Activities of kreb's cycle enzymes in the flight muscles of the tsetse fly (Glossina) and the fleshfly (Sarcophaga)

Insect Bhwhem.. Vol. 9, pp. 85 to 87. Perganum Press Ltd. 1979. Prhlt,'d in Great Britain

ACTIVITIES OF KREB'S CYCLE ENZYMES IN THE FLIGHT MUSCLES OF THE TSETSE FLY (GLOSSINA) A N D THE FLESHFLY (SA R COPHA GA) DAPHNE A. NORDEN and COLUMBUS MATANGANYIDZE Department of Biochemistry, University of Rhodesia, P.O. Box MP 167, Mount Pleasant, Salisbury, Rhodesia (Received 28 June 1978)

Al~tract--The specific activities of enzymes of the tricarboxylic acid cycle have been measured in flight muscle mitochondria of tsetse flies and fleshflies. Similar levels of those enzymes which are involved in the metabolism of both carbohydrate and proline (2-oxoglutarate dehydrogenase, succinate dehydrogenase and fumarase) were found in the mitochondria of the two insects. Enzymes of the cycle not involved in the oxidation of proline (citrata synthase, aconitase, isocitrate dehydrogenase and malate dehydrogenase) had considerably lower specific activities in the tsetse fly. Key Word Index: Enzymes, tricarboxyclic acid cycle, flight muscle mitochonoria, Glossina mortisans. Sarcophaga nodosa

dehydrogenase (NAD*) was assayed by the method described by ALP et al. (1976) with antimycin A replaced by lmM potassium cyanide. Oxoglutarate dehydrogenase (EC 1.2.4.2) was assayed by the method of SANADI and LITTLEEIELD(1951): the assay mixture contained 6.7 mM 2oxoglutarate, 2.5 mM thiamine pyrophosphate, 3.5 mM MgCl 2, 1 mM potassium cyanide, 1.3 mM 2, 6dichlorophenol-indophenol and 50 mM potassium phosphate buffer, pH 6.9. Succinate dehydrogenase (EC 1.3.99.1 ) was assayed by the method described by ARRIGONEand SINGER (1962) using an assay mixture containing 33 mM succinate, 30 FtM 2, 6-dichlorophenol-indophenol, I.l mM phenazine methosulphate, 1 mM potassium cyanide and 50 mM Tris-HC1 buffer, pH 7.8, the enzyme was activated by 15 min,incubation with substrate at 25 C and the reaction started by addition of the phenazine methosulphatedichlorophenol-indophenol mixture. The assay system for fumarase (fumarate hydratase, EC 4.2.1.2) described by HILL and BRADSHAW(1969), contained 50 mM malate and 50 mM potassium phosphate buffer, pH 7.9. Malate dehydrogenase (EC 1.1.1.37) was assayed with oxaloacetate as substrate using a mixture containing 33 #M oxaloacetate, 130 /zM NADH, 8.3 nM rotenone, 50 mM Tris-HCl buffer containing 20 mM phosphate in the case of tsetse flies and 40 mM phosphate for fleshflies, pH 7.5. With malate as substrate the system contained 20 mM malate, 2 m M NAD +, l mM EDTA, l mM potassium cyanide and 50 mM glycine buffer, pH 10.0. 'Malic" enzyme [L-malate-NAD + oxidore--reductase (decarboxylating) EC 1.1.1.39] was assayed as described previously (NORDEN and MATANGANYIDZE,1977). All assays were carried out at 25 C, Mitochondrial protein was determined by the method of LOwRy et al. (1951).

