The oxidase systems of Moniezia expansa (Cestoda)

Comp. Biochem. Physiol., 1967, Vol. 23, pp. 277 to 302. Pergamon Press. Printed in Great Britain

THE

OXIDASE

SYSTEMS

OF MONIEZIA

EXPANSA

(CESTODA) K. S. CHEAH* Department of Zoology, The Australian National University, Canberra, A.C.T., Australia (Received 28 March 1967)

Abstract--1. Moniezia expansa has a branched respiratory chain system with two terminal oxidases, cytochrome oxidase and an o-type cytochrome.

2. The major pathway involving the o-type pigment tentatively designated "Cytochrome 552, 556 (Moniezia expansa)" (77°K), the major terminal oxidase, is shown to be closely associated with fumarate reduction and hydrogen peroxide formation. 3. Two antimycin-A sensitive sites were demonstrated; one blocking the reoxidation of "Cytochrome 556 (Moniezia expansa)" (77°K) and which required a high concentration of the inhibitor, the other acting between cytochromes b and

c x.

INTRODUCTION

ALTHOUOHthere are reports of various cytochrome components in different parasitic helminths (yon Brand, 1966), virtually nothing is known about their function. In general, the dectron transport system in parasites is not as fully understood as that in mammalian tissues (Lehninger, 1964; Lee et al., 1965 ; Green & Tzagoloff, 1966) and in some types of bacteria (Taber & Morrison, 1964; Kikuchi et al., 1959; Taniguchi & Kamen, 1965 ; Motokawa & Kikuchi, 1966). Even though considerable information was available on Ascaris lumbricoides, conflicting results about the respiratory chain have been reported by different groups of investigators. Kmetec & Bueding (1961) concluded that the electron transport system in A. lumbricoides involved a flavoprotein-containing terminal oxidase, a result confirming the earlier investigations of Laser (1944) and Bueding & Charms (1952). These workers found the respiratory activities of Ascaris were markedly dependent on oxygen tension and resulted in the formation of hydrogen peroxide rather than water. Kikuchi et al. (1959), however, proposed a branched-chain system; one branch involved cytochrome oxidase as a terminal oxidase, the other linked a b-type cytochrome directly to oxygen. The situation is also confused in cestodes, van Grembergen (1944) demonstrated the presence of a component with an absorption maximum between 550 and 560 m/~ in Monie¢ia benedeni, and concluded that this was due to a mixture of cytochromes b and c. He also found that p-phenylenediamine stimulated the oxygen uptake in homogenates of M. benedeni and that this was inhibited by * Present address: Research Unit in Biochemistry, Biophysics and Molecular Biology, McMaster University, Hamilton, Ontario, Canada. (Canadian Medical Research Council Fellow.) 277

278

K . S . CrmArI

cyanide. These observations led van Grembergen to conclude that the respiratory chain in M. benedeni resembled that of mammalian tissue. Cheah & Bryant (1966), working with a mitochondria-containing particulate fraction from M. expansa, prematurely concluded that the detected predominant b-type cytochrome and the very low level of an a-type cytochrome were not involved in the electron transport system, and that the terminal oxidase in M. expansa was a flavoprotein. This paper reports the results of further investigations on the oxidase systems of M. expansa. T h e data obtained favour the existence of a branched respiratory chain system with two terminal oxidases. T h e main pathway was demonstrated to participate in fumarate reduction involving a b-type terminal oxidase. MATERIALS AND METHODS Antimycin A (Type III), p-chloromercuribenzoic acid, dicumarol, NADH, a-glycerophosphate, crude horseradish peroxidase (approximately 34 purpurogallin units per rag) were obtained from the Sigma Chemical Corporation, St. Louis; ascorbic acid from the Nutritional Biochemical Corporation, Cleveland, Ohio; 2-thenoyltrifluoroacetone from K and K Laboratories Inc., California; fumarate, guaiacol from the British Drug Houses Ltd., Poole, England; sodium amytal from Eli Lilly and Company Ltd., Basingstoke, England; maieic acid from May and Baker Ltd., Dagenham, England, and all other reagents were analytical grade. The mitochondria-containing particulate fraction and the first and second 12,000g supernatant fractions, hence referred to as SI and SII, were prepared as previously described (Cheah & Bryant, 1966). The particulate fraction was suspended in 50 mM phosphate buffer so that 1"0 ml suspension was equivalent to 5"0 g wet weight of original tissue. Except where otherwise stated, all experiments were carried out with freshly prepared particulate fractions. The oxygen uptake was measured in Warburg manometers with air as the gas phase at 38°C, 0.1 ml KOH in the centre well and a shaking rate of 110 c/min. All manometric changes were recorded at 5-min intervals. Sodium amytal, dicumarol and p-ehloromercuribenzoic acid were dissolved in the minimum volume of 0"5 N NaOH and diluted to the required concentration with 50 mM phosphate buffer (pH 7"4). Antimycin A was prepared as a 2"5 × 10 -8 M solution and diluted with ethanol prior to use. Other inhibitors (azide, cyanide, 2-thenoyltrifluoroacetone) were dissolved in 50 mM phosphate buffer (pH 7"4). These inhibitors were added directly into the main compartment and allowed to preincubate with the particulate fraction for 10 min at room temperature (20°C). The respective substrate was then tipped into the main compartment after the inhibitors had been in contact with the particulate fraction for another 20 min at 38°C. Hydrogen peroxide was detected using the method adapted from Maehly & Chance (1954) except that 0-5 ml guaiaeol (20 mM) and 0"1 ml horseradish peroxidase (0"1%, approximately 3"7 purpurogallin units) were added to the incubation medium at the end of the experiments. The tetraguaiacol was extracted with equal volume of ether, and its spectrum compared with that obtained from authentic hydrogen peroxide with a Beckman DB speetrophotometer. The experiments on the effects qf peroxidase, SI and Sir on the 0t-glycerophosphate oxidase activity were performed as for the phenazine methosulphate and methylene blue methods of Ringler & Singer (1959) except that cyanide and the dyes were omitted, and peroxidase, Sr, $II were in the main compartment of the vessels. The difference absorption spectra of the cytochrome and flavoprotein components of the electron transport system in the particulate fraction were determined with a Cary (Model 14R) spectrophotometer fitted with a scattered transmission accessory containing a

279

OXIDASE SYSTEMS O F MONIEZIA E X P A N S A

RCA-type 6217 photomultiplier. The particulate fraction wa~ suspended in 100 mM phosphate buffer (pH 7.6) so that 1"0 ml suspension was equivalent to 8'0 g wet weight of original tissue. The preparation was placed in both the sample and the reference ceils, and the base-line was recorded between 700 and 390 m/~ wavelength. The contents of the sample cell were treated with various reagents after a satisfactory base-line was obtained, The low-temperature (77°K) spectrophotometric studies on the particulate fraction were based on those of Estabrook (1961). Further details of individual experiments are given in the legends to Figures. Total nitrogen was determined by the method of Johnson (1941). The method was slightly modified by the omission of CuSeOa which did not affect the colour density with the samples used. RESULTS

