Studies on the electron transport system of Moniezia expansa (CESTODA)

Comp. Biochem. Physiol., 1966, Vol. 19, pp. 197 to 223. PergamonPress Ltd. Printed in Great Britain

STUDIES ON THE ELECTRON TRANSPORT SYSTEM OF M O N I E Z I A E X P A N S A (CESTODA) K. S. C H E A H * and C. B R Y A N T Department of Zoology, The Australian National University, Canberra, A.C.T., Australia (Received 15 February 1966; in revised form 31 March 1966)

A b s t r a c t - - 1 . Succinoxidase and NADH oxidase systems in a particulate fraction from MonieMa expansa were studied by manometric and spectrophotometric methods. 2. The use of redox dyes and inhibitors showed that succinate and NADH oxidation were accomplished by mechanisms which differed from those in mammalian tissues. 3. Succinate and NADH were apparently oxidized by different pathways, involving one or more flavoproteins and a cytochrome component, with the production of hydrogen peroxide. 4. A "peroxidase" was detected in a soluble fraction from the worm. 5. Spectrophotometric study showed that the cytochrome component was of the b type, provisionally identified as "cytochrome 557 (Monie~ia expansa)". 6. Only small amounts of cytochrome-a were found; there was some evidence for the presence of cytochrome-c. 7. The oxidation of cytochrome 557 by fumarate in the particulate preparation from Moniezia expansa, following reduction by NADH, has been demonstrated. 8. Experiments with radioactive fumarate showed that it was converted to suecinate more readily in the presence of NADH. 9. Additional reactions resulting in the formation of malate, aspartate, lactate and alanine also occurred. 10. Metabolic schemes to account for these reactions are presented, together with an hypothesis attempting to describe anaerobic respiration in large intestinal parasites.

INTRODUCTION THERE is considerable evidence suggesting that the terminal oxidation mechanisms of helminths have undergone marked modification and divergence (Read, 1961). Although a cytochrome component has been recognized in the tissues of Ascaris lurabricoides for many years (Keilin, 1925) it is only recently that attempts have been made to determine its function, but it has resisted efforts to place it in the pathway of electron transport. * Australian National University Research Scholar. 197

198

K. S. CHEAH AND C. BRYANT

Bueding & Charms (1952) failed to detect any cytochromes in homogenates of muscle and reproductive systems of Ascaris and concluded that the succinoxidase system lacked the classical cytochrome components. Kikuchi et al. (1959) observed an absorption spectrum characteristic of cytochrome-b in cholate extracts of Ascaris muscle pulp, and suggested it was involved in terminal oxidation. They also detected small amounts of cytochrome-c and cytochrome oxidase in their preparations. Kmetec & Bueding (1961), however, working with mitochondria from Ascaris muscle, were unable to confirm this work, and reported that the electron transport system in Ascaris involved several flavoprotein carriers. They observed the presence of a cytochrome-b but stated that it played no part in respiration. Their conclusions were based on the rather unsatisfactory determination of hydrogen peroxide in incubates of Ascaris by the p-phenylene diamine method (Laser, 1944), and on their own observation that catalase and manganese stimulated succinoxidase activity in Ascaris mitochondria. Kikuchi & Ban (1961) disagreed with this interpretation and demonstrated unequivocally the presence of cytochromes-b and -c and cytochrome oxidase in a particulate preparation from Ascaris muscle. Finally, Chance & Parsons (1963), using low-temperature spectroscopy, detected both cytochrome-b and cytochrome-c in suspensions of Ascaris mitochondria. However, they did not state whether these cytochromes were involved in respiration. In other helminths little work has been carried out. Bryant & Nicholas (1966) concluded that respiration in the acanthocephalan Moniliformis dubius involved a cytochrome-b, but the conclusion was based on manometric experiments only. A cytochrome of the b type has been demonstrated in Moniliformis, but its involvement in respiration has yet to be established (Bryant, 1966). The situation is equally confused in the cestodes. Friedheim & Baer (1933), working on Diphyllobothrium latum, concluded that cestodes possessed a special type of respiration. Van Grembergen (1944), however, demonstrated the presence of a cytochrome component in Moniezia benedeni, similar to that observed by Friedheim & Baer, with maximum absorption between 550 m/z and 560 m/x. This he took to be a mixture of cytochromes-b and -c, in disagreement with the earlier workers. Van Grembergen also demonstrated a stimulation of oxygen uptake in homogenates of Moniezia benedeni with p-phenylene-diamine, concluded that cytochrome oxidase was present, and thus saw no reason to diverge from contemporary views of cytochrome oxidation. Other aspects of cestode metabolism have been explored more thoroughly. For example, yon Brand (1933) showed that Moniezia expansa produced succinic acid under anaerobic conditions, and Agosin & Repetto (1965) have recently shown that scolices of Echinococcus granulosus convert pyruvate to succinate by pathways which also probably occur in the Trematoda, Nematoda and Acanthocephala (e.g. Bryant & Williams (1962), Bueding (1960), and Bryant & Nicholas (1965)). In addition, cestodes have been shown to possess a marked succinic dehydrogenase activity. Read (1952), Goldberg & Nolf (1954) and, more recently, Heyneman & Voge (1960) have demonstrated this enzyme in Hymenolepis diminuta

THE E L E C T R O N TRANSPORT SYSTEM OF M O N I E Z I A E X P A N S A

199

and Hymenolepis nana. Succinate metabolism thus appears to play an important role in the biochemistry of parasitic helminths, and the present paper investigates some of the reactions by which succinate and reduced nicotinamide adenine dinucleotide (NADH) may be metabolized in the tapeworm Moniezia expansa.

MATERIALS AND METHODS Materials Cytochrome-c (Type II, from horse heart), nicotinamide adenine dinucleotide (NADH), antimycin A and crude peroxidase (from horseradish; approximately 34 purpurogallin units/mg) were obtained from the Sigma Chemical Corporation, U.S.A.; methylene blue, p-phenylene diamine and guaiacol from British Drug Houses, Poole, Ltd., Dorset, England; and phenazine methosulphate from K and K. Laboratories, Inc., U.S.A. Hydrogen peroxide was stored as a 30% w/v solution and diluted as required. [1 : 4-C14~] fumarate, specific activity 15 mc/mM, was obtained from the Radiochemical Centre, Amersham, Bucks., England. All other reagents were analytical grade and distilled water was used throughout. Methods Adults of Moniezia expansa, from the intestines of freshly slaughtered lambs, were obtained from the Queanbeyan Abattoirs, New South Wales, Australia, and were immediately placed on ice. The worms were used for various preparations within 3 hr of removal from the host. After several washings in distilled water and in Ringer's solution to remove intestinal contents, the particulate fraction was prepared according to the flow chart (Fig. 1). Dry weights of the particulate fractions were determined by vacuum desiccation over P205 at room temperature until constant weights were obtained (about 5 days). Nitrogen content of particulate and supernatant fractions was determined by the method of Johnson (1941). Oxygen uptake by the particulate fractions was measured in a Warburg constant-volume respirometer, with air as the gas phase (except where otherwise stated) at 38°C, with 0.1 ml KOH (20%) in the centre well and shaking at 80 cycles/min, according to the methods described in Umbreit et al. (1957). The particulate fraction was made up in sodium-potassium phosphate buffer (6.7 x 10 -2 M; pH 7.0), so that 1"0 ml of suspension (dry wt. 16-1 + 0.24 mg; total nitrogen 181.2 +_12.9/zg) was equivalent to 1.0 g wet wt. of original tissue. 1.0 ml of this suspension was added to the main compartment of the Warburg flask. Substrate, either succinate, NADH or p-phenylene diamine in sodium-potassium phosphate buffer (6"7 x 10 -2 M; pH 7.0) was added from the side-arm to give final concentrations of 50-0 mM, 0.6 mM and 15.0 mM respectively. When present, phenazine methosulphate, methylene blue or cytochrome-c was dissolved in the ol 0.04% and 0"4% respecsubstrate solution to give final concentrations of 0.1/o, tively. The total volume of the reaction mixture in the flask was 2-5 ml.

