Comp. Biochem. Physiol. Vol. 78B, No. 2, pp. 373-378, 1984 0305-0491/84 $3.00 + 0.00 Printed in Great Britain © 1984 Pergamon Press Ltd

SUBSTRATES RESPIRED BY MITOCHONDRIAL FRACTIONS OF TWO ISOLATES OF THE NEMATODE

A P H E L E N C H U S A VENAE AND THE EFFECTS OF ELECTRON TRANSPORT INHIBITORS

A. H. W. MENDIS* and A. A. F. EVANS Imperial College, Silwood Park, Sunninghill, Ascot, Berks SL5 7DE, England, UK

(Received 4 November 1983)

Abstract--1. Electron transport has been assayed and compared in two isolates (M and F) of the free-living (model) nematode Aphelenchus avenae.

2. Of the substrates tested only ct-glycerophosphate and succinate were utilised to any significant extent by both isolates.

3. Comparative data on respiratory rates, respiratory control ratios and ADP:O ratios for various substrates are given.

4. Succinate oxidation by isolate-F mitochondria was ca 80-90% sensitive to antimycin A while that of isolate M was almost completely refractory to antimycin A.

5. The response to other electron transport inhibitors suggests the operation of (a) azide/cyanide sensitive, (b) azide/salicylhydroxamic acid (SHAM) insensitive but carbon monoxide sensitive and (c) SHAM-sensitive terminal oxidases to varying degrees in the mitochondria of these two isolates of A. avenae.

INTRODUCTION

The oxidative metabolism of mitochondria from free living nematodes has not been extensively studied. A particulate fraction of C. briggsae (prepared by sonic disruption) converted [~4C]succinate to fumarate and malate but the cytochromes in this preparation were not reduced by ~-glycerophosphate or succinate even in presence of antimycin A, cyanide or carbon monoxide (Bryant et al., 1976). Ells and Read (1961) found little evidence for operation of a tricarboxylic acid (TCA)-cycle in T. aceti, but the conversion of [~4C]acetate to TCA cycle-related amino acids and glycerol in another T. aceti preparation (Rothstein, 1963) demonstrated the occurrence of the TCA cycle in this species. The ability of P. redivivus and C. briggsae to convert acetate to glycerol was later demonstrated (Rothstein, 1969) suggesting the pres- ence of an active TCA cycle in these two species also.

It was shown subsequently that mitochondrial preparations of T. aceti readily oxidised succinate, glutamate, pyruvate, fl-hydroxybutyrate and ct-keto- glutarate, with respiratory control ratios between 2 and 6.2 (Rothstein et al., 1970). Succinate-induced state 3 respiration of these T. aceti preparations were completely inhibited by 111/zM cyanide (NaCN) while 74/~M NaCN caused only partial inhibition. Exogenous cytochrome c (cyt c) and 2,4-dinitro- phenol (2,4-DNP) stimulated the succinate oxidation (Rothstein et al., 1970) but the effects of a-glycero-

*Present address: The Wellcome Research Laboratories, Langley Court, Beckenham, Kent BR3 3BS, England, UK (Tel: 01-658-2211).

phosphate as substrate or antimycin A as inhibitor were not reported.

Murfitt et al. (1976) prepared mitochondria from C. elegans and reported that the respiratory chain of this species was similar to the classical mammalian system. The ADP:O ratios were consistent with those reported for mammalian systems; and essentially complete inhibition of state 3 respiration rates were obtained with cyanide, azide, antimycin A and rotenone. The respiratory rates of the preparation achieved with succinate, glutamate/malate and fl-hydroxybutyrate agreed with those reported for T. aceti (Rothstein et al., 1970) although they differed with respect to pyruvate, ~-ketoglutarate and ct-glycerophosphate (Murfitt et al., 1976).