1. Manometric studies of the particulate fraction from M. expansa Table 1 shows the oxidation rates of various substrates by the particulate fraction from M. expansa. T h e ~-glycerophosphate oxidase activity was about three and five times greater than that of succinate and N A D H respectively. T h e results obtained suggested that ~-glycerophosphate might be the most important substrate oxidized by M. expansa, as all other tricarboxylic acid cycle intermediates which are oxidized via the respiratory chain system are linked to N A D and thus oxidized via N A D H . M. expansa thus appears to have an oxidative pattern resembling those of the parasitic protozoon, Trypanosoma rhodensiense (Grant & Sargent, 1960) and insect muscle (Estabrook & Sacktor, 1958). N o significant oxidation of either N A D P H , /~-hydroxybutyrate, malate or a-ketoglutarate was observed with the particulate fraction from M. expansa. TABLE 1--THE

O X I D A T I O N O F VARIOUS SUBSTRATES B Y T H E P A R T I C U L A T E F R A C T I O N F R O M

M. expansa Activity Substrate et-Glycerophosphate Succinate NADH

Number of estimations

Range

Mean

14 13 15

73-7-79.5 23.4-26.5 13.1-14.5

77.4 24.3 14.2

Each vessel contained 50 mM phosphate buffer (pH 7"4 or 7"6), 1"0 ml particulate fraction (1"67 mg N) and 0.5 ml substrate. Final volume, 2.5 ml. Incubation, 60 rain for at-glycerophosphate and suecinate, and 25 rain for NADH. Temperature, 38°C. ~t-Glycerophosphate experiments conducted at pH 7.6, and suecinate and NADH at pH 7"4. Final concentrations: ~,-glycerophosphate, 50 mM; succinate, 50 mM; NADH, 5 raM. Corrections made for endogenous activity. Activity expressed as pl 02 (air) uptake per mg N per hr at 38°C. Table 2 summarizes the effect of 2-thenoyltrifluoroacetone on the succinate and N A D H oxidase activities of the particulate fraction from M. expansa. T h e oxidation of suecinate was inhibited 42 per cent by 2-thenoyltrifluoroacetone (10 -8 M) but similar concentration of this inhibitor had no effect on N A D H

280

K . S . CHEAH

o x i d a t i o n . T h e r e s u l t s i m p l i e d t h a t s u c c i n a t e o x i d a t i o n in M. expansa i n v o l v e d t h e p a r t i c i p a t i o n o f n o n - h a e m iron.

T A B L E 2 - - E F F E C T OF 2-THENOYLTRIFLUOROACETONEON THE SUCCINATE AND

NADH

ACTIVITIES

OF THE PARTICULATE FRACTION FROM M . e x p a n s a

Activity 2-Thenoyltrifluoroacetone Oxidase system (M) Succinate

-1 × 10 -3 1 x 10 -4

NADH

--

1 x 10 -3 1 × 10 -4

Range

Mean

Inhibition (%)

22"4-26"5 11"9-15"9 21"3-28"8 13'8-14-9 12"2-14"6 15.0-16.2

24"3 14"0 24"5 14'2 13"4 16'0

-42"4 -----

Experimental details as described in Table 1 and in Methods. Activity calculated from at least six experiments, and expressed as gl 02 (air) uptake per mg N per hr at 38°C.

T a b l e 3 s h o w s t h e effect o f p - c h l o r o m e r c u r i b e n z o i c a c i d o n t h e d e h y d r o g e n a s e activities o f t h e p a r t i c u l a t e f r a c t i o n f r o m 3I. expansa. A t 2 x 10 -4 M , p - c h l o r o m e r c u r i b e n z o i c a c i d i n h i b i t e d t h e s u c c i n i c a c i d d e h y d r o g e n a s e b y 86 p e r cent, t h e N A D H d e h y d r o g e n a s e b y 89 p e r cent, b u t o n l y i n h i b i t e d t h e a - g l y c e r o p h o s p h a t e d e h y d r o g e n a s e a c t i v i t y b y 39 p e r cent. T h e r e s u l t s i n d i c a t e t h a t free s u l p h y d r y l g r o u p s o f t h e v a r i o u s d e h y d r o g e n a s e s w e r e essential for t h e d e h y d r o g e n a s e activities.

T A B L E 3 - - E F F E C T OF p-CHLOROMF-~CURIBENZOIC ACID ON THE DEHYDROGENASE ACTIVITIES OF THE PARTICULATE FRACTION FROM M. expansa

Activity Substrate

p-Chloromercuribenzoic acid (M)

ct-Glycerophosphate Succinate NADH

2 x 10 -4 1 × 10 -4 -2 x 10 -~ --

2 x 10 -4 1 x 10 -4

Range

Mean

Inhibition (%)

73.9-79.5 44.3-51.2 56.9-62.9 22-4-26.5 5"1- 6.7 13"1-14"5 2-6- 4.5 4"8- 6"8

77"4 47"0 60.4 24.3 5"7 14"2 3"0 5"7

-39-4 22.0 -86.5 -88"9 59"7

Experimental details as described in Table 1 and in Methods. Activity calculated from at least five experiments and expressed as #1 O2 (air) uptake per m g N per hr at 38°C.

281

OXIDASE SYSTEMS OF MONIEZIA EXPANSA

T a b l e 4 illustrates the results obtained f r o m the experiments on the effect of amytal on the particulate fraction f r o m M. expansa. T h e a-glycerophosphate and N A D H oxidase systems showed about the same extent of sensitivity to amytal. TABLE 4-----EFFECTOF AMYTALON THE OXIDASEACTIVITIESOF THE PARTICULATEFRACTIONFROM

M. expansa Activity Oxidase system ~- Glycerophosphate Succinate

NADH

Amytal (M) -4 x 10 -~ 2 x 10 -~ m 2 x 10 -2 1 x 10 -2 1 x 10 -3 1 × 10 -4 --

2 x 10 -~ 1 x 10 -* 1 x 10 -3 1 × 10 -4

Range

Mean

73-9-79"5 0-8- 2"3 16"2-22"0 22"4-26"5 8"1-11-1 13"9-15"1 20"5-27"7 20"5-27'7 13.1-14.5 1.6- 2-2 1.6- 4.4 12.0-13.2 13.9-15.2

77"4 1-9 11-8 24"3 9"7 14-4 24"9 25"0 14.2 1.6 2.6 13-0 14.5

Inhibition (%) -99-2 84'4 -60"1 41"8 -89-0 81-7 8.5

Experimental details as described in Table 1 and Methods. Activity calculated from at least five experiments and e x p r e s s e d a s / ~ 1 02 (air) uptake per mg N per hr at 38°C. At 2 × 10 -2 M, amytal inhibited the oxidation of c~-glycerophosphate and succinate by 84 and 89 per cent respectively, with complete inhibition of the a-glycerophosphate oxidase activity at 4 x 10 -3 M concentration. T h e succinate oxidase pathway appeared to be m o r e resistant, as amytal at 2 x 10 -3 M only gave a 60 per cent inhibition. T h e concentration of amytal required to produce a significant inhibition of the a-glycerophosphate and N A D H and, to a certain extent, succinate was about ten times greater than that normally employed for glutamate oxidation by rat-liver mitochondria (Hochstein et al., 1965), for the succinate and N A D H oxidase systems of dark aerobically grown Rhodospirillum rubrum (Taniguchi & K a m e n , 1965) and for the a-glycerophosphate oxidase activity of T. rhodesiense ( G r a n t & Sargent, 1960). T h e results obtained with M. expansa, however, compared well with those of T a b e r & Morrison (1964), who also found that elevated levels of amytal were required to produce a significant inhibition of the succinate and N A D H systems of Staphylococcus aureus. T a b l e 5 summarizes the effect of dicumarol on the c~-glycerophosphate and succinate oxidase activities of the particulate fraction f r o m M. expansa. Both the oxidase systems were inhibited, but the succinate oxidase pathway was m o r e resistant to dicumarol than was the c~-glycerophosphate oxidase system. T h e result indicated that vitamin K might be the component linking the flavoproteins to the cytochromes in the respiratory chain of M. expansa.