200

K. S. CHEAHAND C. BRYANT

Antimycin A, in ethanol, was added from the side-arm, whereas cyanide and azide, in buffer, were added directly to the main compartment. The final concentrations of these inhibitors are given in the tables. Moniezia expansa 1. 2. 3. 4.

Washed (twice) with distilled water. Washed (4 times) with Ringer's solution--washings assayed for bacterial activity. Weighed. Homogenized (Waring Blendor; 15 see at 4°C) with three times its volume of 0.25 M sucrose. 5. Filtered with bolting silk (60 mesh). Homogenate

I

Centrifuged (1000 g/15 min/O°C)

I I

I

Residue (1000 g)

Supernatant (1000 g)

I

(1) Filtered with bolting silk (60 mesh)

I

(2) Centrifuged (12,000 g/80 min/0°C)

I I

I

Particulate fraction (unwashed)

Supernatant (12,000 g) (with "peroxidase" activity)

I

Resuspended in 2 vol. 0.25 M sucrose at 0°C, and recentrifuged (12,000 g/80 min/O°C)

I

I

Particulate fraction (12,000 g)

I

Supernatant

FxG. 1. Scheme for the preparation of a particulate fraction from Moniezia expansa. The effect of carbon monoxide on the particulate fraction was determined after the contents in the flasks had been gassed for 30 min with a CO (85%) : 02 (15%) mixture. A qualitative test, adapted from Maehly & Chance (1954), was performed to detect hydrogen peroxide in the media at the end of the experiments in which the particulate fraction was incubated with succinate or NADH. 1-0 ml guaiacol (20 raM) was added to the incubation medium followed by 0-3 ml of horseradish

THE ELECTRON T R A N S P O R T SYSTEM OF M O N I E Z I A

EXP.4NSA

201

peroxidase (0.1%; approximately 11 purpurogallin units). The red-brown colour was rapidly extracted with an equal volume of ether, its spectrum analysed with a Beckman DB recording spectrophotometer and compared with that obtained with authentic hydrogen peroxide. The 12,000g supernatant was assayed for peroxidase activity by a method complementary to that described above. To 2.5 ml samples (total nitrogen 545.0+31.18/~g) of supernatant (fresh or boiled for 2min) 0.3 ml hydrogen peroxide (5/~M) was added. After incubation for 2 hr at 38°C, 1.0 ml guaiacol (20 mM) was added together with 0.3 ml peroxidase (0.1%). The reaction mixture was extracted with ether and examined spectrophotometrically as before. Cytochrome and flavoprotein components of the electron transport system were detected spectrophotometrically with a Cary (Model 14) spectrophotometer fitted with a scattered transmission accessory, using methods described by Chance (1957), with a scan speed of 2.5 rn~/sec and a chart speed of 5 in/sec. Details of individual experiments are given with the figures. For electron microscopy, samples of the particulate preparation were treated as described by Bryant & Nicholas (1966). In the isotope studies, aliquots (100/~1) of the particulate preparation were incubated at various time intervals at 20°C with 1.0/~c radioactive fumarate dissolved in either 10/~I sodium-potassium phosphate buffer (6.7x 10 -~ M; pH 7.0) or 10/~1 buffer containing NADH (1.5 x 10 -3 M). At the end of the incubation periods the preparations were inactivated by the addition of 400 #1 ethanol, and the cellular debris removed by centrifugation at 500g for 10 min. The soluble radioactive intermediates were separated by two-dimensional chromatography, visualized by radioautography and their identity and radioactivity determined by the techniques described by Smith & Moses (1960). RESULTS

1. Electron microscopy Electron micrographs (Fig. 2) showed that the particulate fraction consisted of a heterogeneous mixture of vesicles, some membranous material and a few fat globules. Some of the vesicles possessed an outer double membrane but only in very few of them could well-organized cristae be observed. The size and number of these cristae did not resemble those of "classical" mammalian mitochondria (Lehninger, 1964).

2. Manometry The particulate fraction gave a low level of oxygen uptake with succinate or NADH as substrate (Table 1). When phenazine methosulphate was added together with succinate the oxygen uptake was increased nearly tenfold. With methylene blue instead of phenazine methosulphate the stimulation was between two- and threefold. The effect of methylene blue on the oxidation of NADH proved to be inhibitory.

202

K. S. CHEAHAND C. BRYANT

Cyanide (10 -a M) had no effect on succinate oxidation. However, in the presence of succinate and phenazine methosulphate, oxygen consumption was increased by nearly 40 per cent by cyanide, and with methylene blue instead of phenazine methosulphate, the increase was greater than 300 per cent with added cyanide. The oxygen consumption of the particulate fraction with both succinatedye systems in the presence of cyanide was similar. TABLE 1--SuccxNIC

AND N A D H

DEHYDROGENASE ACTIVITY OF THE PARTICULATE FRACTION FROM Moniezia expansa

Activity* Substrate

Succinate

NADH

Electron aceeptor

Cyanide

Number of estimations

Range

Mean

Oxygen

None 10 -8 M

13 12

11' 3-16-9 12'6-16-2

13' 3 13'8

Phenazine methosulphate

None 10-3 M

5 4

93"5-138"5 145"0-179"7

118"9 162"3

M ethylene blue

None 10-* M

5 5

25-2--42'9 93"8-180-1

35"2 146"2

None None

12 6

Oxygen Methylene blue

11'8-17"5 8"2-13-7

14.7 9.2

* Activity expressed in /zl O3 (air) uptake/g wet wt. tissue/hr at 38°C. Corrections made for endogenous activity. When cytochrome-c was added to the preparation, with succinate as substrate, and examined spectrophotometrically at the beginning and end of the experiment, no change in its spectrum was observed; neither did it stimulate the oxygen uptake of the preparation. With p-phenylene diamine as substrate, the oxygen uptake of the particulate fraction was increased (Table 2). The stimulation was greater than that observed with either succinate or NADH, and was inhibited by nearly 80 per cent in the presence of cyanide (10 -3 M). Table 3 illustrates the effects of various concentrations of cyanide, azide and antimycin A on the respiration of the particulate fraction. Cyanide in concentrations up to 10 -3 M had no effect. Azide in concentrations greater than 10 -4 M proved to be stimulatory, and antimycin A had no inhibitory effect in concentrations less t h a n 10 -5 M .

