Indian Journal of Biochemistry & Biophysics Vol. 37, April2000, pp. 130-134

Succinate oxidase and fumarate reductase systems of filarial parasite Setaria digitata

L S Unnikrishnan and R Kaleysa Raj*

Department of Biochemistry, University of Kerala, Thiruvananthapuram 695 581 , Indi a

Received 8 September 1998; accepted 29 December 1999

Activities of succi nate oxidase, fumarate reductase (FR) and succinate dehydrogenase (SOH) under a set of defined conditi ons were determined in the mitochondrial isolate from Setaria digitata , the filarial parasite from the cattle Bos indicus. Presence of only two activities namely SOH and succinate- UQ reductase of the succinate oxidase system could be detected in S. digitata. In the absence of cytochromes, the 3rd enzyme of the complex namely cytochrome oxidase is absent and it is proposed that an alternative oxidase is responsible for completing the succinate oxidation expressed as succinate oxidase activity. Though SOH and FR catalyse reverse reactions, they responded differently to modulators such as oxaloacetate; aspartate, alanine, pyruvate and fumarate . The degree of response of the two act ivities against inhibitors of electron transport was also different. Interestingl y fu marate caused only 50% inhibition of succinate oxidation, while the effect against FR was more convincing.

The pathways of energy metabolism in parasites are unique and are modified to adapt to the environmental conditions. The metabolism in filarial parasites is cons idered to be predominantly anaerobic in nature 1

•

Fumarate reductase (FR), catalysing NADH dependent fumarate reduction coupled with ATP is the major enzyme of anaerobic energy exchange in many parasitic he lminths2

-6

. Succinate dehydrogenase (SOH), the enzyme of aerobic energy exchange converts succ inate to fumarate whereas the conversion of fumarate to succinate is mediated by FR. The relative activities of SOH and FR are cri tical in determining the type of energy exchange of

. 78 parasites · . Setaria digitata , a filarial parasite from the cattle

Bos indicus (cow) is reported to be similar to the parasites causing human filariasis with the only difference that it has different terminal hosts . In addition, it is also one of the model systems recommended by the World Health Organisation9

. S. digitata takes up oxygen in presence of cyanide and lacks characteristic cytochrome system. Unlike the host system it has two lower ubiquinones Q6 and Qs and both these are formed in the parasite itself. Though cytochromes are absent, haem and haem containing compounds are formed in the parasite. Mitochondrial isolates from S. digitata have been designated as mjtochondria like particles (MLP) in the

*Author for correspondence

text. The isolates from Setaria did show typical marker activities such as succinate dehydrogenase. However it was thought best to call them MLP because they lacked complete TCA cycle activities and showed many other distinct differences compared to mammalian mitochondriae. The TCA cycle is incomplete due to the absence of isocitrate and a­ketoglutarate dehydrogenases and succinyl CoA synthase activities. Though terminal oxidase is absent in MLP, rotenone and antimycin sensitive as well as insensitive NADH dehydrogenases, SDH and transhydrogenase acttvtttes are present 10

-17

. The present work is a study of succinate oxidase (SO) and FR activities in the MLP in an attempt to show the difference between the two systems.

Materials and Methods S. digitata, located in the peritoneal cavity of the

cattle, were collected immediately after opening the abdomen in modified Tyrode solution, conta,ining NaCl (0.8%), KCl (0.02%), CaC(z (0.02%), MgC(z (0.01%), NaHC03 (0.015%), NazHP04 (0.05%), glucose (0.5%) and pH was adjusted to 7.2 with NaH2P04 . Worms freed from extraneous material were used.

Preparation of mitochondria like particle ( M LP)

The weighed live worms were suspended in 0.25 M sucrose containing 0.1 % bovine serum alburrun and 5 mM EDT A ( 10 ml per g wet weight of worm) and the

UNNIKRISHNAN & RAJ: SUCCINATE OXIDASE AND FUMARATE REDUCTASE SYSTEMS OF S. DIGITATA 131

mixture was homogenized in a Potter Elvehjem tissue homogenizer. The homogenate was centrifuged in a refrigerated centrifuge. The pellet collected at 5000 g was discarded and that collected at 12,000 g was used as MLP.

Enzyme assays Fumarate reductase (FR), succinate dehydrogenase

(SOH) and SOH-coenzyme Q (SDH-Q) reductase acttvtttes were measured by standard

h . h d 18 19 s . "d spectrop otometnc met o s · . uccmate oxt ase activity (SO) was determined by oxygen uptake in presence of succinate in a Gilson Oxygraph using Clark type oxygen electrode having a cell capacity of I A ml20

. Due to the scarcity of filarial samples, assays for the determination of Km was carried out using partially purified preparation.