Aphelenchus avenae has many properties desirable in a laboratory model (Evans and Womersley, 1980) and since preliminary studies showed the existence of differential responses of the QO2 of whole nematode suspensions of different isolates of A. avenae to cya- nide, azide, and the nematicide ethylene di-bromide (Mendis and Evans, 1984b,c) the mitochondrial respiratory metabolism of 2 of these isolates were further investigated.

Isolation procedures for mitochondria from free- living nematodes reported previously differed significantly (Rothstein et al., 1970; Murfitt et al., 1976), so a modified isolation procedure was devel- oped to study the oxidative capacities of the mito- chondrial fractions of two A. avenae isolates (desig- nated M and F; Mendis and Evans, 1984a,b) with reference to respiratory control ratios (RCRs), ADP:O ratios, sensitivity to respiratory electron transport inhibitors and the respiratory uncoupler 2,4-DNP.

373

374 A . H . W . MENDIS and A. A. F. EVANS

M A T E R I A L S A N D M E T H O D S

Isolation medium .[br mitochondria

The isolation medium consisted of 225 m M mannitol, 7 5 m M sucrose, 0 . 2 m M EGTA and 16mM Tris -HCl (pH 7.0) with l mg/ml bovine serum albumin.

Preparation o f mitochondrial J?actions

Nematodes from each isolate were harvested from 30 to 40 day old mass cultures (Evans, 1970), extracted as de- scribed earlier (Mendis and Evans, 1984a) and rinsed twice with sterile distilled water. The washed nematode pellet (10-15g wet wt) was placed in a sterile, chilled (4"C) mortar. A volume of pre-chilled, acid-washed sand, equal in volume to the nematode pellet, and 7 ml of chilled isolation medium were added and the mixture was ground by hand for two 5-min periods using a prechilled pestle at a temp of 1 2~C.

The resulting homogenate , diluted in 15 ml of isolation medium was collected in pre-chilled centrifugation tubes. The remaining nematode/sand mixture was homogenised for a further 2 minutes and the supernatant collected and pooled with the first. The pooled homogenate was centri- fuged at 380g for 7 min and further purified according to the procedure in Fig. 1. All centrifugations were carried out in an MSE high-speed refrigerated centrifuge at 1-2' C. The final pellet was resuspended in 1.5-2.0mi of isolation medium and used for all polarographic assays.

Respirator)' assays

A closed Clark-type oxygen electrode (Rank Bros) was used for all polarographic assays (details in Mendis and Evans, 1984a). The reaction medium (RM) used contained 5 0 m M Tris, 12mM NaH2PO4, 2 2 5 m M sucrose, 1 .0mM EGTA, 10mM KC1 and 5 m M MgCI 2 adjusted to pH 7.4 with half strength HCI. A vol of 1.8 ml of RM equilibrated to 25"C and 0.2 ml of the mitochondrial preparation (usu- ally containing 0.5-0.6 mg protein) was used throughout the polarographic studies. A saturated oxygen concentration of 2 4 0 # M was employed in all calculations, since 2 ml of air-saturated reaction medium (at 25°C) contained 960.0 a toms of dissolved oxygen. All assays were carried out at 25°C.

Protein estimation

The mitochondrial protein concentration was determined using the method of Lowry et al. (1951). Since the isolation medium employed contained mannitol which interferes with the assay, the method was modified as described by Murfitt et al. (1976). The sample protein was precipitated with 5!'<i TCA centrifuged (500g, 7 min), the supernatant discarded, and the pellet was redissolved in 0.1 M NaOH. This effectively removed the mannitol and the final mixture was neutralised with 0.1 M HC1 before the Lowry protein assay was made.