Carbon monoxide did not inhibit the oxidation of succinate or NADH by the particulate fraction. In fact, an apparent stimulation was observed (Table 4), but this was found to be significant in only one experiment in three when the data were examined by the application of "Student's" t-test, because of the range of values obtained.

FIG. 2.

Electron micrograph of the particulate preparation from Moniezia M - - m i t o c h o n d r i o n ; c--crista; v--vesicular material.

expansa.

203

THE ELECTRON TRANSPORT SYSTEM OF M O N 1 E Z I A E X P A N S A TABLE

2--COMPARISON

p-PHENYLENE

OF S U C C I N I C ACID DEHYDROGENASE~

NADH

DEHYDROGENASE A N D

D I A M I N E OXIDASE A C T I V I T I E S I N A P A R T I C U L A T E P R E P A R A T I O N F R O M

Moniezia

expansa Activity*

Substrate

N u m b e r of estimations

Cyanide

Range

Mean

Inhibition (%)

p-Phenylene diamine

None 10 -3 M

6 6

27'4 43"0 4.2-11-7

33"8 7"6

-77'5

Succinate

None 10 -3 M

13 12

11-3-16-9 12.6-16.2

13" 3 13.8

-0

None

12

11.8-17'5

14'7

NADH

* A c t i v i t y expressed i n / z l 0 2 (air) u p t a k e / g wet wt. t i s s u e / h r at 38°C. C o r r e c t i o n s m a d e fl}r e n d o g e n o u s activity.

TABLE 3--EFFECT

OF V A R Y I N G THE C O N C E N T R A T I O N S OF I N H I B I T O R S

ON S U C C I N I C DEHYDROGENASE A C T I V I T Y OF A P A R T I C U L A T E PREPARATION

FROM Moniezia expansa Inhibitor Concentration (M)

Cyanide

10 -s 10 -7 10 -6

Azide

Antimycin A 0

0

10-5 10 -a

10 -s 10 -~

0 0 0 0

0 0 0 + 16

0

+39

0 - 45 - 65

Effect expressed as p e r c e n t a g e i n h i b i t i o n ( - ) or s t i m u l a t i o n ( + ).

TABLE

4---EFFECT

OF

CARBON

MONOXIDE

ON

SUCCINIC

A C T I V I T I E S OF A P A R T I C U L A T E PREPARATION FROM

AND

Air

Substrate Succinate NADH

NADH

DEHYDROGENASE

Moniezia expansa C O / O 2 (85 : 15)

N u m b e r of estimations

Range

Mean

N u m b e r of estimations

Range

Mean

7 6

15'0-21"5 8'0-17"5

18-4 14"8

9 8

17'5-32'0 8"0-17"5

25.4 13"4

Activity expressed in /zl 0 2 u p t a k e / g w e t wt. t i s s u e / h r at 38°C. e n d o g e n o u s activity.

C o r r e c t i o n s m a d e for

204

K . S . CHEAH AND C. BRYANT

3. Hydrogen peroxide formation and peroxidase activity When guaiacol and horseradish peroxidase were added to the medium in which the particulate fraction had been incubated for 2 hr with succinate or NADH, the characteristic red-brown colour of tetraguaiacol was observed, indicating the presence of hydrogen peroxide. The colour was extracted with ether and its absorptkm spectrum was found to correspond with that obtained using authentic hydrogen peroxide (Fig. 3). Because tetraguaiacol is unstable, it was not possible to determine accurately the concentration of hydrogen peroxide formed.

0.03,

A

B

0.0~

R ao

0.01

520

480

440 Wavelencjth,

400

360

320

m/J.

FIG. 3. Test for hydrogen peroxide in the incubation medium of the particulate

fraction from Moniezia expansa--absorption spectra of tetraguaiacol. (A) Control-compound formed with authentic hydrogen peroxide extracted with ether. (B) Test--spectrum of compound formed with incubation medium, after extraction with ether. With a modification of the same technique, peroxidase activity was detected in the 12,000 g supernatant. However, this activity was removed when the supernatant was boiled.

4. Spectrophotometry Figures 4-11 are difference absorption spectra of cytochromes and flavoproteins obtained with various preparations from Moniezia expansa. The endogenous reduction of the whole homogenate resulted in maximum absorption peaks at 425 m/~ and 557 m/~, with "shoulders" between 440 m/~ and 450 m/~, and at 527 m/~. Dithionite enhanced the absorption peaks at 425, 527

THE ELECTRONTRANSPORTSYSTEMOF MONIEZIA EXPANSA

205

and 557 m/x, revealed an absorption m i n i m u m or " t r o u g h " at about 500 nag (Fig. 4), b u t r e m o v e d the shoulder between 440 m/z and 450 nag.

0.10,

425

0 "08 I

0-06"

557

[

o

0-04"

527

f tr~,

\

.

.,f.d,,,v,¢ \

0.02-

Oql= 410

450

490

530

Wavelength,

570

610

650

rn/x

Fro. 4. Difference spectra of the cytochrome and flavoproteins in whole homogenates of Moniezia expansa. Dotted line--endogenous reduction; oxygen bubbled through reference sample, test sample stagnant. Continuous line-dithionite reduction; 1-0 mg sodium dithionite added to the test sample. Homogenate prepared in 0"25 M sucrose; 2"0 g wet wt. of tissue/ml. Volume of cuvette 0"2 ml, light path 2-0 ram. Readings carried out at room temperature. Closer examination of the dithionite-reduced s p e c t r u m showed only a single absorption peak at 557 m/~ (Fig. 5A). T h e same absorption b a n d was observed

206

K . S. CHEAH AND C. BRYANT

when the reference cell was oxygenated, but 10 m i n were required for the peak to attain the same absorbance as that observed with the dithionite-reduced system (Fig. 5B). T h e r e was no difference observed in the spectra obtained with the dithionite, and the dithionite--ferricyanide reduced systems (Fig. 5C).

