABLE 3--RESPIRATORY CONTROL AND OXIDATIVE PHOSPHORYLATION IN Ascaris MUSCLE MITOCHONDRIA: ( C ) ASCORBATE-TMPD OXIDASE SYSTEM
Additions Ascorbate + TMPD Ascorbate + TMPD + ADP Ascorbate + TMPD + ATP (crude) Ascorbate + T M P D + ATP (crude) + oligomycin Ascorbate + TMPD + ADP + ATP (crude) Ascorbate + T M P D + ADP + ATP (crude) + oligomycin Ascorbate + TMPD + ADP + ATP (crude) + oligomycin + FCCP
Respiratory rate (%) 100 (7) 162 (5) 169 (3) 127 (1) 192 (4) 138 (3)
Respiratory control index (RCI)
1"33 1"39
198 (3)
Experimental details as described in the legends to Table 1 except that antimycin A (0"5/~g/mg protein) was added prior to the addition of ascorbate plus TMPD. Final concentration of additions: ascorbate, 4 mM; TMPD, 0.4 mM; ADP, 300/~M; crude ATP, 5000/~M containing 300/~M ADP. based on either A D P or the crude A T P - i n d u c e d oxygen uptake rate divided by the rate of oxygen uptake in the presence of oligomycin was about 1.3. T h e data in Table 3 thus favour the presence of a loosely coupled Site I I I in Ascaris muscle mitochondria. T h e average State 3 rate based on seven estimations from four different mitochondrial preparations was 66 natoms O/rain per mg protein at 25°C (pH = 7.2), and this rate was comparable with the State 3 rate of 52 natoms
OXIDATIVE P H O S P H O R Y L A T I O N I N .~,.qCAIUS MUSCLE M I T O C H O N D R I A
241
O/min per mg protein for Moniezia muscle mitoehondria (Cheah, 1971a). It thus appears that the State 3 rate for a coupled functional Site I I I in large intestinal parasites like .dscaris and Moniezia is about 25-30 per cent of the State 3 rate for Site I I I in mitochondria isolated from aerobic skeletal muscle (Cheah, 1973a). T h e present data on oxidative phosphorylation and respiratory control in Ascaris do not support claims (Kmetec & Bueding, 1961; Saz, 1970, 1972) that .dscaris possesses a predominantly anaerobic type of metabolism. Our present findings together with previous work on other large intestinal parasites strongly suggest that mitochondria from these parasites (Cheah, 1967; Cheah & Chance, 1970; Cheah, 1971a) have functional cytochromes in their respiratory chain systems, and also that oxidation of the various substrates is linked to oxidative phosphorylation (Cheah, 1971a). Acknowledgements--The author would like to thank Mrs. A. M. Cheah for technical assistance.
REFERENCES BARRETTJ. & BEIS I. (1973) Studies on the glycolysis in the muscle tissue of Ascaris lumbricoides (Nematoda). Comp. Biochem. Physiol. 44B, 751-761. CHANCE B. & PARSONSD. P. (1963) Cytochrome function in relation to inner membrane structure of mitoehondria. Science 142, 1176-1180. CHANCEB. & WI~-LXAMSG. R. (1956) The respiratory chain and oxidative phosphorylafion. In Advances in Enzymology (Edited by NO~D F. F.), Vol. 17, pp. 65-134. Interseienee, New York. CHWH K. S. (1967) Spectrophotometric studies on the succinate oxidase system of Taenia hydatigena. Comp. Biochem. Physiol. 20, 867-875. CrlEAH K. S. (1971a) Oxidative phosphorylation in Moniezia-muscle mitochondria. Biochim. biophys..4cta 253, 1-11. CH~H K. S. (1971b) Effect of loss of cytochrome c following storage of mitochondria in situ. F E B S Lett. 19, 105-108. CrmAH K. S. (1972) Cytochromes in Ascaris and Moniezia. In Comparative Biochemistry of Parasites (Edited by VAN DEN Bosscrm H.), pp. 417-432. Academic Press, New York. CHEAH K. S. (1973a) Comparative studies of the mitochondrial properties of Longissimus dorsi muscle of Pietrain and Large White pigs. ft. Sci. Fd .4gric. 24, 51-61. CHEAH K. S. (1973b) Purification and properties of Ascaris cytochrome bseo. ft. biol. Chem. 248, 4101-4105. C~AH K. S. & CHANC~ B. (1970) The oxidase systems of .4scaris-muscle mitoehondria. Biochim. biophys. Aeta 223, 55-60. CHEArI K. S. & CHWH A. M. (1971) Post-mortem changes in structure and function of ox muscle mitoehondria--1. Electron microscopic and polarographic investigations. J. Bioenerg. 2, 85-92. HXLL G. C., P~.RKOWSKXC. A. & MAXHEWSONN. W. (1971) Purification and properties of cytochrome c55o from Ascaris lumbricoides var. suum. Biochim. biophys. Acta 236, 242245. KIRUCrlI G. & BAN S. (1961) Cytochrome in the particulate preparation of the Ascaris lumbricoides muscle. Biochim. biophys. Acta 51, 387-389. KXRUCm G., RAMIm~XJ. & BAa~aONE. S. G. (1959) Electron transport system in Ascaris lumbricoides. Biochim. biophys. Acta 36, 335-342. KMEX~C E. & BtmDXNO E. (1961) Succinic and reduced diphosphopyridine nueleotide oxidase system of Ascaris muscle. J. biol. Chem. 236, 584--591.
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Loway O. H., ROSF.BllOUGHN. J., FARR A. L. & RANDALLR. J. (1951) Protein measurement with the Folin phenol reagent, j~. biol. Chem. 193, 265-275. OYA H., KXKUCm G., BANDO T. & HAYASm H. (1965) Muscle tricarboxylic acid cycle in .4scaris lumbricoides vat. suis. Expl Parasit. 17, 229-240. SAZH. J. (1970) Comparative energy metabolisms of some parasitic helminths..7. Parasit. 56, 634-642. S~z H. J. (1972) Comparative biochemistry of carbohydrates in nematodes and cestodes. In Comparative Biochemistry of Parasites (Edited by VAN DZN BOSSCHZ H.), pp. 33-47. Academic Press, New York. SLAT~ E. C. (1967) Application of inhibitors and uncouplers for a study of oxidative phosphorylation. In Methods of Enzymology (Edited by ESTABROOKR. W. & PULLMAN M. E.), Vol. X, pp. 48-57. Academic Press, New York. SMXTHM. H. (1969) Do intestinal parasites require oxygen ? Nature, Lond. 223, 11291132.
Key Word Index---.dscaris lumbricoides muscle mitocbondria; oxidative phosphorylation; respiratory control; succinoxidase; cytochrome oxidase (as).