RESULTS

Resp i ra tory control ratios ( R C R s )

T h e e n d o g e n o u s r e sp i r a to ry ra te (QO2) was r eco rded for 2 - 3 m i n be fore s u b s t r a t e s were a d d e d , (all to a final c o n c h o f 5 m M un l e s s o the rw i se m e n - t ioned) , the first s t a t e 4 QOz was o b t a i n e d , fo l lowing w h i c h 150/2 M o f A D P was a d d e d to o b t a i n the s ta te 3QO2. R C R va lue s were ca l cu l a t ed for the s e c o n d s ta te 4 to s t a te 3 t r a n s i t i o n fo l lowing a s econd a d d i t i o n o f 1 5 0 / 2 M A D P , i.e. a f te r the first cycle o f s t a te 3 o x i d a t i o n . All a s s a y s were ca r r i ed o u t wi th in 2 - 3 h r o f p r e p a r a t i o n o f the m i t o c h o n d r i a .

T h e r e sp i r a t i on ra tes o f m i t o c h o n d r i a l f r a c t i ons o f b o t h i so la tes ( M a n d F) in the p r e sence o f v a r i o u s s u b s t r a t e s is g iven in T a b l e 1; the e n d o g e n o u s ra te was s imi la r in each isolate . T h e a d d i t i o n o f N A D H in s u b s t r a t e a m o u n t s resu l t ed in little o r no inc rease in the Q O 2 ove r the e n d o g e n o u s ra te i nd i ca t i ng g o o d m i t o c h o n d r i a l in tegr i ty . F u r t h e r o b s e r v a t i o n s were on ly m a d e on p r e p a r a t i o n s like these .

Succ ina t e a n d ~ - g l y c e r o p h o s p h a t e (c~-GP) were the T C A - c y c l e i n t e r m e d i a t e s m o s t read i ly ox id i sed by b o t h i so la tes u n d e r g o o d r e s p i r a t o r y c o n t r o l (i.e. e x h i b i t i n g c lear s t a te 3 to s t a te 4 t r ans i t i ons ) . T h e s ta te 3 ra te o f i so la te M p r e p a r a t i o n s wi th s u c c i n a t e was s ign i f ican t ly h ighe r t h a n t hose o b s e r v e d wi th

Table 1. Oxidation of substrates by mitochondrial fractions of A. avenae isolates F and M. For succinate and ~-GP the results are the means of 12 14 assays; 3 replicates each from at least 4

different batches

0 2 utilisation (natoms/min/mg protein) Respiratory

Substrate With added control alone ADP ratio ADP:O

Substrate (State 4) (State 3) (RCR)* ratio*

Isolate F Succinate 85 -+ 8.1" "194 -+ 27.4 '~ 2.3 + 0.3 h [.9 ± 0 I'; Glutamate/malate 29 + 8.0 31 + 7.2 /3-Hydroxybutyrate 26 + 1.8 34 + 5.8 (1.3 + 0.2) Pyruvate 29 + 5.0 36 _+ 5.3 (I.3 + 0.2) NADH 26 + 4.8 Unaltered ~-Glycerophosphate 67_+7.1' 142_+ 13.8 t 2.1 _+(I.2' 2.2_+0.1 Endogenous 23.1 _+ 5.3 Unaltered Isolate M Suceinate 119 + 4.4" *409 ± 25.9'; 3.4 -+ 0.2 ~ 1.4 -+ 0. I" Glutamate/malate 30 -+ 4.8 33 _+ 5.9 ( 1.1 _+ 0.1) /~-Hydroxybutyrate 26 _+ 1.3 27 + 1.6 Pyruvate 26 _+ 3.4 34 _+ 3.8 (I .3 _+ 0.2) NADH 26 _+ 3.7 Unaltered ~-Glycerophosphate 118_+ 10.9" 310 40.1 ~ 2.6 0.2" 2.2 t 0.1 Endogenous 24.3 _+ 3. I Unaltered

Means + S.D. of a minimum of 6 assays from 4 different batches: means with superscript "e" differ significantly from the endogenous rate at P < 0.05.

Superscripts a d and f: between isolates the means with the same superscripts differ significantly at P < 0.01.

*The RCRs and ADP:O ratios were obtained with reference to the second state 4/state 3 cycle. RCRs in parentheses are (QO: in presence/QO 2 in absence) of ADP due to lack of any observable

state 3 state 4 transition.