282

K . S . CHEAn

TABLE 5--EFFECT OF DICUMAROL ON THE (X-GLYCF.ROPHOSPHATEAND SUCCINATE OXIDASE ACTIVITIESOF THE PARTICULATEFRACTIONFROM m . expansa Activity Oxidase system ~x-Glycerophosphate

Succinate

Dicumarol (M)

Range

Mean

Inhibition (%)

-2 x 10 -4 1 x 10 -4 -2 x 10 -4 1 x 10 -4

73'9-79"5 19'1-20'8 31"8-33"5 22.4-26.5 11"3-13"5 17"5-19'2

77-4 20-2 32"4 24"3 12.4 18.4

-73'9 58'3 -48"0 24"3

Experimental details as described in Table 1 and Methods. Activity calculated from at least six experiments and expressed as/~1 02 (air) uptake per mg N per hr at 38°C. T h e effects of a n t i m y c i n A o n the e l e c t r o n t r a n s p o r t s y s t e m of M . expansa w i t h c~-glycerophosphate, s u c c i n a t e a n d N A D H are s u m m a r i z e d i n T a b l e 6. C o m p l e t e i n h i b i t i o n was n o t achieved e v e n w i t h a high c o n c e n t r a t i o n of the i n h i b i t o r (10 -a M). A p p r o x i m a t e l y 50 per c e n t i n h i b i t i o n of the oxidase s y s t e m s was o b t a i n e d w i t h c o n c e n t r a t i o n s of a n t i m y c i n A b e t w e e n 10 -4 M a n d 10 - s M. T h i s c o n c e n t r a t i o n of the i n h i b i t o r was a b o u t f o u r t h o u s a n d t i m e s greater t h a n that expected to give a 50 p e r c e n t i n h i b i t i o n o n a m a m m a l i a n - t y p e of r e s p i r a t o r y c h a i n ( L i g h t b r o w n & Jackson, 1956; B u e t o w & B u c h a n a n , 1965). T h e lack of sensitivity of t h e p a r t i c u late fraction to a n t i m y c i n A could perhaps, at the p r e s e n t m o m e n t , be best e x p l a i n e d T A B L E 6----EFFECT OF A N T I M Y C I N A ON THE OXIDASE ACTIVITIES OF THE PARTICULATE FRACTION FROM

M. expansa Activity

Oxidase system a-Glycerophosphate

Succinate

Antimycin A (M) -1 x 10 -I 1 x 10 -5 1 x 10 -6 1 x 10 -7 1 x 10 -s -1 x 10 -4 1 x 10 -s

NADH

-1 x

10 -4

1 x 10 -5

Range

Mean

Inhibition (%)

73"9-79"5 29"2-32-3 41"3-43-4 39"7-49"0 59.4-65.2 62.9-68"7 22"4-26"5 8"2-11"0 10"5-14"4 13"1-14"5 5"3- 6"4 7 " 5 - 8"4

77"4 29"8 42"2 44.4 60"1 67-5 24.3 8"5 13"4 14"2 5'6 7"8

-61"5 45'4 42'6 22'3 12.7 -65"0 45-0 -60"6 45.1

Experimental details as described in Table 1 and Methods. Activity calculated from at least five experiments and expressed as/zl 02 (air) uptake per mg N per hr at 38°C.

OXIDA,SE SYSTEMS OF MONIEZIA E X P A N S A

283

either by the presence of a very low level of a mammalian-type of cytochrome b or by the existence of an antimycin A insensitive pathway. Table 7 illustrates the effects of azide and cyanide on the oxidation of succinate and NADH by the particulate fraction from M. expansa. Azide (10 -s M) did not show any effect on the oxidation of both succinate and NADH even though the inhibitor had been preincubated with the particulate fraction. Cyanide, on the other hand, exerted different quantitative effects on suceinate and NADH oxidation. The oxidation of succinate was inhibited by 57 per cent and NADH by 18 per cent with I0 -8 M cyanide, a concentration which normally would give a 100 per cent inhibition in a mammalian type of electron transport system. T A B L E 7 - - E F F E C T S OF AZIDE AND CYANIDE ON THE OXIDASE ACTIVITIES OF THE PARTICULATE FRACTION FROM M. expansa

Activity Oxidase system

Azide (M)

Succinate 1 x 10 -s 1 x 10 -4 1 x 10 -5 -NADH

w 1 x

10 -s

Cyanide (M) ----1 x 10-3 1 x 10-4 1 x 10 -5 ~

-l x 10-s

Range

Mean

22'4-26"5 18"4-21"3 23"5-26"4 22"3-26"6 8"0-13"6 18"5-28"4 19"6-28"8 13"1-14"5 13"8-14"9 11-2-12"6

24"3 19"6 24"9 24"7 10"4 23"3 23"6 14'2 14"2 11"7

Inhibition (%) -8"0 -w 57-2 -17"0

Experimental details as described in Table 1 and in Methods. Activity calculated from six experiments and expressed as pl Os (air) uptake per mg N per hr at 38°C. Several possible explanations for this difference in inhibition due to cyanide between the succinate and NADH oxidation, and the overall insensitivity to both cyanide and azide could be given. The particulate fraction from M. expansa could contain a terminal oxidase which was relatively insensitive to both azide and cyanide. An alternative explanation would be the existence of a branched-chain system with two terminal oxidases, one of which was cytochrome oxidase. The major electron pathway, not involving the mammalian-type cytochrome oxidase, would thus be azide and cyanide insensitive. Tetraguaiacol was formed when guaiacol and horseradish peroxidase were added to the medium in which the particulate fraction had been incubated with ~-glycerophosphate. The extracted tetraguaiacol spectrum corresponded with that obtained using authentic hydrogen peroxide. Figure 1 illustrates the spectrum of tetraguaiacol, indicating hydrogen peroxide formation by the various preparations, with a-glycerophosphate as substrate. These results suggest that the oxidation of e~-glycerophosphate resulted in hydrogen peroxide formation. Cheah & Bryant

284

K . S . CI-mAH

(1966) also r e p o r t e d t h a t t h e o x i d a t i o n o f b o t h s u c c i n a t e a n d N A D H b y t h e p a r t i c u l a t e f r a c t i o n f r o m M . expansa r e s u l t e d in h y d r o g e n p e r o x i d e f o r m a t i o n .

t

TRANSMITTANCE (20 ~)

l

~ I

560

I

520

C B A m

I

4BO

I

440

I

400

WAVELENGTH

I

360

(rap)

FIG. 1. Spectra of tetraguaiacol. Method of detection of hydrogen peroxide formation employing guaiacol and horseradish peroxidase as described in Methods. Other experimental details of individual experiment as given in Table 1. A - - T y p i c a l result obtained from incubation medium in which no hydrogen peroxide was formed. B - - S p e c t r u m obtained using authentic sample of hydrogen peroxide. C - - S p e c t r u m obtained from incubation medium in which hydrogen peroxide was formed. 10 m m light-path cells used. TABLE 8--FACTORS AFFECTINGHYDROGENPEROXIDEFORMATIONBY THE PARTICULATEFRACTION FROM M. expansa Additions Without c~-glycerophosphate None Amytal (4 x 10 -~ M)-(2 x 10 -~ M) Antimycin A (1 x 10 -4 M)-(1 x 10 -s M) Fumarate (0"1 M) Peroxidase (0-020/o) Supernatant (SI) Supernatant (SII)

Tetraguaiacol + +

Each vessel contained 50 m M phosphate buffer (pH 7.6), 1-0 ml particulate fraction (1.67 mg N) and 50 m M ~-glycerophosphate. Final volume, 2.5 ml. Incubation, 60 rain. Other experimental details as described in Methods. Production of hydrogen peroxide detected by subsequent tetraguaiacol formation. Tetraguaiacol formation: + = detected; - = not detected.

