Cytosolic malate dehydrogenase in muscle extracts of Toxocara canis

Comp. Biochem. Physiol. Vol. 75B, No. 1, pp. 147 to 152, 1983

0305-0491/83/050147-06503.00/0 © 1983 Pergamon Press Ltd

Printed in Great Britain

CYTOSOLIC MALATE DEHYDROGENASE IN MUSCLE EXTRACTS OF T O X O C A R A C A N I S C. M. ANDRADE*,M. F. A. FERREIRAtand LUIZ P. RIBEIRO Departamento de Bioquimica, lnstituto de Quimica, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brasil (Received 2 September 1982)

Abstrael--1. Malate dehydrogenase (L-malic acid:NAD + oxydoreductase, EC 1.1.1.37t was partially purified from muscle extracts of Toxocara canis by means of gel chromatography in Sephadex G-150 and affinity chromatography in Sepharose-4B-Blue dextran. 2. The purified enzyme was very active in reducing oxalacetate and less active in oxidizing e-malate. It was inhibited by excess oxalacetate but not by L-malate. 3. The kinetic parameters of the enzyme were obtained and these included: pH and temperature optima and apparent Michaelis constants for the substrates. 4. The results suggest that the enzyme from Toxocara canis behaves like the enzyme of the model helminth Ascaris lumbricoides.

INTRODUCTION It has been shown that a number of nematodes accumulate succinate as a product of fermentation and this is true not only for nematodes but for cestodes and trematodes as well (Saz & Bueding, 1966). In this respect, the intestinal parasite Ascaris lumbricoides has been quite successfully used as a model organism to study the energy metabolism of helminths (Saz & Hubbard, 1957; Saz, 1971). F r o m the results obtained in a series of investigations it was evident that the metabolism of A. lumbricoides mitochondria was essentially anaerobic, that is, quite different from the metabolism of the mitochondria of the host organism (Saz, 1972). The pathway of carbohydrate dissimulation in Ascaris muscle has been established as follows: first, it was shown that malate is generated in the cytoplasm, which in turn, is used as substrate in the mitochondria (Bueding & Saz, 1968; Saz & Lescure, 1969). The cytosolic reaction is catalyzed by a very active malate dehydrogenase (EC 1.1.1.37) while in the mitochondria one equivalent of malate is decarboxylated via the NAD+-linked malic enzyme (EC 1.1.1.39) thus generating N A D H (Kmetec & Bueding, 1961; Rew & Saz, 1974). Also, it was shown that the mitochondria contained a inner membrane bound N A D H : N A D ÷ transhydrogenase (EC 1.6.1.1) system that would be responsible for the hydride ion translocation (Fioravanti & Saz, 1976; K6hler & Saz, 1976). Recently, the distribution of enzymes of malate metabolism in muscle cell fractions of the helminth Toxocara canis has been reported (Ribeiro et al., 1981). The results obtained were quite similar to those

obtained in A. lumbricoides (Rew & Saz, 19741. Cytosolic malate dehydrogenase was also found to be very active. T. canis besides being an intestinal parasite of dogs is the causative agent of Larva migrans visceralis in man. Also, it is quite evident that a perfect knowledge of the properties of the enzymes of the parasite as compared with those of the host organism, may lead to the use of specific enzyme inhibitors as antihelminth agents. Therefore, we now report on the partial characterization of malate dehydrogenase of muscle extracts of T. canis. MATERIALS AND METHODS Chemicals

Bovine serum albumin (Fraction V), cis-oxalacetic acid, )malic acid (monsodium salt1, o( + )malic acid, e( + Itartaric acid, NADH, NAD ÷ and Tris(hydroxymethyl)aminomethane were all from Sigma Chemical Company (St. Louis, Mo.). SephadexG-150, CNBr-activated Sepharose4B-Blue dextran and the chromatographic columns were obtained from Pharmacia Fine Chemicals, Inc. (Uppsala, Sweden/. All other reagents were of the highest purity commercially available and mostly obtained from E. Merck Darmstadt (Germany). e( -