Electron transport in free-living nematode Aphelenchus avenae

PROCEDURE FOR THE ISOLATION OF A.avenae MITOCHONDRIA

375

I Supernatant

r Supernatant

Supernatant

Supernatant

I [POO'EOSOPERNATANTSJ

12,000g/7mins. 1 - 2°C

Supernatant Pellet

10 -- 15g wet wt. nematodes w/w pre-chilled land (BDH) Homoglnise 2 x 5 mini. in 7ml IM. 0 o _ 4Oc Homogenate diluted with 15ml IM.

~pernatant

380g/7mins. 1 - 2°c

3ml IM. Homogenised for 2 mira.

Diluted Homogenate

I i

Residue ~ Discard

I Pellet Resuspend in IM (7ml)

I Dounce-type Homogeniser 10 - 12 passes

I 380g/7mins. .1 - 2°C

I Pellet Resuspend in IM (7ml)

L Dounce-type Homogeniser 10 - 12 passes)

] 38~/7mins. 1 - 2°C

I Pellet ~ Discard

[with cuticular fragments and ruptured mitochondria]

Discard

Supernatant 4 - Discard

Resuspend in IM (8ml) with "cold-finger"

12,0009/7mins. 2°C

I P ,,:E-fl I-us ,or 1 Resuspended in . . . . ~ /Respi ra tory / 1.5ml IM. L Assays _1

Fig. 1.

isolate F (P < 0.05) as were the RCRs. However, the A D P : O ratio for isolate F utilising succinate (1.9 ___ 0.1) was significantly higher than that observed for isolate M (1.4 ___ 0.1).

The state 3 rate of isolate M mitochondria in the presence of ~-GP was approximately double that observed with isolate F preparations and the RCRs for the 2 isolates (RCR(n= 2.1 +0.2; RCR(M)= 2.6__+0.2) were significantly different (P <0.05). However, the ADP:O ratios for the two isolates were similar for ~t-GP.

Although the state 4 QO2 of isolate F utilising ~-GP as substrate (67 _ 7.1 natoms/min/mg protein) was significantly lower than the state 4 QO 2 observed with succinate (85 ___ 8.1 natoms/min/mg protein) the RCRs for the two substrates were similar, but the

ADP:O ratio with ~-GP as substrate was higher than that with succinate (see Table 1).

In the case of isolate M mitochondria both ~-GP and succinate showed similar state 4 rates, although the RCR for succinate (3.4 + 0.2) was significantly higher than the RCR for ~t-GP (2 .6_ 0.2). The ADP: O ratio for ~t-GP was significantly higher than that for succinate (Table 1).

Effect of inhibitors With ct-GP as substrate and an excess of ADP

(final concn 3 mM) a steady state 3 rate was achieved and 2.0 mM rotenone (in 1.3 v/v e thanol :RM) was added. Succinate (5 mM) was then added followed by antimycin A (2 #g/mg protein) and the QO2 recorded. Finally, ascorbate (5 mM) and TMPD (0.5 raM) were

376 A. H. W. MENDIS and A. A. F. EVANS

added to obtain the ascorbate/TMPD oxidase activ- ity followed by NaCN (1 raM) or azide (3 mM) to assess cytochrome aa~-mediated oxidation. When incomplete inhibition was observed, 3 mM salicyl- hydroxamic acid (SHAM) and/or 890 nmoles carbon monoxide (CO) was added.

The CO was prepared by saturating 1 ml of dis- tilled water with pure CO at 25'~C. The amount of dissolved CO was calculated using the Bunsen coefficient (25~tI of this CO solution contained 890 nmoles CO), and was injected into the reaction chamber via the capillary port in the usual way.

The results of the respiratory inhibitor studies observed with mitochondria from isolates M and F are given in Table 2.

Effect ofrotenone. 2 rnM rotenone (a concentration much greater than that required to inhibit mam- malian state 3 QO2 inhibited at less than half the state 3 rate of both isolates when utilising ~-GP. The rotenone inhibition was completely bypassed by 5raM succinate in both isolates. Controls were unaffected by ethanol which was the solvent for rotenone.