OXIDASE SYSTEMS OF MONIEZIA E X P A N S A

285

Table 8 shows the various factors affecting hydrogen peroxide formation by the particulate fraction from M. expansa using a-glycerophosphate as substrate. Hydrogen peroxide was not detected in the presence of amytal, an inhibitor of a-glycerophosphate oxidation (Table 4), peroxidase, SI, Su and fumarate (see Section 2 for fumarate addition). Antimycin A did not prevent hydrogen peroxide formation, a result not unexpected from the previous observation that antimycin A within the range of concentration used did not completely inhibit a-glycerophosphate oxidation (Table 6).

2. Spectrophotometricstudies of the particulatefraction from M. expansa (A) Room temperaturespectrophotometricstudies (20°C). The dithionite-reduced minus oxidized (oxygen or ferricyanide) difference spectrum showed three distinct maxima at 555-557, 526--528 and 425 m/z. A small shoulder at 440--450 m/z, suggesting an a-type cytochrome(s), and a trough at about 460 m/z probably contributed by fiavoprotein(s) were also observed. The substrate-reduced (a-glycerophosphate, succinate or NADH) difference spectra were identical with those obtained by dithionite except that cytochrome reduction level was only 80-90 per cent of that achieved with dithionite. No difference was observed in the dithioniteor substrate-reduced minus oxidized spectra when either oxygen or ferricyanide was used to oxidize the particulate fraction in the reference cell. Oxygen, being more convenient to use than ferricyanide, was therefore employed throughout the spectrophotometric studies of the particulate fraction from M. expansa. On the subsequent addition of fumarate to the particulate fraction pretreated with either a-glycerophosphate or NADH, 60-65 per cent of the cytochrome component(s) contributing the a-peak (555-557 m/z) was oxidized. Two broad bands also appeared at 550-557 and 521-527 m/z in place of the 555-557 and 526-528 m/z peaks respectively, 10 rain after fumarate addition, together with an additional peak at 442 A,A,6m/z, and a small shoulder component at 562 mix. A band at 600 m/z was also occasionally detected following fumarate addition, and this was probably due to cytochromes a(+a3) in the particulate fraction. This method was devised to give a better demonstration of the y-peak of cytochromes az(+ a) and to show that fumarate oxidized one or more of the cytochrome components, which would normally prevent the detection of the cytochromes a3(+ a) peak at 442--446 m/z. Similar effects were not observed with either maleate or crotonate instead of fumarate. This interesting observation suggested that the cytochrome component(s) with c~(555-557 m/z), /g (526-528 m/z) and V (425 m/z) peaks was only associated with the reduction of fumarate to succinate. Both maleate (a c/s-isomer of fumarate) and crotonate are C4-acids having an unsaturated bond in the same position as fumarate. Figure 2 illustrates the reduction of the respiratory pigments in M. expansa by a-glycerophosphate (A) and the fumarate effect (B). Figure 3 illustrates the effect of antimycin A on the particulate fraction from M. expansa. A reduced cytochrome b with its Soret band at 431 m/z and its a-band at 560-562 m/z was observed when antimycin A was added to a sample of the

286

K . S . CHEAH

particulate fraction. The ratio of the difference in absorbance changes between the wavelength pairs 4 3 0 4 1 0 and 562-575 m/~ was 10 : 1 ; this corresponds fairly well with those reported for liver mitochondria and heart muscle succinic oxidase (Chance, 1957). This observation suggests a classical antimycin effect on the small amount of classical cytochrome chain present in the particulate fraction, i.e. s u b s t r a t e ~ c y t b-->cyt c t ~ c y t c-+cy,t (a + a3).

.~ . antlmycm A

oxygen

/ 425

1

555-557

440-4S0

1

L/

S2~S2~

/ 425

4112-446

\ I

I

440

I

I

I

I

480 520 560 600 WAVELENGTH (mr )

I

640

F l a . 2. Effect of fumarate on the ct-glycerophosphate-reduced particulate fraction from M. expansa (20°C). Both cells (2-0 ram light-path) contained 0-24 ml (0.47 mg N) particulate fraction. A--~-Glycerophosphate-reduced minus oxidized difference spectrum. Recorded at 10rain after substrate addition. B - - F u m a r a t e added to sample pretreated with ~-glycerophosphate (A), and difference spectrum recorded at 10 rain after fumarate addition. Final concentrations: ~t-glycerophosphate, 10 m M ; fumarate, 5 mM.

OXIDASESYSTEMSOF MONIEzIA EXPANSA

287

ABSOIIBANCY = 0.015

1

431

1

I 400

I 440

I

I

I

4110

520

560

....

I

I

600

640

WAVELENGTH(mu) FIQ. 3. Effect of antirnycin A on the particulate fraction from M . expansa (20°C). T h e preparation was similar to that described in Fig. 2. Antimycin A added to the sample cell and oxygen bubbled through the reference cell for 2 rain. Final concentration: antimycin A, 10 -e M.

Figure 4 shows the effect of cyanide on the particulate fraction previously reduced with succinate. Addition of cyanide (10 -3 M) to the particulate fraction pretreated with suceinate resulted in 25 per cent oxidation of the cytochrome component(s) (B). A further 25 per cent oxidation was achieved by the subsequent treatment with oxygen (C), suggesting that 50 per cent of the reduced cytochrome component(s) was oxidized in the presence of cyanide. The difference spectrum (suecinate+ cyanide minus suceinate) showed a trough at 555-557 m/~. The data suggested that cyanide could either cause the oxidation of the reduced pigment(s) with an s-peak at 555-557 m# or form a complex with the reduced eytochrome component(s). The oxidation of the cytochrome(s) in the presence of cyanide could also be due to cyanide blocking a component preceding the cytochrome(s) under investigation. Figure 5 illustrates the CO-binding pigment detected in the particulate fraction. Three maxima at 569-570, 536 and 419 m/~ with two troughs at about 556 and

288

K.S. CHEAH

ABSORBANCY= 0.02 555-557

1

B

A

I

I

I

I

I

480

520

560

600

640

WAVELENGTH

(m~)

FIG. 4. Effect of cyanide on the succinate-reduced particulate fraction from M. expansa (20°C). The preparation was similar to that described in Fig. 2. except that 0"48 ml (0"94 mg N) particulate fraction in 4"0 m m light path cells used. A--Succinate-reduced minus oxidized difference spectrum. B - - S u c c i n a t e + cyanide minus oxidized spectrum. C--Oxygen bubbled through the sample pretreated with succinate + cyanide (B). Final concentrations : succinate, 10 m M ; cyanide, 10 -3 M.