Helminths Adult T. canis were collected from the intestine of young

dogs. After dissection, the internal organs were all discarded and the muscle tissue separated by published procedures (Rhodes et al., 19631. The tissue was washed with cold saline, blotted, weighed and kept in ice for immediate homogenation. Enzyme extraction

Present addresses: *Departamento de Ci~ncias Biol6gicas, Escola Nacional de Safde Pflblica, Fundag'~o Oswaldo Cruz, Rio de Janeiro; -tDepartamento de Biologia Celular, Instituto de Biologia, Universidade do Estado do Rio de Janeiro. Rio de Janeiro, Brasil. 147

All steps described were carried out at 4°C. The muscle was homogenized with 10mM Tris-HCl buffer, pH 7.5 using a volume of buffer of 4 times the weight in grams of the muscle sample. The homogenates were prepared in a Omni-Mixer (Ivan Sorvall, Inc., USAI at 4~C and trans-

C.M. ANDRADE et al.

148

ferred to a Potter-Elvehjem glass homogenizer with a Teflon pestle for further homogenization. Cell debris and most particles were removed by centrifugation at 5000 g for 30min in a refrigerated centrifuge. The pellet was discarded and the supernatant removed and centrifuged at 100,000g for 1 hr in an ultracentrifuge (Beckman L-2 65B with a Ti 65 rotor). The supernatant was lyophilyzed and stored at - 2 O C if not used immediately. This represented the crude enzyme preparation.

100, eo sO

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40

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Sephadex G- 150 chromatography The Sephadex G-150 column (2.5 × 40 cm) was packed essentially as described previously (Ribeiro et al., 1981). However, in the present case the column was equilibrated with 4vol of Tri~HCI buffer pH 7.5 and the proteins eluted with the same buffer at a column flux of 15 ml/hr. Affinity chromatograph.v CNBr-activated Sepharose-4B-Blue dextran was used for binding the dehydrogenase. The column (1.0 × 10 cmJ was prepared essentially as described by Ryan & Vestling (1974). The activated gel was suspended in 10-3M HC1, washed with carbonate buffer pH 10 and the Blue dextran was left to react for 18 hr at 4 C . Excess Blue dextran was removed by washing with 1 M KCI. The column was equilibrated with Tri~HCI buffer pH 7.5. Non-linked proteins were removed with the Tris-HC1 buffer pH 7.5 and the bound enzymes were eluted stepwise with 1 mM NADH. Enzyme assays (a) Reduction of oxalacetate. Malate dehydrogenase activity using oxalacetate as substrate was assayed as described previously (Ribeiro et al., 1981). (b) Oxidation of L-malate. Malate dehydrogenase activity using L-malate as substrate was assayed essentially as described by Yoshida (1969). The reaction mixture contained: 2.5ml of Tris-HCl buffer pH 8.8, 0.1 ml of 0.1 M L-malate, 0.1 ml of 10 mM NAD + and 0.1 ml of enzyme (1 #g of protein/ml). The reduction of the pyridine nucleotide was followed at 340 nm in a Cary 17 spectrophotometer (Varian, Palo Alto, CA) for 3min. The specific activity was expressed as #mol of NADH oxydized/mg of protein/min. Protein concentration was determined by the method of Lowry et al. (1951) using bovine serum albumin as standard. Column eluates were followed at 280 nm.

~E ,,i

20

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,b

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Fig. 1. Rates of thermal inactivation of T. canis cytosolic malate dehydrogenase at 55'C. O• enzyme preparation: O O enzyme preparation plus 0.281~mol NADH.