Antimycin A. The succinate dependent state 3 QO2 of isolate M preparations were little affected by high

concentrations (2 #g/mg protein) of antimycin A (in ethanol) and a mean inhibition of 7.8~ was observed. This was in marked contrast to a mean inhibition of 87.8°/ of the succinate dependent state 3 QO2 of isolate F with identical concentrations of the inhibitor (Table 2). These differences were highly significant (P < 0.01). Again ethanol (5 #1) alone produced no effects on controls.

Effect of NaCN. The state 3 QO2 inhibited by antimycin A was completely relieved by ascorbate/ TMPD in both isolates but the ascorbate/TMPD oxidase activity of isolate M preparations were only partially sensitive to 1 mM NaCN showing a mean inhibition of about 63~, (Table 2). This differed significantly (P < 0.01) from the 88% inhibition of isolate F preparations.

Effect of SHAM and CO on the residual respiration. The NaCN insensitive mitochondrial QO2 of isolate M obtained with added 7-GP was 21~ inhibited by 3mM SHAM (Table 2) but NaCN and SHAM together produced 71~, inhibition of ascorbate/ TMPD oxidation (Table 3). Carbon monoxide (890 nmoles) inhibited more than 80~ of the remaining (NaCN/SHAM insensitive) respiration of isolate M. In the presence of all three inhibitors the respiration

Table 2. The effect of inhibitors on the mitochondrial respiration of A. avenae isolates M and F

Substrate or Inhibitor and Number of Inhibition (%) electron donor concentration assays Mean Range

Z~olate M (A) :~-GP (state 3) (B) Rotenone (2.0 mM) 10 38.6 34.0-43.7 (C) Succinate (D) Antimycin A 12 7.82 0-19.0

(state 3) (2 #g/mg protein) (E) Ascorbate/TMPD (F) NaCN (1 raM) 12 63.0 b 60.4~66.0 ~-GP* SHAM (3 mM) 8 21.2 18.9-24.2 Ls'olate F (A) ~-GP (B) Rotenone (2.0 mM) I 1 39.4 25.2-44.6 (C) Succinate (D) Antimycin A 9 87.8" 85.6-90.6

(2/Lg/mg protein) (E) Ascorbate/TMPD (F) NaCN (1 mM) 11 88.6 b 88.7 91.3 7-GP* SHAM (3 mM) 10 21.8 18.9 25.1

Percentage figures angularly transformed for statistical analysis. Means with same superscripts differ significantly at P = 0.05 according to Student's ' t '-test (two-tailed). (A)-(F) represents the sequence in which ~-GP, rotenone, succinate, antimycin A, ascorbate/TMPD, NaCN

were added to each replicate assay. *The response to SHAM was assessed in the absence of all other inhibitors with only ~-GP as substrate.

Table 3. The inhibition of ascorbate/TMPD induced QO~ in mitochondrial fractions of A. avenae isolates M and F by NaCN, SHAM and carbon monoxide (CO)

Inhibitor(s)

Isolate M NaCN (3 mM)* NaCN (3 mM)* + SHAM (3 mM)

NaCN (3 mM)* + SHAM (3 mM)t CO (890 nmoles)

Isolate F NaCN (3 mM) NaCN (3 mM) + CO (890 nmoles)

% Inhibition % Inhibition range mean (n = 12)

60.,~ 65.9 63.0 ~ 62.1 73.7 71.0 SHAM inhibited

23.5~ o of cyanide insensitive QO2

93.0-95.9 CO inhibited 80.4~ of NaCN/SHAM

insensitive QO~

88.6-91.3 88.6" 91.5 98.4 93.0 CO inhibited

37.7% of NaCN insensitive

QO2

Percentage figures angularly transformed for statistical analysis. Means with same superscript differ significantly at P = 0.05, according to Student's ~t'-test

(one tailed). *3 mM cyanide was used to conveniently differentiate between the cyanide sensitivity of the

2 isolates. Usually 1 mM NaCN completely inhibits Complex IV, i.e. cyt aa 3 terminal oxidation in most mitochondria.