5F7

0 05-

557

-0.05

557

I 0 04

O.04m

g o oo3a~

003

0-02-

0 02

001-

0 0~

I

530

;

570

:

610

I

530

I

570

Wavelength,

:

610

:

530

I'

570

I

610

m/.L

FIc. 5. Difference spectra in the range 520-620 m/z of whole homogenates of Moniezia expansa. (A) Dithionite reduction--l'0 mg sodium dithionite added to the test sample; reference sample bubbled with oxygen. (B) Endogenous reduct i o n - t e s t sample stagnant; reference sample bubbled with oxygen. Lower curve--0min. Upper curve--10min. (C) Reduction with dithionite plus ferricyanide---l'0 mg sodium dithionite added to the test sample, together with 20/zl potassium ferricyanide (5"8 x 10 -s M) in sodium/potassium phosphate buffer (6"7 x 10 -~ M; pH 7.0); reference sample bubbled with oxygen after the addition of 20/zl buffer. Other experimental conditions as in Fig. 4. T h e supernatant f r o m the intial 12,000g centrifugation was also examined spectrophotometrically (Fig. 6). Only a weak absorption band at 557 m/z was detected when the reference cell was oxygenated. I n the presence of dithionite a trough appeared at 440 m/z, together with a new absorption peak at 420 mtz, but the initial peak at 557 m/z was unaffected. T h e absorption peaks observed in the homogenate were m u c h more pronounced in the particulate fraction (Fig. 7). I n the presence of dithionite, absorption bands at 425 m/~, 527 m/~ and 557 m/~ were again detected. T h e shoulder

THE ELECTRONTRANSPORTSYSTEMOF MONIEZIA EXPANSA

207

between 440 m/z and 450 m/~ was also present, together with a small peak at about 600 m/x. T h e trough at 470 m/z was very marked.

001 0-03--

- 0 03

0.02--

"0

001-

-

0-03

--

u

02

=001

,1o

0,02--

A

-003

~

- 0.02

0~31 -

,-0,01

: 400

I

440

I

480

I

520 Wovelength,

I

560

I

600

I 'o

640

m~

FIG. 6. Difference spectra of components in the 12,000g supernatant from (A) Endogenous--test sample stagnant; reference sample bubbled with oxygen. (B) Dithionite reduetion--l'0mg sodium dithionite added to the test sample. Volume of cuvette 3"0 ml, light path 10 ram. Readings carried out at room temperature. Moniezia expansa.

As a preliminary to the investigation of the ability of succinate or N A D H to produce a similar reduction pattern, oxygen was bubbled through the reference cell to determine whether any endogenous reduction occurred in the sample (Figs. 8 and 9). No absorption bands were detected in the regions indicated by the dithionite experiments. W h e n either succinate or N A D H was added to the test sample, however, the same peaks and troughs appeared, but maximum absorption was reached after 15-30 min. T h e r e was little or no difference in the heights the peaks attained with either dithionite or succinate.

208

K. S. CHEAH AND C. BRYANT i

.0.10

557 I

- 0'08

.006

.004

-0.02

527

=o o .o i J

0.5. 0'4,

0.3. 0.2. 0.1. 0.

!

425

/

,0

• -0.02

f

i

j

I J • -0'04

J , -0.06

|

i 400

440

480

520

W0velength,

I

:

560

600

'

~40

mF

FIG. 7. Difference s p e c t r u m o f the d i t h i o n i t e - r e d u c e d cytochromes and flavoproteins i n the particulate fraction f r o m Moniezia expansa. 1 2 , 0 0 0 g pellet suspended i n 6 . 7 x 1 0 - 2 M s o d i u m / p o t a s s i u m phosphate buffer, p H 7"0; final

concentration equivalent to 5-0 g wet wt. unhomogenized tissue/ml (total nitrogen 1037+ 65 mg). 1"0 mg sodium dithionite was added to the test sample. T h e reference sample was bubbled with oxygen. Volume of cuvette 0"5 ml, light path 4.0 ram. Readings carried out at room temperature. Vertical dashed line indicates change of scale.

THE ELECTRON

TRANSPORT

209

SYSTEM OF MONIEZIA EXPANSA

B

•0

I0

557 • 0.09

.0'08

425 0.5

J/

0.4.

0"04

-

0'3" O-Z. , , ~

0'1

0'01 0

005.

A

,.

0 ' 0 4 " ~ ~ ~ ~ ~ / ~

0"04

0,05"I 400

0.05

I 003 440

480

520 Wavelength,

560 m/L

600

640

FIG. 8. Difference spectra of the succinate-reduced cytochromes and flavoproteins in the particulate fraction from Moniezia expansa. Experimental details as in Fig. 7 except that instead of dithionite, 10 F1 sodium succinate (0"25 M) was added to the test sample. T h e reference sample was bubbled with oxygen after the addition of 10 F1 phosphate buffer. (A) Endogenous (without succinate). (B) Succinate reduction. U p p e r curve--5 rain after the addition of succinate. Lower curve--15 rain after the addition of succinate. (C) Succinate reduction--Soret region, reduced scale.

210

K . S . CHEAH AND C. BRYANT

Neither antimycin A nor cyanide had any effect on the spectra obtained with either substrate once a steady state had been attained.

0 08-

~5

0 07, 557

0.06, 527 t

0 05,

440-450

1 ~

'~V~'V~

0 04, 0 03,

8 0

02,

.1o 001, 0, 00200I 0

A

0.01 •

O, Wavelength,

m~.

FIG. 9. Difference spectra of the NADH-reduced cytochromes and flavoproteins in the particulate fraction from Moniezia expansa. Experimental details as in Fig. 7, except that instead of dithionite, 10/4 N A D H (1"5 x 10 -a M) was added to the test sample, volume 0"2 ml, light path 2"0 mm. The reference sample was bubbled with oxygen after the addition of 10/~1 phosphate buffer. (A) Endogenous (without NADH). (B) N A D H reduction--5 min after addition of NADH. (C) As (B), 25 rain later. W h e n the test sample was first reduced by dithionite and then gassed with carbon monoxide, the spectrum was considerably modified. T h e peak in the Soret region (425 m/z) was displaced to 420 m/~, and a trough appeared at 443 m/~. T h e absorption band at 557 mtz disappeared (Fig. 10).

T H E E L E C T R O N T R A N S P O R T S Y S T E M OF .~IONIEZIA E X P A N S A

211

0.18. 420

0"16'

0-14.

0 12.

010c o

JD <~

008,

006.

0 04.

0.02-

V. _ _ 0

I 400

I 440

445 I 480

I 520

Wavelength,

I 560

I 600

I 640

m/z

FIG. 10. Difference spectrum s h o w i n g the effect of carbon m o n o x i d e on the particulate fraction from Moniezia expansa. Experimental details as in Fig. 7, except that b o t h the test and reference samples were reduced w i t h 1"0 m g dithionite. Carbon m o n o x i d e w a s b u b b l e d t h r o u g h the test cell.

Finally, reduction of the particulate fraction with succinate, succinate plus dithionite, and succinate plus dithionite plus ferricyanide was followed by examination of the spectra at 77°K (Fig. 11). With succinate a small absorption peak was observed at 600 m/z, and the absorption band at 556 m/z was observed to possess a

212

K. S. C H E A H A N D C. B R Y A N T

small shoulder at 553 m/~. The smaller band at about 525 m/z split into subsidiary peaks at 525 m/~, 520 m/z and 514m F. Addition of dithionite decreased the absorption band at 600 mp and removed the subsidiary peaks at 520 m F and 514 m F. Addition of ferricyanide enhanced the absorption peak at 556 m/z and removed the shoulder at 553 m F. A

556

1

0.$0.