Antimycin A, 0-hydroxy diphenyl (OHO), 2-theonyl trifluoroacetone (TTFA), salicyl hydroxamic acid (SHAM) and other modulators on enzyme activities were analysed after pre-incubation of the inhibitor/modulator with washed MLP for 5 min. All biochemicals used were purchased from Sigma Chemical Co, USA.

Results The activities of NAOH dependent FR, SOH,

SDH-Q and SO are given in Table I. It is clear that the SO activity is considerably less than that of FR activity . Concentration of different inhibitors required for 50% inhibition of SOH and FR activities is given in Table 2 . Tables 3 and 4 respectively show the effect of different inhibitors on SDH-Q reductase and SO activities . A comparison of the activities of SOH and FR have been made in presence of selected

Table !-Activities of fumarate reductase, succinate dehydrogenase, SDH-Q reductase and succinate oxidase in MLP

of S. digitata [Values are ±SD of 6 different experiments]

Enzymes

Fumarate reductase* Fumarate reductase Succinate dehydrogcnaset SDH-Q reductase** Succinate oxidase:j:

Coenzyme

NADH NADPH

Sp. ac tivi ty

357±3.8 No activity 356±3.26 53±1.3 18±0.63

*Activi ty expressed as n moles of NADH oxidized/minlmg protein . t Activity expressed as n moles of DCIP reduced/min/mg protein. **Activity expressed as flmoles of succinate oxidized/minlmg protein. :j:Activity expressed as n atoms of oxygen consumed/minlmg protein.

modulators and the result is shown in Table 5. Figures I to 3 show the Lineweaver-Burk plot of FR, SOH and SO.

Discussion Conversion of fumarate to succinate mediated by

FR is important in the energy transduction systems of anaerobic organisms and is the terminal step of the

Table 2-Effect of inhibitors on the activities of fumarate reductase and succinate dehydrogenase

[Values are average of 5 different experiments. p<O.OI]

Inhibitor Cone of inhibitor producing 50% inhibition Fumarate Succinate

reductase* dehydrogenaset

Rotenone 15 pM 50mM Antimycin A 0.002 J.1M 1.84 flM OHD 75 f1M ND TTFA 0. 1 J.1M 0.6 flM MPA 0.034 flM No inhibition Sodium malonate 211M llflM

NO-Not detected *Activity expressed as n moles of NADH oxidized/min/mg protein t Activity expressed as n moles of DCIP reduced/min/mg protein

Table 3- Effect of inhibitors on the activity of SDH-Q-reductase [Values are average of 6 different experiments. I ml reaction mixture contained 200 11g MLP protein activity expressed as

11moles of succinate oxidized/minute/mg protein]

Inhibitor Concentration % Inhibition

(11M) (max)

TTFA 0.1 57 OHD 40 93 Ant imycin A 0.36 89

P<O.O l

Table 4-Effect of inhibitors on the activi ty of succinate oxidase [Specific acti vity expressed as n atom of oxygen consumed/min/mg protein. Values are ±SD of 6 different experiments p<O.OI (determined by one tailed test for t distribution). 1.6 ml assay mi xture contained 250 11g mitochondri al protei n]

Inhibitor Concen- Sp. activity % tration Inhibition (11M)

No inhibitor 18±0.63 Nil Sodium malonate 12 9±0.28 50 Sodium fumarate 5 15.7±0.47 13

10 10.57±0.47 13 Sodium malate 6 10.08±0.28 44 Sodium azide 10 15.8±0.44 12 SHAM 0.031 9.72±0.41 46 Antimycin 0.46 9±0.28 50 OHD 50 9.9±0.44 45

132 INDIAN J. BIOCHEM . BIOPHYS., VOL. 37, APRIL 2000

Table 5-Effect of modul ators on succinate dehydrogenase and fumarate reductase activities

[V alues are average of 5 different experiments p<O.O l]

Modulator

C02 OA Aspartate Alan ine Pyruvate Fumarate

Concen-tration (mM)

2.5 1.33 0.67 1.33 1.33 1.33

% % Inh ibition Stimulation

SOH FR

24 12 100 Nil 100 Nil 24 13 24 NO 78 NO

-1.8-1.6 -1.4-1.2 -1 -0.8 -0.6 -0.4-0.2 0 0.2 0.4 0.6 0.8

1/s Fig. 1-Lineweaver-Burk plot for succinate hydrogenase

-1.6 -1 .4 -1.2 -1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0.6 0.8 1

1/s

Fig. 2-Lineweaver-Burk plot for succinate oxidase

. 4 5 21 phosphoenol pyruvate (PEP)-succmate · · pathway. FR activity has been reported in many parasite systems including certai n filarial parasites22

.

Comparati ve study using different inhibitors show differences between SOH and FR, suggesting bas ic differences between the two enzyme activities. Such differences have been recently reported in Nipposrrogyles brassiliensis23

. Mcthoxy phenyl acetic acid. (MPA), a specific inhibitor of FR, does not affect the SOH acti vi ty of S. digitata24

• Though SOH and FR catalyze forward and reverse reactions, it is clear

-0.6 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.2 2.4 1/s

Fig. 3-Lineweavcr-Burk plot for fumarate reductase

from the results that they are reasonably independent of each other and are perhaps distinct enzymes.