Electron transport in free-living nematode Aphelenchus avenae

Table 4. The inhibition of ct-glycerophosphate dependent state 3 QO 2 of mito- chondrial fractions of A. avenae isolates M and F by SHAM and sodium azide

Mean % inhibition of % Inhibition % Inhibition SHAM insensitive

Inhibitor(s) range mean QO2 by azide

Isolate M (n = 8) SHAM (3 mM) 18.9-24.2 21.2 SHAM (3 mM) + 67.7-76.5 73.1 a 65.9 b azide (3 mM) (60.0-69.4) Isolate F (n = 19) SHAM (3 mM) 18.9-25.1 21.8 SHAM (3 mM) + 83.0-95.3 87.9" 84.5 b azide (3 mM) (74.4-88.3)

Percentage figures angularly transformed for statistical analysis. Means with same superscript differ significantly at P = 0.05 according to Student's

"F-test (one tailed).

377

of isolate M mitochondria was almost completed inhibited (94~). The ascorbate/TMPD oxidase activ- ity of isolate F which was 88~o inhibited by I mM NaCN was further repressed by the addition of 890 nmoles CO and almost complete inhibition (93~) was observed. In isolate F the CN- and CO combination produced the same degree of inhibition which was only produced by the presence of SHAM/CN-/CO in the case of isolate M.

The effect o f S H A M and azide on ~t-GP dependent respiration

The effect of SHAM and azide were recorded in the absence of other inhibitors. With 5 mM ct-GP and excess ADP (3mM) SHAM produced 18-25~ inhibition of the state 3 respiration of both isolates (Table 4). Addition of 3 mM sodium azide brought about 66~ further inhibition of the SHAM- insensitive respiration of isolate M preparations and 85~o inhibition of the SHAM-insensitive respiration of isolate F. SHAM and azide together inhibited 73~ of the state 3 ct-GP dependent QO2 of isolate M and approximately 88~ of isolate F (Table 4).

Effect o f 2 ,4-DNP

A steady, state 4 rate was obtained with 5 mM ct-GP; addition of 50/~ M 2,4-DNP increased oxygen consumption by 182~o in isolate F and by 151~o in isolate M preparations.

Effects o f exogenous cyt c

Added mammalian ferrocytochrome c (10/~M) in the absence of added ADP or inhibitors had no apparent effect on the state 4 QO2 of either isolate induced by 5 mM ~-GP. However in the presence of ascorbate (5 mM) and TMPD (0.05 mM), exogenous cyt c increased the QO2 of isolate F preparations by 77~ whereas that of isolate M was increased by 44~o.

DISCUSSION

Isolation of mitochondria from free-living nema- todes has proven difficult, largely due to their tough cuticle, and procedures used for C. elegans by Murfitt et al. (1976) differed significantly from those of Rothstein et al. (1970) with T. aceti. A modification incorporating elements of both these methods was used in the present study to isolate mitochondria from A. avenae except that Nagarse (proteinase) was omitted to avoid any interference with cyanide sensi-

tivity as was found by Weinbach and von Brand (1970). That the method produced intact mito- chondria is suggested by the high activity of the preparations when respiring the two most favoured substrates, succinate and ct-GP. The RCR of mito- chondria from isolate F respiring succinate (2.3 + 0.2) agrees well with those of T. aceti (1.9+2.2 in Rothstein et al., t970). Although RCRs for isolate M were higher still (3.4 + 0.2) they were comparable to C. elegans (3.6 in Murfitt et al., 1976). The ADP:O ratios were also significantly different between iso- lates and higher than observed in C. elegans. With ct-GP as substrate, isolates again differed in RCR and were higher than in C. elegans; T. aceti was unable to use ct-GP (Rothstein et al., 1970).