556 556

553 55:5

] .O.OB

0.08,

0"06,

"0'06

600

c

525

600 525 ,0.04

,0'0~

0.02-

0-

490

540

590

640 490

540

590

Wovelength,

640 490

540

590

640

m/J.

FIG. 11. Low-temperature (77°K) difference spectra of reduced cytochromes and flavoproteins in the particulate fraction from Moniezia expansa. (A) Reduction with 10/zl succinate (0.25 M) only. (B) Reduction with succinate plus dithionite (1 "0 rag). (C) Effect of addition of ferricyanide after reduction with succinate plus dithionite. Concentration of the preparation as in Fig. 7. Volume of cuvette 0"2 ml, light path 2"0 ram. The reference sample was bubbled with oxygen. Both cells quenched in liquid nitrogen before reading. Figure 12 shows the progressive reduction, with time, of cytochrome in the particulate preparation from Moniezia expansa after the addition of NADH (final concentration: 4 x 10 -5 M). The progress of the reaction was followed by measuring the absorbance of the cytochrome at 425 m/z. After an initial lag of about 10 min, absorbance rapidly increased reaching a maximum in about 25 min. At this time, the addition of fumarate (final concentration: 1.2 x 10 -2 M) resulted in the sudden decrease of the absorption maximum at 425 m F to its value at zero time. This was followed by a slow increase in the reduction of the cytochrome,

213

THE ELECTRON TRANSPORT SYSTEM OF M O N I E Z I A E X P A N S A

stabilizing ~fter 50 min at a level which was only 14 per cent of that observed in the presence of N A D H alone.

z

I

0

5

I0

15

20

25 Time,

50

35

40

45

50

min

FIG. 12. T h e reduction of cytochrome 557 by N A D H , and its reoxidation by fumarate. Reaction followed by measurement of extinction at 425 m/z. Experimental details as in Fig. 9,

5. Isotope studies W h e n t h e p a r t i c u l a t e f r a c t i o n w a s i n c u b a t e d w i t h [1 : 4-C14~] f u m a r a t e , r a d i o -

carbon was detected in succinate, malate, lactate, aspartate and alanine. Table 5 shows the distribution of radioactivity in these intermediates at two selected time TABLE 5--Tm~ INTO

THE

INCORPORATION OF RADIOCARBON FROM

SOLUBLE

INTERMEDIATES

OF

THE

[1

: 4-C142] FUMARATE

PARTICULATE

FRACTION

FROM

Moniezia expansa 20 min Intermediate Fumarate Succinate Malate Aspartate Alanine Lactate Total

Control 10"9 4"1 28-0 0"8 5'1 0"2 49-1

180 min

+ NADH

Control

+ NADH

13"5 13.9 16'0 3"0 6"3 0.6 53.3

1-6 2"3 4"4 1-1 13"6 0.8 23"8

0"1 6"0 0"2 0"3 12"4 1 '1 20"1

Fumarate initially added = 72-3. Results expressed as counts/rain x 10 -3.

214

K.S.

C H E A H AND C . BRYANT

intervals in the presence and absence of NADH. The major components in each case were succinate, malate and alanine. The total recovery of radiocarbon was considerably less at the end of 180 min. The behaviour of the pools of radioactive intermediates during the experiment is shown in Figs. 13 and 14. Figure 13(1) shows that the percentage utilization of fumarate by the preparation proceeded at a slightly greater rate in the presence of 100

I

8O

6O

4O

20

"~

-

I

"5 25'

I

"

I

d-

. _~

4"

I 2

,o/ e

/

~

l e I0

FIG. 13.

expansa.

•

I 20 Time,

I 50 mln

I 180

The metabolism of fumarate by a particulate preparation from Moniezia (1) The utilization of fumarate. (2) The formation of succinate. O--phosphate buffer only. I--phosphate buffer + NADH.

THE ELECTRON TRANSPORT SYSTEM OF MONIEZIA EXPANSA

215

NADH. The percentage incorporation of radiocarbon into sueeinate, however, took place at a greatly increased rate from the beginning of the experiment. Although this declined with time, the percentage of radiocarbon in succinate after 180 min was much greater with NADH [Fig. 13(2)]. IOO

I

8o

6o

%% ``9. 20 0

/

"

%

%

-

.

%.

.

%

> ~

% •

%. 2o

i

,

,

II

"

'

2

~-

20

it

15

I I I I i I I I Alonine

f-,

I0

o

io

20

i I i I i I t I

30

Time,

180

min

FIG. 14. T h e m e t a b o l i s m of f u m a r a t e b y a p a r t i c u l a t e p r e p a r a t i o n f r o m M o n i e z i a expansa. (1) T h e f o r m a t i o n of malate. (2) T h e f o r m a t i o n of a l a n i n e a n d aspartate. ©, O - - p h o s p h a t e buffer only.

[], i - - p h o s p h a t e

buffer+NADH.

216

K. S. CHEAH AND C. BRYANT

The incorporation of radiocarbon into malate reached a maximum, in both cases, after 15 min. Subsequently there was a steady decrease in the amount of isotope in this intermediate. Throughout the period of the experiment, incorporation into malate was greater in the absence of NADH [Fig. 14(1)]. Similar-shaped curves were obtained with aspartate, although in this case the maxima were reached at about 30 min, and incorporation of radiocarbon was greatest in the presence of N A D H [Fig. 14(2)]. Finally, the rate of incorporation of isotope into alanine did not differ markedly in the presence or absence of NADH, in each case achieving a maximum at the end of the experiment [Fig. 14(2)]. DISCUSSION Monlezia expansa mitochondria in situ have not been examined with the electron microscope. However, studies on the tegument of taeniiform cestodes (Morseth, 1965; Barton & Smyth, 1965) have shown that their mitochondria consist of vesicular structures rather deficient in cristae, similar to those observed in other parasitic helminths (e.g. Bj6rkman & Thorsell, 1964; Nicholas & Mercer, 1965). The vesicular nature of the present preparation from Moniezia, coupled with its ability to oxidize succinate and NADH (Table 1) and also ~-glycerophosphate (Cheah & Bryant, 1966), suggests that it consists largely of mitochondria whose properties are similar to those observed in the mitochondria of other helminths. The very low uptake of oxygen by the Moniezia preparation, when either succinate or N A D H was present, shows that the terminal oxidase is either present in very small amounts or operating under sub-optimal conditions. The different stimulatory effects of methylene blue and phenazine methosulphate also suggest that there are one or more oxidative steps involving flavoproteins during succinate oxidation. The rate of NADH oxidation, however, was not increased by methylene blue; thus, this process must take place by a different pathway. High concentrations of cyanide (Tables 1 and 3) had no effect on succinate oxidation, demonstrating that a "classical" cytochrome oxidase, as found in mammalian systems, was absent. On the other hand, oxygen uptake by the preparation in the presence of succinate and the dyes was increased by the addition of cyanide. Singer & Kearney (1956) have described a similar effect in mammalian succinic dehydrogenase systems, which is due to the removal by cyanide of an inhibition of the flavoprotein enzyme caused by the accumulation of hydrogen peroxide in the presence of dyes. Thus, there is additional evidence confirming the presence of one or more flavoproteins in the Moniezia preparation. The stimulation of the oxygen uptake of the preparation in the presence of p-phenylene diamine (Table 2) may be interpreted as being due to the presence of a cytochrome oxidase, especially as it was inhibited by cyanide, an observation in agreement with that of van Grembergen (1944) in 31oniezia benedeni. However, p-phenylene diamine is very unspecific in its action, and the stimulation may be due to other cyanide-sensitive oxidases. Certainly, it is not consistent with the