INTRODUCTION IN CONTRAST to other Diptera which utilize carbohydrate as the principal substrate for mitochondrial oxidations, in the case o f the tsetse fly the oxidation of proline has been shown to provide energy for flight (BuRSELLand SLACK, 1976). Proline oxidation requires some enzymes of the Kreb's tricarboxylic acid cycle but not all. However in the fleshfly (another Diptera), all the enzymes of the cycle are involved in the oxidation o f carbohydrate. The present study compares the activities of the enzymes of the cycle in the flight muscle of these two insects and considers the possibility that the differences found might reflect differences in the metabolic pathways of the two insects. MATERIALS AND METHODS Tsetse flies (Glossina morsitans Westwood) and fleshflies (Sarcophaga nodosa Engels) were obtained from laboratory colonies. Both sexes were used. Substrates were obtained from Sigma Chemical Co. and all chemicals used were A.R. grade. Mitochondria were isolated by the method of CHAPPELand HANSFORD (1972), and exposed to three 15 sec bursts of sonication using a soniprobe (Dawe Instruments Ltd. England) tuned at approximately 21 KHz, during which time the mitochondrial suspensions were cooled in ice. Mitochondria to be assayed for isocitrate dehydrogenase (EC 1.1.1.41) activity were suspended in a solution similar to the extraction medium described by ALP et al. (1976) except that 50 mM Hepes (N-2-hydroxyethylpiperazine-N-2~-ethane sulphonic acid) replaced Pipes (piperazine--NN~-bis-ethane sulphonic acid) buffer. Citrate synthase (EC 4. 1.3.7) was assayed by the method of SrU~RE (1969). For the assay of aconitate hydratase (EC 4.2.1.3) the sonicated mitochondria were first incubated with mercaptosuccinic acid and ammonium ferrous sulphate to activate the enzyme, as described by FANSLER and LOWEt~STEIN(1969) and the enzyme activity was determined at pH 7.8 by the method of ANF1NSEN(1955). Isocitrate I.B, 9 . 1 - - 1 :

RESULTS The specific activities of the enzymes assayed are shown in Table 1. As far as possible conditions o f assay (pH, concentration o f substrate, activators, etc.) giving maximum velocities were chosen. Since it was not feasible to discover these conditions in the case o f . citrate synthase because of the high cost of the

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DAPHNEA. NORDENANDCOLUMBUSMATANGANYIDZE Table I. Specific activities (itmole rain- ~mg protein - ~) of mitochondrial enzymes from the flight muscles of tsetse flies and fleshflies Enzyme

Tsetse f l i e s

F l e s h f l i e s Ratio A/B

(A)

(B)

2-Oxyglutarate dehydrogenase Succinate dehydrogenase Fumarase

0.012±0.003 0.93±0.36 2.05±0.27

0.013±0.002 0.80±0.26 1.98±0.22

Citrate synthase Aconitase Isocitrate dehydrogenase Malate dehydrogenase (Oxaloacetate substrate) Malate dehydrogenase (Malate substrate)

0.92±0.19 0.33±0.06 0.~5±0.015 1.04±0.29 1.9±0.5

7.7±3.3 1.2±0.1 0.38±0.06. 6.9±1.9 20.9±7.2

0.1 0.2 0.1 0.1 0.09

0.18+0.06

0.03+0.003

6.0

Malic enzyme

0.9 1.1 1.0

Enzyme activities were determined as described in Materials and Methods. Activities are given as the mean + the standard deviation of twelve determinations. substrate, the conditions already described for the pig heart enzyme were employed (SRERE, 1969). Succinate dehydrogenase activity was found to be slightly higher in Tris buffer than in the phosphate buffer described bu ARRIGONI and SINGER (1962). As in the case of malate dehydrogenase from pig heart, at pH 7.5 oxaloacetate itself inhibited the reaction of the enzyme from both insects; a maximal rate of reduction of oxaloacetate was obtained with 33/~M substrate. The activity of malate dehydrogenase at pH 7.5 was increased by adding inorganic phosphate, maximum velocities being obtained in the presence of 20 mM and 40 mM phosphate with the tsetse and fleshfly enzymes respectively. On comparison of the activities in the two insects, it can be seen that there is one group of enzymes present at very similar levels in both insects, a second group of enzymes have considerably lower activity in the tsetse fly and malic enzyme, the only representative of a third group, is much more active in the tsetse fly than in the fleshfly. DISCUSSION Tsetse flies feed solely on blood and the use of the blood meal has been outlined by BURSELLet al. (1974). After meeting requirements for growth and reproduction, lipid reserves are synthesized from the amino acids resulting from hydrolysis of the proteins which are quantitatively by far the most important constituents of the diet. Proline plays the central role in energy provision when the fly is at rest as well as during flight and is readily oxidized by the mitochondria, whereas the capacity for pyruvate oxidation is very low. Fleshfly flight muscle metabolizes carbohydrate to provide energy and the pyruvate produced by glycolysis is oxidised by the mitochondrial enzymes of the tricarboxylic acid cycle. Proline, on the other hand, is oxidized by tsetse fly flight muscle mitochondria to glutamate which, through an amino-transferase reaction with pyruvate, is converted to 2-oxoglutarate and finally to malate by the action of enzymes of one section only of the tricarboxylic acid cycle (BURSELLe t al., 1974). The pyruvate required as substrate for the aminotransferase reaction is regenerated from malate