Very clear state 3-state 4 transitions were observed with ~-GP, giving ADP:O ratios greater than 2.0 (significantly greater than with succinate, P < 0.05). A similar high state 4 rate with ~t-GP was reported for T. taeniaeformis mitochondria which Weinbach and von Brand (1970) attributed to the action of a mitochondrial ~-glycerophosphate oxidase (ct-GPO).

Neither A. avenae isolate showed high respiration with pyruvate, fl-hydroxybutyrate or glutamate/ malate but it appears that real metabolic differences between the isolates do exist and that they differ from other nematode species (despite the differences in preparation techniques).

Further evidence for the integrity of the mito- chondria comes from the use of cyt c and 2,4-DNP. The failure of added cyt c to stimulate succinate or ~-GP respiration suggests that no endogenous cyt c had been lost through membrane damage. However, ascorbate/TMPD respiration in A. avanae was increased with added cyt c as in T. aceti which Rothstein et al. (1970) interpreted as evidence for an active cytochrome oxidase.

The response to inhibitors provides further evi- dence of difference between the isolates. The rotenone inhibition of both isolates was almost completely relieved by succinate but isolate M was much less inhibited by antimycin A, even at 2 pg/mg protein, a concentration five times the amount required for complete inhibition of the classical mammalian sys- tem (Cheah, 1976) which produced 85~ inhibition in isolate F. This suggests that while much of the electron transport in isolate F reaches cytochrome oxidase, the antimycin-insensitive respiration shown by isolate M is on an alternative pathway having a separate terminal oxidase, possibly cyt o (Mendis and

378 A . H . W . MENDIS and A. A. F. EVANS

Evans, 1980, 1984a), and that A. avenae may thus have a branched-chain electron transport system containing varying amounts o fcy t aa3 and cyt o; such pathways are widely distributed among helminths (von Brand, 1979), Further differences between the isolates were evident in response to ascorbate where l mM cyanide inhibited 8~°'~/o of the respiration of isolate F but only 62% of isolate M, results consistent with the existence of a terminal oxidase relatively less sensitive to cyanide such as cyt o (Lemberg and Barrett, 1973; Bowman and Flynn, 1976). Evidence from carbon monoxide inhibition is incomplete with- out photo dissociation data to separate its effects of cyt aa3 from those of alternative CO-binding oxidases.

The percentage inhibition of ~ -GP respiration due to S H A M was identical in both isolates of A. avenae at only 21% indicating that either a S H A M inhibited ~-GP oxidase could not account for all the respira- tion of : t-GP or that it was only partially sensitive to SHAM. Although ~-GP oxidase was claimed to be specifically inhibited by S H A M (Hill, 1976), this has been questioned (see Palmer, 1982). Azide inhibition of SHAM-insensit ive respiration with : t-GP is further evidence for higher amounts of cyt aa3 in isolate F.

From the evidence presented it may be concluded that several oxidases operate in both isolates of A. aL'enae to varying degrees; (a) an azide and cyanide sensitive oxidase, (b) an azide- and SHAM-insensit ive but CO-sensitive oxidase, and (c) a SHAM-sensi t ive oxidase, which may respectively be equated with a cytochrome oxidase, a cyt o type oxidase and an ~-GPO. Of these the cyt o-mediated terminal oxidation results in the production of hydrogen peroxide (Cheah, 1968, 1972, 1976) and a high cata- lase activity (which would be necessary to break down this toxic product) has been observed in several isolates of A. avenae (Evans, 1971; Mendis and Evans, unpublished). Further work is required to link the catalase activity with production of hydrogen peroxide by the putative cyt o-type terminal oxidase but since this pathway probably also exists in para- sitic helminths such studies offer possibilities for the development of selective chemotherapy in parasitic infections.

Acknowledgements This work ibrmed part of a thesis submitted by A. H. W. Mendis for the degree of Doctor of Philosophy, University of London, Imperial College, and was generously supported by financial assistance from his family.

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