THE ELECTRON TRANSPORT SYSTEM OF M O N I E Z I A E X P A N S A

217

lack of effect of cyanide in the succinoxidase system. If cytochrome oxidase is present, it appears to play no part in the aerobic oxidation of succinate. Examination of the effects of three other inhibitors (Table 3) used in studies on electron transport also produced results which suggested that cytochrome oxidase was absent. Antimycin A, which inhibits the transfer of electrons between cytochromes-b and -c, had only a partial effect at concentrations considerably greater than those reported to cause 95 per cent inhibition in mammalian preparations (Potter & Reif, 1952). This may indicate that a cytochrome-c is not involved in aerobic respiration in Moniezia. Azide in high concentrations was stimulatory, perhaps due to its ability to combine with, and render non-toxic, inhibitory metal ions in the preparation. Carbon monoxide did not inhibit either succinate or NADH oxidation, confirming the absence of a functional cytochrome oxidase. The accumulation of hydrogen peroxide when mitochondrial preparations were incubated with succinate or N A D H shows that the oxidase involved in aerobic respiration may be a flavoprotein. It seems unlikely that such an inhibitory substance as hydrogen peroxide is a normal end-product--in a largely anaerobic environment, such as the ruminant gut, it would probably not be formed during electron transport--but an artefact of the experimental procedure. However, there is present in Moniezia a peroxidase which is inactivated by boiling (so that its effect cannot be due to heavy metal catalysis) which could remove hydrogen peroxide, should it be formed. T h e evidence derived from electron microscopy, from the manometric experiments with dyes and inhibitors and from the demonstration that hydrogen peroxide accumulated during oxidation (coupled with the fact that added cytochrome-c did not stimulate this oxidation, nor was it reduced during the progress of the experiment) indicates that the mitochondrial particle from Moniezia differs markedly from the mammalian pattern. These experiments suggest that succinate oxidation is mediated by one or more flavoproteins; that cytochrome-c and cytochrome oxidase are either absent or present in small amounts and not involved in the aerobic oxidation of succinate and N A D H by the mitochondria; that cytochrome-b may or may not be present, but if present is probably not involved in aerobic respiration; and, finally, that N A D H oxidation proceeds by a separate pathway. These results are essentially in agreement with those obtained by Kmetec & Bueding (1961) in Ascaris, but it must be stressed that conditions under which the experiments were carried out are thoroughly atypical of the animals' normal environment, where the mechanism of electron transport may differ markedly. Further investigation of the problem was carried out by the spectrophotometric methods developed by Chance (1957) employing scattered transmission through thick mitochondrial suspensions. When an oxygenated sample of a whole homogenate of Moniezia was compared with a stagnant one, the ~ (557 m/z), fl (527 m/~) and y (425 m/z) absorption bands of a b-type cytochrome were detected in the latter [see Morton (1958), for a review of the properties of cytochromes] ; oxidation of the reduced cytochrome was complete within 10 rain--that is, the absorption bands in the stagnant cell attained maximum levels in that time. Thus, in Moniezia

218

K . S . CHEAH AND C. BRYANT

homogenates there is an autoxidizable cytochrome-b which can be reduced by some unknown endogenous substrate. The presence of flavoprotein was also indicated by a trough at about 490 m/z, but no traces of cytochrome-c or cytochrome oxidase were detected (Fig. 4). Addition of dithionite accentuated the characteristics of the spectrum, but cytochrome-c and cytochrome oxidase remained undetectable. Further addition of ferricyanide did not alter these observations (Fig. 5). The first supernatant obtained during the preparation of the mitochondrial fraction was also examined (Fig. 6) and was found to contain no cytochrome components (which were thus shown to be associated with particulate material), although with dithionite a peak and trough at about 420 m/~ and 400 m/~ respectively suggested the presence of some reducible material. This supernatant fraction has been shown to possess peroxidase activity, and it is possible that the dithionitereduced spectrum is clue to this enzyme. The cytochrome component of the homogenate was concentrated in the particulate fraction, providing further evidence of its mitochondrial nature. No endogenous reduction was observed in the preparation, indicating that all endogenous substrate had been removed during the preparatory procedures. Addition of succinate (Fig. 8) or N A D H (Fig. 9) to the test cell resulted in the same absorption maxima detected in the whole homogenate. The cytochrome-b was therefore shown to be implicated in oxidation of these substrates. In addition, however, small deflexions in the spectrum at 600 m/z and 400 m/z--450 mt~ suggested that small amounts of cytochrome-a might be present. The time taken for maximum absorption to be reached (15-30 min) was rather long, when compared with mammalian systems. Complete reduction with dithionite gave a spectrum similar to those obtained with these two substrates. The addition of cyanide or antimycin A did not alter this pattern in the steady state which again suggested that neither cytochrome-c nor cytochrome-a was involved in the oxidation of the substrates. Carbon monoxide (Fig. 10), however, combined with the cytochrome-b, causing the removal of the ~ peak and a shift in the ~, peak from 425 m/z to 420 mt~. In view of the lack of inhibitory effect of carbon monoxide on the oxygen uptake of the mitochondrial preparation from Moniezia, this indicates that the cytochrome-b is not acting as an oxidase in the aerobic oxidation of succinate or NADH. Further examination of selected experiments, at 77°K to afford greater resolution of the ~ and/3 peaks, confirmed these conclusions (Fig. 11). The preparation reduced by succinate gave a spectrum in which the absorption peak of cytochrome oxidase (600 m/z) was clearly visible, although it was considerably smaller than the peak (536 m/~) of cytochrome-b, differing remarkably from the mammalian pattern. The ~ peak of the cytochrome-b possessed a slight shoulder at 333 m/~, which remained in the presence of dithionite, but which was removed by ferricyanide. The behaviour of this component which absorbs at 553 mtz is thus similar to that of a haemoprotein (designated cytochrome 553) which is isolated together with cytochrome-b from pig heart muscle (Stotz, 1955).