443 mtz were observed. When this was repeated at liquid-nitrogen temperature (77°K), the CO difference spectrum showed maxima at 571-572, 538-540 and 420421 m/z with two troughs at 441--442 and 554-556 m/z. These bands are characteristic of the CO-complex of cytochrome o, an oxidase which has been found in various types of bacteria (Castor & Chance, 1959; White et aL, 1962; Taber & Morrison, 1964; Taniguchi & Kamen, 1965) and algae (Webster & Hackett, 1965, 1966) although not in animal tissues. The trough in the Soret region was probably contributed by the sum of the troughs of cytochrome as-CO complex (445 m/z) and cytochrome o-CO (432433 m/z) difference spectra. The successful formation of the cyanide and CO-complexes with approximately identical

OXIDASE SYSTEMS OF M O N I E Z I A

289

EXPANSA

troughs in their difference spectra strongly suggested that about 50 per cent of the (555-557 m/~) peak was probably contributed by a terminal oxidase which resembled an o-type cytochrome.

419

,,,,

536

I ABSORBANCY = 0.015

ABSORBANCY = 0.1

1

443 556

I

I

I

I

I

I

I

400

440

480

520

560

600

640

WAVELENGTH

(rap)

Fic. 5. Room temperature (20°C) CO difference spectrum of the particulate fraction from M. expansa. Both cells contained 0"5 ml (1"04 mg N) particulate fraction. A--Base-line. 1.0 mg dithionite added to both cells. B--CO difference spectrum (dithionite + CO minus dithionite). Figure 6 illustrates the effect of amytal and p-chloromercuribenzoic acid on the ~-glycerophosphate oxidase system. Amytal (2 x 10 -3 M) inhibited the reduction of cytochromes by about 72 per cent (B), while p-chloromercuribenzoic acid (2 x 10 -4 M) suppressed it to the extent of approximately 81 per cent (C). Similar concentrations of both these inhibitors were also effective against the N A D H and succinate oxidase systems, showing the same degree of inhibition as that previously described with ~-glycerophosphate as substrate. (B) Low temperature spectrophotometrie studies (77°K). Figure 7 shows the NADH-reduced minus oxidized difference spectrum (A) of the particulate fraction at liquid-nitrogen temperature. The e~(600 m/~) peak of cytochromes a(+ as) and the shoulders representing cytochromes b (562 m/z), c (546 m/~) were observed together with a predominant double-headed peak (552, 556 m/L). Identical results were obtained when the particulate fraction was reduced with other substrates (a-glycerophosphate, succinate) and dithionite. This shows that the oxidation of a-glycerophosphate, succinate and NADH involved the same pathway. With dithionite reduction, the shoulders contributed by cytochromes b and c were mostly obscured by the predominating component(s) with the doubled-headed peak. On the subsequent addition of antimycin A (10 -4 M) and oxygen to the sample cell, IO

290

K . S . CnEAH 425

555-557

1

/*

',& ........ 7-"-:; " ' / ' -

y

'~" . ....

\

"I

c

:'4 I

i i, I'

V . . . .

I

I

I

I

I

I

I

4OO

44#

~10

52O

560

600

640

WAVELENGTH (,~1

FIG. 6. Effect of amytal and p-chloromercuribenzoic acid on the c~-glycerophosphate-reduced particulate fraction from M . expansa (20°C). The preparation was similar to that described in Fig. 2. A-~-Glycerophosphate-reduced minus oxidized difference spectrum. B--~-Glycerophosphate+amytal minus oxidized difference spectrum. C--o~-Glycerophosphate +p-chloromercuribenzoic acid minus oxidized difference spectrum. Final concentrations: ct-glycerophosphate, 10 mM; amytal, 20 raM; p-chloromercuribenzoic acid, 2 × 10 -4 M. an ~ (557 m/~) and a fl (526-527 m/z) peak were observed (B) following the disappearance of the other cytochrome components previously detected with N A D H (A). A similar result was also obtained when the same concentration of antimycin A was added to the particulate fraction which had been allowed to react with either ~-glyeerophosphate or succinate. Under these conditions, one would also expect cytochrome b (562 m/z) which was previously demonstrated (Fig. 3), to be more reduced. As only a single maximum at 557 m/~ was observed, it appeared that the (562 m/z) peak of cytochrome b was probably overlapped by the predominant

OXIDASE SYSTEMS OF

MONIEZIA EXPANSA

291

552

/

--T

S46

557

I

480

I

55t0

I

960

I

600

I

640

I

WAVELENGTH (sp)

FIC. 7. Effect of antimycin A on the NADH-reduced particulate fraction from described in Fig. 2. A--NADH-reduced minus oxidized difference spectrum. B----NADH+antim~,cin A minus oxidized difference spectrum. Final concentrations: NADH, 10 mM; antimycin A, 10-4 M. M. expansa (77°K). The prepaxstion was similar to ~ t

pigment (557 mF), the reduction of which was sensitive to high antimycin A concent.ration. Addition of CO to the sample cuvette did not alter the 557 m/z band, showing that the cytochrome component responsible for this peak could not form a CO-complex. This observation suggested that the cytochrome component responsible for the 557 m/~ peak was a b-type cytochrome, on the basis of its sensitivity to antimycin A and the position of the s-peak in the difference spectrum. Thus, approximately 35 per cent of the initially observed double-headed peak (552, 556 m~) was contributed by this b-type cytochrome, which, normally, was obscured by the predominant double-headed component(s), and thus escaped detection.

292

K.S. CnFa~H

An interesting result was obtained following fumarate addition (Fig. 8B) to the ~-glycerophosphate-reduced particulate fraction (Fig. 8A). Approximately 65 per cent of the cytochrome component(s) responsible for the double-headed peak (552, 556 m/~) was oxidized (~-glycerophosphate reduced level taken as 100 per cent).

552

556

546

~

A

ss~ssl6~°t/~ B

I I I I I 480 520 560 600 640 (rn~s} WAVELENGTH

FIa. 8. Effect of fumarate on the ct-glycerophosphate-reduced particulate fraction from M. expansa (77°K). The preparation was similar to that previously described in Fig. 2. A---et-Glycerophosphate-reduced minus oxidized difference spectrum. B--ct-Glycerophosphate+fumarate minus oxidized difference spectrum. Final concentrations : at-glycerophosphate, 10 mM; fumarate, 5 mM. This suggested that 65 per cent of the double-headed peak might be contributed by a single component, oxidizable by fumarate. The small shoulder at 548 m/~ and the peak at 552 m/~ (Fig. 8B)were probably due to cytochromes c and ct respectively, as none of these bands were detected in the presence of antimycin A (10 -4 M).

OXIDASE SYSTEMS OF MONIEZIA E X P A N S A

293

The addition of antimycin A also shifted the 556 m~ peak to 557 m#, and this pigment was not affected by CO. Thus, the 556 m# component observed following fumarate addition was identical with that previously detected with similar concentration of antimycin A (Fig. 7).