Oxygen dependent succinate oxidation is detected in this parasite. This is generally catalysed by the succinate oxidase system, considered to be composed of succinate-ubiquinone and succ inate cytochrome c oxidoreductases and cytochrome c oxidase activities. Such systems are reported to be present in the lung fluke, Paragonimus westermani, intestinal nematode Ascaris lumbricoides and mammal ian ti ssue such as bovine heart25

. However as S. digirata is devoid of cytochromes and the oxygen uptake is cyanide insensitive, the nature and composition has to be definitely different from the systems just mentioned. The comparison of the effect of inhibitors on SO activity differs from that on SOH and SOH-Q reductase activities. The effects of sodium malonate and antimycin A on SOH activity are similar. Inhibition of SO activity by the cytochrome oxidase inhibitor sodium azide however shows the possibility of a low level of typical or modified mamrnaliam type respiration in S. digitata. However the absence of cyctochrome systems in Setaria rules out such a possibi lity. Succinate oxidation should naturally result in the formation of fumarate . However the effect of fumarate on SO is insignificant and hence is interesting. In fact, an increase in concentration above 5 mM does not even affect the oxygen uptake. At the same time malonate did show appreciable inhibition. Similar level of inhibition was also shown by electron transfer complex I inhibitor antimyc in A, complex II inhibitor OHO, SOH inhibitor malonate and alternative oxidase (AOX) inhibitor SHAM26

'27

. Evidence collected so far suggest that the SO system of S. digitata is probably linked to AOX system. (unpublished data).

UNNIKRISHNAN & RAJ : SUCCINATE OXIDASE AND FUMARATE REDUCTASE SYSTEMS OF S. D!G!TATA 133

C02 fixation with PEP results in the formation of oxaloacetate (OA), a competitive inhibitor of SOH, which inhibits succinate oxidation but not FR activity thus favouring C02 fixing direction from PEP to succinate. PEP to succinate pathway is known to be present in S. digitata :tlso 13

• OA caused 100% inhibition of SOH acti vity but no inhibition of FR activity. Similar effect is noted with aspartate amino transferase. Alanine inhibits SOH activity and stimulates FR activity. The stimulation of FR by alanine may be because it is converted to pyruvate by alanine dehydrogenase with the formation of NADH which enhances the FR activity. Pyruvate can be converted to OA by pyruvate carboxylase, which inhibits the SOH activity.

It is clear from Fig. I that SOH is biphasic in appearance and hence drawn to give two Km values. The plot for SO shown in F ig. 2 is also biphasic in nature. The Km for fumarate by FR is shown in Fig. 3. The latter is 2.4 times less than the Km for succinate by SOH . The low Krn fo r succinate by SOH is found to be comparable to the Km for succinate by mammalian SOH suggesting si milar affinity in the formation of the complex. It appears that the low Km for succinate is associated with SOH acti vi ty and that it is this activity which is inhibited on additi on of fumarate. The high Km for succ inate by SOH must be therefore associated with FR activity and the low K 111 for succinate by SOH mu st in al l probability be the one associated with SO acti vi ty. The differenti ation of the two enzyme sys tems cou ld not be achieved by inhib it ion studies on acti vity alone . However the presence of suc h a complex system with a c lass ical SOH activi ty and also showi ng functionally independent FR activity cannot be ruled out.

SOH 0.63 m M, 3.57 m M

Succinate ......._ fumarate

"""""" FR 1.04 m M

It is to be remembered that the filarial parasite is always in constant motion. This motion called wriggling movement must be demanding lot of energy and hence it is only natural to assume that oxygen is indeed used for this purpose. The lymphatics where the adult filarial parasite is located has oxygen all the time. The detailed oxidative metabolism of parasites is being worked out. Even anaerobic metabolism of parasites differs from that of free living invertebrates in that, it is continuous, not transient and persists in the presence of oxygen28

.

The results of the present study suggest the presence of an active FR system in preference to the SO system. The inhibition studies show basic differences between these two enzyme systems. Though we could not identify SOH and FR as two separate enzyme systems in the present work, the data indicate that even in the absence of typical cytochrome systems these enzymes play a major role in the maintenance of both aerobic and anaerobic electron transfer system in S. digitata. Further, it could be shown that the SO system of S. digitata is different from similar systems present in parasites such as A. lumbricoides.

Acknowledgement The authors thank the Department of Science and

Technology, New Delhi for financial assistance, Council of Scientific and Industrial Research, New Delhi for the award of Senior Research Fellowshi p to LSU and The Indian Council of Medical Research for the award of Emeritu s Scientist's posi tion to RKR.

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