THE ELECTRON TRANSPORT SYSTEM OF M O N 1 E Z I A E X P A N S A

219

It is interesting to compare these results with observations, albeit very few, on other helminths. In Ascaris, the cytochrome-b described by Kikuchi et al. (1959) has similar spectral properties, although it is reported not to combine with carbon monoxide (Kikuchi & Ban, 1961). This is obviously the same cytochrome described by Chance & Parsons (1963). In other helminths, a similar cytochrome has been observed in the Acanthocephala (Bryant, 1966) and, surprisingly, in a freeliving nematode, Caenorhabditis briggsae (Bryant et al., 1966). A cytochrome-c has been identified in Ascaris preparations with a 7-absorption peak at 417 m/z (Kikuchi & Ban, 1961) or 419 m/z (Chance & Parsons, 1963). The latter workers reported that the ~ peak of the cytochrome-c in Ascaris was situated at 547 m/~ while Kikuchi & Ban (1961) place it at 549 m/z. The shoulder observed in Moniezia at 553 m/z, which may possibly be attributed to cytochrome-q, differs, therefore, from the Ascaris cytochrome-c. The observations so far discussed may be summarized by Fig. 15, where it is postulated that, under aerobic conditions and in the absence of added fumarate, the oxidation of NADH and succinate by the particulate fraction from Moniezia expansa is mediated by flavoproteins with the accumulation of hydrogen peroxide. Cytochrome 557, although capable of reduction and autoxidation, does not play a great part in aerobic respiration, which agrees with the findings of Kmetec & Bueding (1961). Figure 15, however, describes an aerobic system. It seems likely that a respiratory system in which oxygen is the terminal acceptor is probably not normal to the organism, and that some other mechanism operates in its normal, relatively anaerobic, environment. Von Brand (1933) showed that Moniezia expansa accumulated succinate under anaerobic conditions, and Kmetec & Bueding (1961) demonstrated that a particulate fraction from Ascaris muscle converted fumarate to succinate by a reaction dependent on NADH. They suggested (but did not demonstrate) that this was mediated by electron transport enzymes. The latter workers also detected a cytochrome of the b type in their preparation, but concluded that it was not involved in the oxidation of succinate and NADH. Figure 12 shows the rate of reduction of cytochrome 557 in the particulate fraction from Moniezia by NADH (measurements of the reaction rate were made at 425 m/z as greater differences were observed in unit time at this wavelength) and that the addition of fumarate brought about its reoxidation. Cytochrome 557 remained in the oxidized state after fumarate addition for the duration of the experiment. It thus appears that the sequence of reactions was, firstly, the reduction of cytochrome 557 by N A D H and, secondly, the reduction of fumarate by cytochrome 557. The reduction product of fumarate is succinate. This seemed to offer a possible mechanism for anaerobic respiration in the parasite, and one which could readily be tested. Incubation of the particulate fraction with radioactive fumarate in the presence or absence of N A D H proved that succinate was formed more rapidly and in greater quantities in the presence of the coenzyme (Fig. 13). Additional reactions also occurred which perhaps

NADH

NAD

FUMARATE

SUCCINATE

II

02 [Monlezla expansa)

CYTOCHROME 557

ili~i!i!!i ii:iI~IIg~

iiiiiiiiil, :::::::::: :::::::::: iii!!i!il

I

~-PHENYLENE DIAMINE

'CYTOCHROME a'

FIG. 15. E l e c t r o n t r a n s p o r t in Moniezia expansa: A e r o b i c system.

FLAVOPROTEIN 2

H2 0z

II FLAVOPROTEIN

/ METHYLENE BLUE

PHENAZINE METHOSULPHATE

Jl

O~

:YANIDE INHIBITION

SYSTEM

DISCONTINUITY IN CYTOCHROME

@

,q

t~

THE ELECTRON TRANSPORT SYSTEM OF M O N I E Z I A E X P A N S A

221

provide some insight into the control mechanisms of the respiratory process described above (Table 5, Fig. 14). Figure 16 shows the metabolic pathways which we think probably occur in vivo, deduced from the experiments described in the present paper. Fumarase is present in the particulate preparation, and also, presumably, malic dehydrogenase. Their presence is indicated by the detection of radiocarbon in malate and in aspartate, as the latter can be derived from a transamination reaction involving oxaloacetate. A decarboxylase was also present, as the isotope was found in alanine in large ASPARTATE I

NADi

LACTATE

ATE AiEf l ~

s['OXALO

NADH

!

=l STARTINGMATERIALS

r"

~'1 INTERMEDIATESNOT DETECTED, BUTTHEPRESENCEOF WHICH IS INFERREDFROMDERIVATIVES

m

~:'NADH .NAD

(

ALANINE NAD{PJH

MALATE

!1

[FUMARATEI

REDUCED

FIG. 16. Electron transport in Moniezia expansa: Anaerobic system. quantities, and in lactate. Alanine and lactate were presumably formed from pyruvate, although pyruvate was not detected on the chromatograms. The absence of radiocarbon in oxaloacetate and pyruvate is probably because of their low biological half-life and their volatility during chromatography. Incorporation of radiocarbon into malate was greater in the absence of NADH. This was probably due to the increased conversion of fumarate to malate, with a decreased utilization of malate once it was formed, and the partial block of succinate formation. Evidence for this is found in the smaller amount of radiocarbon in aspartate in the absence of NADH. This also suggests that the decarboxylase involved is a true "malic enzyme". If it were an oxaloacetic decarboxylase it would be expected that the incorporation of radiocarbon into alanine would be greater with NADH. A metabolic cycle thus appears to operate. The NAD formed by the reduction of fumarate is itself reduced by malic dehydrogenase, and is then available for further reaction with fumarate. The "malic enzyme" may also be NAD-dependent,