If cytosolic malate dehydrogenase was the only enzyme to be recovered in the purification procedure, the c h r o m a t o g r a p h y columns could be eluted with T r i > H C 1 buffer pH 7.5. The results were equivalent to those obtained previously (Ribeiro et al., 1981) in which a 10 m M imidazole buffer pH 6.5 containing 7 m M glycerol, 10 m M 2-mercaptoethanol and 1 m M E D T A was used. However, imidazole buffer was necessary if the mitochondrial enzyme or the malic enzyme were also to be recovered. After the c h r o m a t o g r a p h y in S e p h a d e x G - 1 5 0 the fractions containing the peak of malate dehydrogenase activity were pooled, concentrated a n d applied to the CNBr-activated Sepharose-4B-Blue dextran affinity c h r o m a t o g r a p h y column. Protein and enzyme activity were determined in all steps. The data from several experiments are summarized in Table 1. It shows a 58-fold increase in the specific activity of cytosolic malate dehydrogenase as c o m p a r e d to the activity of the initial homogenate.

Enzyme properties

RESULTS

Partial purification of cytosolic malate dehydrogenase In a previous report it was shown that in muscle extracts of T. canis malate dehydrogenase activity was located mainly i n the cytosol (Ribeiro et al., 1981). However, some activity was also found in the mitochondria. These previous experiments also demonstrated that S e p h a d e x G - 1 5 0 gel c h r o m a t o g r a p h y could be used for the separation of enzymes of malate metabolism. Therefore, it was used as the first step in the purification procedure.

The cytosolic enzyme was characterized with respect to several parameters. First, the enzyme showed a p H o p t i m u m of 7.5 under the assay conditions described for oxalacetate reduction. The stability of the enzyme to p H changes was studied next. In these experiments the enzyme was incubated at different pH values for 2 0 m i n a n d the activity determined at pH7.5. W h e n the enzyme was preincubated at pH > 8.7 or <6.2, irreversible enzyme inactivation was observed once full activity was not recovered when measured at pH 7.5.

Table 1. Partial purification of cytosolic malate dehydrogenase of muscle extracts of T. canis Purification step 1. Tissue extract 2. Sephadex G-150 3. Sepharose-4B-Blue dextran

go

MINUTES

Total protein Total units (mg) (llmol/min) 38 24 0.24

171,000 64,800 62,856

Specific activity (units/mg)

Purification factor

4,500 27,000 261,900

1 6 58.2

Cytosolic malate dehydrogenase in T. canis

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Fig. 2. (A) Rate curve and (B) double-reciprocal plot of kinetic data for T. canis cytosolic malate dehydrogenase. Initial rates of NADH oxidation were measured at a saturating concentration of the coenzyme, v = #mol/min. The cytosolic malate dehydrogenase was incubated at different temperatures during the assay. It was maximally active at 55°C. Thermal inactivation of T. canis cytosolic malate dehydrogenase at 55°C in the presence and in the absence of saturating concentrations of NADH is shown in Fig. 1. The results obtained show that NADH protected the enzyme against heat inactivation to some extent. After 40 min of incubation with the added pyridine nucleotide the enzyme retained 70% of its initial activity while in its absence only 30~o of the initial activity remained. The free energy of activation calculated from an Arrhenius plot was 11,500 cal/mol. The partially purified malate dehydrogenase displayed typical hyperbolic kinetics with increasing oxalacetate concentration. However, inhibition by substrate was observed at oxalacetate concentrations about 190 ~M. The double-reciprocal plot of the data yielded a K,, value of 54 ~tM with respect to oxalace~

tate (Fig. 2). Changing N A D H concentrations gave hyperbolic rate curves. However, inhibition by excess NADH was not observed. The double-reciprocal plots yielded a K,, value of 25 pM with respect to N A D H (Fig. 3). The effect of L-malate concentration on the activity of the enzyme (the reverse reaction) of T. canis was also studied. Inhibition by substrate was also observed in this case. The K,, value for L-malate was 1.8raM at pH 8.8. Hyperbolic response to NAD ÷ concentrations were also observed for the reverse reaction. Double-reciprocal plots of the data yielded a Km value of 0.11 mM. Inhibition ,studies

(a) Effect of inorganic salts. The malate dehydrogenase of T. canis muscle was shown to be strongly inhibited by silver ions, 100~o inhibition being observed at 33/~M AgNO 3. From the other divalent

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6.0

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cytosolic malate

dehydrogenase. Initial rates for NADH oxidation were measured at 0.76/amol oxalacetate. v =/lmol/min.