568-S71

T ABSORBANCY= 0.02 l

538-540

552

/

556

7o

538-$40~-~

556

552 - ~

I

480

I

520

I

560

I

I

600 640 WAVELENGTH (mlJ)

FIG. 9. Effect of fumarate and CO on the ~-glycerophosphate treated particulate fraction from M. expansa (77°K). Preparation similar to that described in Fig. 2. A--~-Glycerophosphate+fumarate minus ~-glycerophosphate difference spectrum. Recorded at 10 rain after fumarate addition. B--~-Glycerophosphate+ CO minus ~-glycerophosphate difference spectrum. Final concentrations: c~-glycerophosphate, 10 raM; fumarate, 5 raM. Figure 9 illustrates the oxidation of the respiratory chain of the particulate fraction pretreated with a-glycerophosphate by fumarate. The (~-glycerophosphate +fumarate minus a-glyeerophosphate) difference spectrum recorded at 10 min after fumarate addition shows the appearance of a split trough (552, 556 mg) with

294

K.S. CHEAH

two corresponding peaks at 538-540 and 568-571 m/z (Fig. 9A). Addition of CO to the c~-glycerophosphate-reduced particulate fraction (Fig. 9B) also caused the same effect as fumarate (Fig. 9A), but the peaks at 538-540 and 568-570 m/z were more enhanced. Furthermore, following CO addition to the sample cuvette, the a-glycerophosphate-reduced minus oxidized difference spectrum of the particulate fraction showed the same extent of loss of the double-headed component (552, 556 mtz) as that treated with fumarate instead of CO. This suggested that fumarate caused the oxidation of the component (552, 556 mtz) and that CO formed a complex with the reduced form of this pigment. Thus, it seemed probable that the component (552, 556 mt~) that was oxidized by fumarate and also capable of corn-

I

ABSORBANCY = 0.02

549 439-441

~553

\/

427

I

400

I

440

I

480

I

520

/

I

560

600

1

600

WAVELENGTH (mp)

FIG. 10. Ascorbate-reduced minus oxidized difference spectrum of the particulate fraction from M. expansa (77°K). Preparation similar to that described in Fig. 2. Ascorbate + cyanide minus oxidized difference spectrum. Final concentrations: ascorbate, 10 mM ; cyanide, 10 -3 M.

OXIDASESYSTEMSOF MONIEZIA EXPANSA

295

bining with CO was one of the terminal oxidases in M. expansa. The result suggested that this component, probably a b-type cytochrome, could also transfer electrons to fumarate converting it to succinate. The results in Fig. 8B demonstrated that M. expansa contained a very low level of cytochromes c and c t. Both of these cytochromes were observed in the ascorbate + cyanide minus oxidized difference spectrum of the particulate fraction. Figure 10 shows maxima indicative of cytochrome c (549 m/~) and c1 (553 m/~). The presence of a-type cytochrome(s) was illustrated by the ~(600 m/~) and the 7(439--441 m/~) peaks. 2-Thenoyltrifluoroacetone is a potent inhibitor of the succinate oxidase system, and the mechanism of its inhibition was suggested to be due to the chelation of

552

ABSORBtNCY= 0.02

$56

552 556

I

480

I

520

I

560

I

6QO

I

640

WAVELENGTH (m~) FIG. 11. Effect of 2-thenoyhrifluoroacetone on the succinate-reduced particulate fraction from M . expansa (77°K). Preparation similar to that described in Figs. 2, 4. A--Succlnate-reduced minus oxidized difference spectrum. B---Succinate + 2-thenoyltrifluoroacetone minus oxidized difference spectrum. Final concentrations: succinate, 10 r a M ; 2-thenoyltrifluoroacetone, 10 -s M.

296

K.S. CnEAH

non-haem iron (Singer et al., 1956). It was previously shown that 2-thenoyltrifluoroacetone (10-3M) inhibited the succinate oxidase activity by about 42 per cent (Table 2); Fig. 11 shows the effect of this inhibitor (B) on the succinate-reduced particulate fraction (A). Only 50 per cent of the component (552, 556m/z) was reduced, and no reduction of cytochromes b (562m/z), c (547 m/~) and the a-type cytochrome(s) (594-601 m/~) could be detected in the presence of 2-thenoyltrifluoroacetone (10 -8 M). This suggested the presence of non-haem iron in the succinate pathway. DISCUSSION Figure 12 illustrates the proposed electron transport system in M. expansa, a conclusion based on manometric and spectrophotometric studies using respiratory inhibitors and electron donors. Amytal inhibited the electron flow from the various substrates to their respective flavoproteins, which were demonstrated by the appearance of a trough at about 460 mk~ (Figs. 2, 6) and the inhibition by p-chloromercuribenzoic acid (Table 3). Of interest was the observation that amytal inhibited the succinate oxidase pathway (Table 4). It is generally accepted that amytal will only block electron transfer to NAD-linked flavoprotein, but could also inhibit succinate oxidation when the preparation was capable of phosphorylation (Chance & Hollunger, 1963 ; Pumphrey & Redfearn, 1963). The results suggested that the particulate fraction was probably capable of oxidative phosphorylation. The inhibition by dicumarol of the oxidase activities (Table 2) suggested that vitamin K might be the probable component linking the flavoproteins to the branched-chain system. This conclusion was strengthened by the unsuccessful attempt to extract any ubiquinone from the particulate fraction using the methods of Pumphrey & Redfearn (1960). Antimycin A (10 -6) blocked the electron transfer between cytochromes b and c 1. p-Phenylenediamine was previously reported to stimulate the oxygen uptake of the particulate fraction from M. expansa, and that this activity was inhibited by cyanide (10 -8 M), suggesting the probable presence of cytochrome oxidase (Cheah & Bryant, 1966). This was confirmed by the difference absorption peaks observed using the fumarate technique (Fig. 2B), by substrate reduction of the particulate fraction at liquid-nitrogen temperature (77°K) (Fig. 7A), and also by ascorbate reduction (Fig. 10). The CO-complex (Fig. 5) detected at room temperature (20°C) and that at liquid-nitrogen temperature (Fig. 9B) were probably contributed by the same pigment. At 20°C, this pigment had an a-peak at 555-557 m~ (Figs. 2A, 4A) when reduced with either dithionite or substrates, but showed two maxima at 552 and 556 m/z at 77°K. The results show that M. expansa possessed two terminal oxidases in its branched respiratory chain system. The major pathway appeared to involve two b-type cytochromes, which were detectable only at 77°K. These have been tentatively designated "Cytochrome 556 (Moniezia expansa)" (77°K) and "Cytochrome 552, 556 (Moniezia expansa)" (77°K), following the recommendations of the Commission on Enzyme Nomenclature (see Florkin & Stotz, 1965). The predominant

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carbon monoxide cyanide

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: SITE

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MINOR

: MAJOR

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oxygen

INHIBITION

u

PATHWAY

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cyt b _ . . _ _ . ~ cyt c! --..-..~ cyt c . . . . _~ cyt o ~.'~1, cyt a 3 p ~ . . . ~ _ _ . . _ ~

(Ixl0"6MI

FIG. 12. Proposed pathway for the electron transport system of M. expansa.