222

K . S . CHEAHAND C. BRYANT

as in Echinococcus granulosus (Agosin & Repetto, 1965). However, as the incorporation of radiocarbon into alanine and lactate was not greatly affected by N A D H it suggests that either the dehydrogenase has a greater affinity for N A D or the decarboxylase is N A D P - d e p e n d e n t . Lactic dehydrogenase also competes for N A D H , but the low level of radioactivity in lactate suggests that malic dehydrogenase and "fumarate reductase" are closely associated. Cytochrome 557 provides the important link between this N A D cycle and the enzyme responsible for the reduction of fumarate. Thus, the significance of the accumulation of succinate in Moniezia (von Brand, 1933) and of its decarboxylation products in Ascaris (Saz & Weil, 1962) can, perhaps, be explained. During the metabolism of carbohydrates, fumarate is formed by a pathway involving glycolytic reactions to yield pyruvate which is then converted to malate by malic enzyme (cf. Bueding, 1960). During this process N A D H is also formed, which can be reoxidized by an electron transport system involving cytochrome 557 and flavoprotein carriers, with fumarate as the terminal electron acceptor. Thus, one of the major problems of a relatively anaerobic environment is overcome, especially as, by analogy with the mammalian system, this anaerobic electron transport pathway involves at least one phosphorylation step. A similar system is found in some micro-organisms, where the reduction of fumarate to succinate provides a mechanism for energy-yielding oxidations in the absence of oxygen (Peck et al., 1957). Acknowledgements--The authors wish to thank Dr. C. A. Appleby and the Division of Plant Biochemistry, C.S.I.R.O., Canberra, A.C.T. for much valuable help, and access to their Cary spectrophotometer; and the Electron Microscope Unit, John Curtin Medical School, The Australian National University, for permission to use their facilities. REFERENCES AGOSlN M. & REPETTOY. (1955) Studies on the metabolism of Echinococcus granulosus V I I I. The pathway to succinate in E. granulosus scolices. Comp. Biochem. Physiol. 14, 299-309. BARTON M. • SMYTHJ. D. (1965) Personal communication. BRYANTC. (1966) Unpublished data. BRYANT C. 8g NICHOLAS W. L. (1965) Intermediary metabolism in Moniliformis dubius (Acanthocephala). Comp. Biochem. Physiol. 15, 103-112. BRYANTC. ~ NICHOLASW. L. (1966) Studies on the oxidative metabolism of Moniliformis dubius (Acanthocephala). Comp. Biochem. Physiol. 17, 825-840. BRYANTC., NICHOLASW. L. & JANTUNENR. (1966) Some aspects of the respiratory metabolism of Caenorhabditis briggsae (Nematoda). (In preparation.) BRYANT C. ~ WILLIAMSJ. P. C. (1962) Some aspects of the metabolism of the liver fluke, Fasciola hepatica L. Expl Parasitol. 12, 372-376. BJ6RKMAN N. & THORSELLW. (1964) On the fine structure and resorptive function of the cuticle of the liver fluke, Fasciola hepatica L. Expl Cell Res. 33, 319-329. BUEDING E. (1960) Concluding remarks. In Host Influence on Parasite Physiology (Edited by STAUBERL. A.). Rutgers University Press, New Jersey. BUEDING E. & CHARMS B. (1952) Cytochrome-c, cytochrome oxidase and succinoxidase activities of helminths. J. biol. Chem. 196, 615-627. CHANCE B. (1957) Techniques for the assay of the respiratory enzymes. In Methods in Enzymology (Edited by COLOWICK S. P. • KAPLAN N. O.), Vol. 4. Academic Press, New York.

T H E 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 O N I E Z I A E X P A N S A

223

CHANCE B. & PARSONS D. P. (1963) Cytochrome function in relation to inner membrane structure of mitochondria. Science, N . Y . 142, 1176-1180. CHEAH K. S. & BRYANT C. (1966) Unpublished data. FRIEDI-mIM E. A. H. & BAER J. C. (1933) Untersuchungen tiber die Atmung von Diphyllobothrium latum (L.). Biochem. Z. 265, 329-337. GOLDBERG E. & NOLF L. O. (1954) Succinic dehydrogenase activity in the cestode, Hymenolepis nana. Expl Parasitol. 3, 275-284. HEYNEMAN D. & VOGE M. (1960) Succinic dehydrogenase activity in cysticercoids of Hymenolepis, measured by the tetrazolium technique. Expl Parasitol. 9, 14-17. JOHNSON M. J. (1941) Isolation and properties of a pure yeast polypeptidase. ,7. biol. Chem. 137, 575-586. KEILIN D. (1925) On cytochrome, a respiratory pigment common to animals, yeast and higher plants. Proc. R. Soc. B 98, 312-339. KIKtYCH! G. & BAN S. (1961) Cytochromes in the particulate preparation of the Ascaris lumbricoides muscle. Biochim. biophys. Acta 51, 387-389. KIKUCHI G., RAMIREZ J. • GUZMAN BARRON E. S. (1959) Electron transport system in Ascaris hlmbricoides. Biochim. biophys. Acta 36, 335-342. KMETEC E. & BUEOING E. (1961) Succinic and reduced diphosphopyridine nucleotide oxidase systems of Ascaris muscle. J. biol. Chem. 236, 584-591. LASER H. (1944) T h e oxidative metabolism of Ascaris suis. Biochem. J. 38, 333-338. LEHNINGER A. L. (1964) The Mitochondrion. Benjamin, New York. MAEHLY A. C. & CHANCE B. (1954) T h e assay of catalases and peroxidases. In Methods of Biochemical Analysis (Edited by GLICK D.), Vol. 1, pp. 357-424. Interscience, London. MORSETH D. J. (1965) T h e ultrastructure of taeniid tapeworms. Ph.D. thesis, University of Otago, Dunedin, New Zealand. MORTON R. K. (1958) T h e cytochromes. Rev. pure appl. Chem. 8, 161-220. NICHOLAS W. L. & MERCER E. H. (1965) T h e ultrastructure of the tegument of Moniliformis dubius (Acanthocephala). Quart. ft. micr. Sci. 106, 137-145. PECK H. D., SMITH O. H. & GEST H. (1957) Comparative biochemistry of the biological reduction of fumaric acid. Biochim. biophys. Acta 25, 142-147. POTTER V. R. & REIF A. E. (1952) Inhibition of an electron transport component by antimycin A. 7. biol. Chem. 194, 287-297. READ C. P. (1952) Contributions to cestode enzymology--I. T h e cytochrome system and succinic acid dehydrogenase in Hymenolepis diminuta. Expl Parasitol. 1, 353-361. READ C. P. (1961) T h e carbohydrate metabolism of worms. In Comparative Physiology of Carbohydrate Metabolism in Heterothermic Animals (Edited by MARTIN A. W.). University of Washington Press, Seattle. SAz H. J. & WEIL A. (1962) Pathway of formation of a-methyl valerate by Ascaris lumbricoides. ,7. biol. Chem. 237, 2053-2056. SINGER T. P. & KEARNEY E. B. (1956) Determination of succinic dehydrogenase activity. In Methods of Biochemical Analysis (Edited by GLICK D.), Vol. 4, pp. 307-333. SMITH M. J. It. & MOSES V. (1960) Uncoupling reagents and metabolism. Biochem..7. 76, 579-585. STOTZ E. (1955) Cytochrome-b (mammals). In Methods in Enzymology (Edited by COLOWICK S. P. & KAPLAN N. O.), Vol. 4. Academic Press, New York. UMBREIT W. W., BURRIS R. H. & STAUFFERJ. F. (1957) Manometric Techniques. Burgess, Minneapolis, U.S.A. VAN GREMBERGEN G. (1944) T h e respiratory metabolism of the cestode, Moniezia benedeni (Moniez, 1879). Enzymologia 11, 268-281. VON BRAND T. (1933) Untersuchungen fiber den Stoffbestand einiger Cestoden und den Stoffwechsel yon Moniezia expansa. Z. vergl. Physiol. 18, 562-596.