by an oxidative decarboxylation catalysed by malic enzyme, also localized in the mitochondria (NORDEN and MATANGANVIDZE, 1977). Thus the products of proline metabolism enter the tricarboxylic acid cycle as 2-oxoglutarate and leave the cycle as malate. From the results in Table 1 it can be seen that the enzymes of the cycle (2-oxoglutarate dehydrogenase, succinate dehydrogenase and fumarase).which are part of the common pathway of metabolism in tsetse flies and fleshflies, have similar activities in both insects. However, the second group of enzymes of the cycle (malate dehydrogenase, citrate synthase, aconitase and isocitrate dehydrogenase) are not involved in the metabolism of the products of proline oxidation and are much less active in the tsetse fly. Malic enzyme, which has a vital role in the regeneration of pyruvate for the aminotransferase reaction, is about six times as active in tsetse fly mitochondria as compared to fleshfly mitochondria. Earlier work (NORDEN and PATERSON, 1969, 1970) revealed differences in activity of the glycolytic enzymes in tsetse fly and fleshfly flight muscle of a similar order, fleshfly enzymes being between three and eighteen times as active as tsetse fly enzymes. Thus, taken as a whole, quantitative differences between these two insects in the activity of enzymes involved in the metabolism of carbohydrate and proline, result in a qualitative difference in metabolic pathway and reflect the metabolic adaptation of the tsetse fly to a diet consisting chiefly of protein, as compared to the mainly carbohydrate diet of the fleshfly.

Acknowledgements--Our thanks are due to the Department of Zoology for its provision of insects and to Professor T. WOODand Professor E. BU~ELL for helpful critism of the manuscript.

REFERENCES

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Enzymes in Flight Muscle ANFINSEN C. B. (1955) Methods in Enzymology (Ed. by COLOWlCK S. P. and KAPLAN N. O.) Vol. !, 695. Academic Press, New York. ARRIGONI O. and SINGER T. P. (1962) Limitations of the phenazine methosulphate assay for succinic and related dehydrogenases. Nature, Lond. 193, 1256--1258. BURSELLE., BILLINGK. C.. HARGROVEJ. W., MCCABEC. T. and SLACK E. (1974) Metabolism of the bloodmeal in tsetse Ilics .4ct, trop. 31, 297-320. BURSELLE. and SLACK E. (1976) The oxidation of proline by sarcosomes of the tsetse fly. Glossina morsitans, lnsc(t Biochem. 6, 159-167. CHAPPEL J. B. and HANSFORD R. G. (1972) In Subcellutar Components (Ed. by BIRNIEG. D.), p. 77. Butterworths, London. FANSLER B. and LOWENSTEIN J. M. (1969) Methods in En:ymology (Ed. by LOWENSTEINJ. M.) Vol. 13, p. 26. Academic Press, New York. HILL R. L. and BRADSHAW R. A. (1969) Methods in Enzymology (Ed. by LOWENSTEINJ. M.) Vol. 13, p. 91. Academic Press, New York.

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LOWRYO. H., ROSEBROUGHN. J., FARRA. L. and RANDALL R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol; Chem. 193 265-275. NORDEN D. A. and MATANGANYIDZE C. (1977) Some properties of a mitochondrial malic enzyme from the flight muscle of the tsetse fly (Glossina). Insect Biochem. 7, 215-222. NORDEN D. A. and PATERSON D. J. (1969) Carbohydrate metabolism in flight muscle of the tsetse fly (Glossina) and the blowfly (Sarcophaga). Comp. Biochem. Physiol. 31,819--827. NORDEN D. A. and PATERSON D. J. (1970) Carbohydrate metabolism in flight muscle of the tsetse fly (Glossina) and the blowfly (Sarcophaga)--II. Int. J. Biochem. 1, 81-84. SANADI D. R. and LITTLEFIELD J. W. (1951) Studies on ct-ketoglutaric oxidase--I. Formation of "active' succinate. J. hioL Chem. 193, 683-689. SRIiRI- P. A. (1969) M('thod~ in Enz.t'mohJgy (Ed. by Lt)wliNSllilN J. M.) Vol. 13. p. 3. Academic Press. New York.