dlcumaro|

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298

K.S. CH~.A~

b-type component (552, 556 m/z) hence referred to as "Cytochrome 552, 556 (Moniezia expansa)" (77°K) appeared as a single band at 555-557 m/z at room temperature (Fig. 2A) and overlapped "Cytochrome 556 (Moniezia expansa)" (77°K), the latter component could only be observed with high concentration of antimycin A (10-4 M). Evidence was presented showing that fumarate, acting as a terminal acceptor, could accept electrons from "Cytochrome 552, 556 (Moniezia expansa)" (77°K). Under such a condition, no hydrogen peroxide was formed (Table 8). This suggested that the transference of electrons from "Cytochrome 552, 556 (Moniezia expansa)" (77°K), the major terminal oxidase, to oxygen resulted in hydrogen peroxide formation rather than water, as was demonstrated in the absence of fumarate (Table 8). The branched-chain electron transport system of M. expansa with two terminal oxidases, cytochrome oxidase and "Cytochrome 552, 556 (Moniezia expansa)" (77°K), using oxygen and fumarate as the terminal electron acceptors and the presence of peroxidase (Cheah, 1967) presents a remarkable example of adaptation in nature for organisms living in an environment where the oxygen tension is low

OXIDIZED

REDUCED

FUMARATEJ A ~ I ~ % ~~MQ .~.UJJ~~, (EXPANSA) ~

NAD(DIHYDROXYACETONE )

EXP~~,~m ~ ox;Pnm)

'FU~kRA~E I~DUCTA~E '

NADH(0C.-GLYCSltOIqtOSiq)tATE

DEHYDR~ENAt

FIG. 13. Proposed pathway for the reduction of fumarate to succinate by M.

expansa.

and variable but not necessarily strictly anaerobic. In the absence of fumarate under high or low oxygen tension, water and hydrogen peroxide are the endproducts of metabolism. The hydrogen peroxide, being toxic to the tissues, is probably destroyed by peroxidase. In a strictly anaerobic environment, one would expect that the minor terminal oxidase, cytochrome oxidase, is not functioning. Electrons derived from the oxidation of various substrates are then transferred probably via a "fumarate reductase" to fumarate resulting in the formation of succinate (Fig. 13). This appears to be the most likely explanation for succinate production and its secretion by M. expansa under anaerobic conditions (yon Brand, 1933). Under aerobic conditions and in the presence of fumarate one would also expect succinate formation, but no hydrogen peroxide accumulation if this theory

OXIDASE SYSTEMS O F MONIEZ1A E X P A N S A

299

of the function of the branched respiratory chain system is correct. This was confirmed by the demonstration with radioactive fumarate, where three to four times as m u c h radioactive succinate was formed in the presence of N A D H , the electron donor (Cheah & Bryant, 1966) and by the absence of hydrogen peroxide formation in the presence of fumarate (Table 8). It is also interesting to note that the four main observations reported for M. expansa: 1. 2. 3. 4.

The The The The

existence of a branched respiratory chain system; reduction of fumarate to succinate; production of hydrogen peroxide from the oxidation of various substrates; occurrence of peroxidase,

have also been made for Ascaris (Kikuchi et al., 1959; von Brand, 1966). Unfortunately, these observations reported for Ascaris were made from various groups of investigators who have so far not attempted to make any correlation between these findings and the biological significance to Ascaris or other helminths. The significance of the four main observations discussed for M. expansa could perhaps also be applied to Ascaris. The accumulation and excretion of sueeinate in Ascaris and perhaps other helminths (von Brand, 1952, 1966) and the metabolic end-products of Ascaris (yon Brand, 1966) can thus be explained. During the metabolism of carbohydrate, pyruvate (derived from glyeolysis) is converted to malate probably by malic enzyme (Saz & Vidrine, 1959; Agosin & Repetto, 1965) and subsequently to fumarate. Fumarate, acting as a terminal electron acceptor, is then converted to succinate (probably by a mechanism similar to that described for M. expansa) which then forms the key intermediate for the formation of other metabolic end-products. For example, sueeinate can be decarboxylated to give propionate which can then be excreted as one of the organic metabolites as reported for various helminths (von Brand, 1966). Propionate can form the important intermediate for the formation of ~-methylbutyrate and ~-methylvalerate, the two branched-chain fatty acids excreted by adults of .dscaris (Saz & Weil, 1962). The results obtained from M. expansa also reveal an interesting pigment, "Cytochrome 552, 556 (Moniezia ex'pansa)" (77°K), closely associated with fumarate reduction and hydrogen peroxide formation. This cytochrome, apparently a b-type, could form a CO-complex resembling cytochrome o, an oxidase so far found only in bacteria and algae, but have not been found in animal tissues. The successful extraction of some of this pigment by deoxycholate (Cheah, 1967a) could perhaps provide a method for the isolation and purification of this most interesting pigment in order to study its properties more thoroughly. Previous studies on Taenia hydatigena also showed that this cestode possessed a branched respiratory chain system with two terminal oxidases, cytoclirome oxidase and an o-type cytochrome (Cheah, 1967b). It is probable that this o-type pigment reported for T. hydatigena might resemble the "Cytochrome 552, 556 (Moniezia expansa)" (77°K). The existence of a mechanism for succinate production is not restricted to M. expansa and other parasitic helminths, but also appears to be widely spread among ruminant bacteria. White et al. (1962) reported that Bacteroides rurainicola could reduce fumarate to succinate by a mechanism involving a fumarate reductase. They suggested that a b-type cytochrome and flavoprotein formed an electron transport system for the reduction of fumarate to succinate by NADH, which was generated by glycolysis. An electron transport system with several terminal oxidases is also common among bacteria. White (1963) very elegantly demonstrated that Hemophilus parainfluenzae, when grown under poor aeration conditions, formed new oxidases (aa, a~), expanded and branched its electron transport system "in order to maintain the maximal respiratory rate under such a condition". White's hypothesis could also perhaps be applied to M. expansa and other intestinal parasites living in an environment with very low oxygen tension.

300

K . S . CHEAH

SUMMARY 1. T h e a-glycerophosphate, succinate and N A D H oxidase systems of M. expansa were investigated using a mitochondria-containing particulate fraction. 2. M a n o m e t r i c and spectrophotometric studies using respiratory inhibitors and electron donors suggested that M . expansa possessed a branched respiratory chain system with two terminal oxidases, cytochrome oxidase and an o-type cytochrome. 3. T h e major o-type terminal oxidase, tentatively designated " C y t o c h r o m e 552, 556 (Moniezia expansa)" (77°K) was demonstrated to be closely associated with hydrogen peroxide formation and fumarate reduction. 4. T h e major pathway with two b-type pigments was shown to participate in the reduction of fumarate to succinate, probably involving a " f u m a r a t e reductase". 5. T h e significance of the branched electron transport system in relation to its parasitic environment and to other intestinal helminths was discussed. 6. C o m p a r i s o n of some aspects between r u m i n a n t bacteria and intestinal helminths was also made.

Acknowledgements--The author wishes to express his thanks to Dr. C. A. Appleby for helpful discussions, and the Division of Plant Industry, C.S.I.R.O., Canberra, for access to the Cary spectrophotometer. REFERENCES AGOSlN M. & REPETTOY. (1965) Studies on the metabolism of Echinococcusgranulosus--VIII. The pathway to succinate in E. granulosus scolices. Comp. Biochem. Physiol. 14, 299-309. BUEDING E. & Ct-IARMSB. (1952) Cytochrome c, cytochrome oxidase and succinoxidase activities of helminths. J. biol. Chem. 196, 615-627. BUETOWD. E. & BUCHANANP. J. (1965) Oxidative phosphorylation in mitochondria isolated from Euglena gracilis. Biochim. biophys. Acta 96, 9-17. CASTORL. N. & CHANCEB. (1959) Photochemical determinations of the oxidases of bacteria. J. biol. Chem. 234, 1587-1592. CHANCE B. (1957) Techniques for the assay of the respiratory enzymes. In Methods in Enzymology (Edited by COLOWlCKS. P. & KAPLANN. O.) Vol. 4, pp. 273-329. Academic Press, New York. CHANCE B. & HOLLUNGERG. (1963) Inhibition of electron and energy transfer in mitochondria--I. Effects of amytal, thiopental, rotenone, progesterone and methylene glycol. J. biol. Chem. 238, 418431. CHEAH K. S. (1967) Histochemical and spectrophotometric demonstration of peroxidase in Moniezia expansa (Cestoda). Comp. Biochem. Physiol. 21, 351-355. CHEA~ K. S. (1967a) Unpublished observations. CrmAI-IK. S. (1967b) Spectrophotometric studies on the succinate oxidase system of Taenia hydatigena. Comp. Biochem. Physiol. 20, 867-875. CX-mAHK. S. & BRYANT C. (1966) Studies on the electron transport system of Moniezia expansa (Cestoda). Comp. Biochem. Physiol. 19, 197-223. EASTABROOK R. W. (1961) Spectrophotometric studies on cytochromes cooled in liquid nitrogen. In Haematin Enzymes (Edited by FALK J. E., LEMBERC R. & MORTONR. K.) Vol. 2, pp. 436-460. Pergamon Press, New York. F-,STABROOKR. W. & SACKTORB. (1958) The respiratory metabolism of insect flight muscle--III. Low-temperature spectra of the cytochromes of flight muscle sarcosomes. Archs Biochem. Biophys. 76, 532-545.

OXIDASESYSTEMSOF MONIEZIA EXPANSA

301

FLORKXN M. & STOTZ E. H. (1965) Comprehensive Biochemistry. VoL 13 (second edn.), Enzyme Nomenclature, Appendix C. Elsevier, Amsterdam. GRANT P. T. & SA~O~'~TJ. R. (1960) Properties of L-u-glycerophosphate oxidase and its role in the respiration of Trypanosoma rhodesiense, j~. Biochem. 76, 229-237. GREEN D. E. & TZAGOLOFFA. (1966) The mitochondrial electron transfer chain. Archs Biochem. Biophys. 116, 293-304. HOCHSTEIN P., LASZLO J. & MmLER D. (1965) A unique, dicumarol-sensitlve, nonphospborylating oxidation of DPNH and T P N H catalyzed by streptonigrin. Biochem. biophys. Res. Commun. 19, 289-295. JOHNSON M. J. (1941) Isolation and properties of a pure yeast polypeptidase, ft. biol. Chem. 137, 575-586. KXKUCHI G., RaMIm~z J. & GUSMANB ~ O N E. S. (1959) Electron transport system in Ascaris lumbricoides. Biochim. biophys. Acta 36, 335-342. KMETEC E. & BUEDING E. (1961) Succinic and reduced diphosphopyridine nucleotide oxidase systems of Ascaris muscle. J. biol. Chem. 236, 584-591. LnSER H. (1944) The oxidative metabolism of .4scaris suis. J. Biochem. 38, 333-338. LEE C. P., EST~ROOKR. W. & CHANC~B. (1965) Spectrophotometric and kinetic studies of the reconstituted succinate oxidase system. Biochim. biophys. Acts 99, 32-45. LE~rNXNGERA. L. (1964) The Mitochondrion. Benjamin, New York. LIGHTBROWN J. W. & JACI~ON F. L. (1956) Inhibition of cytochrome systems of heart muscle and certain bacteria by the antagonists of dihydrostreptomycin: 2-alkyl-4hydroxyquinoline N-oxide..y. Biochem. 63, 130-137. MAEHLYA. C. & CHANCEB. (1954) The assay of catalases and peroxidases. In Methods of Biochemical Analysis (Edited by GLXCKD.) Vol. 1, pp. 357-424. Interscience, New York. MOTO~WA Y. & K x ~ c n I G. (1966) Cytochrome systems in dark-aerobicaUy grown Rhodopseudomonas spheroides. Biochim. biophys. Acts 120, 274-281. PUMPHm~YA. M. & I ~ D ~ E. R. (1960) A method for determining the concentration of ubiquinone in mitochondrial preparations..7. Biochem. 76, 61-64. PUMI'm~Y A. M. & RED~Am~ E. R. (1963) Inhibition of succinate oxidation by barbiturates in tightly coupled mitochondria. Biochim. biophys. Acta 74, 317-327. RINGLER R. L. & SINGERT. P. (1959) Studies on the mitochondrial ~t-glycerophosphate dehydrogenase--1. Reaction of the dehydrogenase with electron acceptors and the respiratory chain. ~. biol. Chem. 234, 2211-2217. SAz H. J. & VIDRI~ZEA., JR. (1959) The mechanism of formation of succinate and propionate by Ascaris lumbricoides muscle. ~t. biol. Chem. 234, 2001-2005. S~. H. J. & WElL A. (1962) Pathway of formation of ~t-methylvalerate by Ascaris lumbricoides. J. biol. Chem. 237, 2053-2056. SINGERT. P., I ~ v E. B. & B~m~ATnP. (1956) Studies on succinic dehydrogenase---II. Isolation and properties of the dehydrogenase from beef heart. ~t. biol. Chem. 233, 599613. TABERH. W. & Mom~ISONM. (1964) Electron transport in Staphylococci. Properties of a particle preparation from exponential phase Staphylococcus aureus. Archs Biochem. Biophys. 10~5, 367-379. TANIGUCHI S. & KAMEN M. D. (1965) The oxidase system of heterotropically-grown Rhodospirillum rubrum. Biochim. biophys. Acta 96, 395-428. VAN Gm~wraERGm~rG. (1944) The respiratory metabolism of the cestode, MonieMa benedeni (Moniez, 1879). Enzymologia 11, 268-281. YON BRANDT. (1933) Untersuchungen tiber den Stoffbestand einiger Cestoden mad den Stoffwechsel yon Moniezia expansa. Z. vergl. Physiol. 18, 562-596. YON BRANDT. (1952) Chemical Physiology of Endoparasitic Animals. Academic Press, New York. YON BRANDT. (1966) Biochemistry of Parasites. Academic Press, New York.

302

K . S . CI-mAB

WEBSTERD. A. & HACKETTD. P. (1965) The respiratory chain of colorless algae--I. Chlorophyta and Euglenophyta. P1. Physiol. 40, 1091-1100. WEBSTERD. A. & HACKETTD. P. (1966) Respiratory chain of colorless algae--I I. Cyanophyta. P1. Physiol. 41, 599-605. WHITE D. C. (1963) Factors affecting the a f ~ i t y for oxygen of cytochrome oxidase in Hemophilus parainfluenzae. J. biol. Chem. 238, 3757-3761. WHITE D. C., BRYANTM. P. & CALDWALLD. R. (1962) Cytochrome-linked fermentations in Bacteroides ruminicola. J. Bact. 84, 822-828.