150

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100

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80

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Fig. 4. Non competitive inhibition of T. canis cytosolic malate dehydrogenase by ~-----O L(+)tartrate (7.5 raM), and • • D(+)malate (7.5 mM). (>----O Initial rates for NAD + reduction measured at a range of L-malate concentration from 1.7 to 10.2mM in the abscence of inhibitors, v = ,umol of NAD + reduced per rain.

metal ions tested (Cu 2 +, Zn z+, Ca 2+, Cd z+, Co 2+ and Mg 2+) only Cd 2 + gave appreciable inhibition. At 1 mM CdCI2 60~o inhibition was observed, (b) Effect of organic acids. Two substrate analogs were tested as possible inhibitors of the malate dehydrogenase of T. canis muscle. Noncompetitive type of inhibition was obtained with L(+)tartrate and D(+)malic acid for the oxidation of e-malic acid (Fig. 4). Small effect was obtained for the reduction of oxalacetate. DISCUSSION

Malate dehydrogenase is a key enzyme in the pathway of carbohydrate dissimulation in the model organism A. lumbricoides. It is the most active enzyme found in the cytosol of the muscle cells of this organism (Saz, 1971). In a previous report the cytosolic malate dehydrogenase of T. canis was found also very active (Ribeiro et al., 1981). However, the properties of the enzyme were not described. T. canis is a parasitic helminth which is phylogenetically related to Ascaris. Therefore, we expected that some of the properties of malate dehydrogenase of both organisms would be similar. The distinction between the helminth enzyme and that of the host organism could be deduced from the analyses of the properties of the enzyme of the parasitic nematode. The enzymological characteristics of the muscle cytosolic malate dehydrogenase are also apparently adapted to the low oxygen tension environment of the T. canis, the metabolism of this parasitic nematode being essentially anaerobic. Although the physiological role of the cytosolic malate dehydrogenase has not been established in T. canis, it is reasonable to assume that the production of malate from oxalacetate may allow the organism to

obtain the necessary reducing power and ATP formation in the mitochondria via the fumarase reductase and malic enzyme reactions. An analysis of the properties of the enzyme found in the present investigation indicated that the pH optimum of 7.5 is very similar with the value found for the enzyme of A. lumbricoides (Rhodes et al., 1964). It is of interest that the enzyme from Bacillus subtilis presents also a similar behavior with respect to the resistance to pH changes. The enzyme of this organism like that of T. canis, also looses activity when preincubated at pH > 7.7 or <6.8 (Yoshida, 1965). Good agreement with malate dehydrogenase of A. lumbricoides was also found with respect to the temperature optimum of reaction (Rhodes et al., 1964). This is also reflected in the energy of activation: for the T. canis enzyme a value of ll,5000cal/mol was found while the reported value for the enzyme of A. lumbricoides was of the order of 12,000 cal/mol (Barret & Fairbairn, 1971). The catalytic properties of the enzyme from T. canis are, in general, equivalent to those reported for the enzyme of other organisms. Substrate inhibition seems to be a characteristic of malate dehydrogenase of several different organisms and has been observed for A. suum (Rhodes et al., 1964; Zee & Zinkham, 1968); Pseudomonas testosteroni (You & Kaplan, 1975); Fasciola hepatica (Probert & Lwin, 1977) and for Neothunnus macropterus (Kitto & Lewis, 1967). It seems that the soluble malate dehydrogenase is less sensitive to oxalacetate inhibition in several organisms such as Euglena 9racilis (Peak et al., 1972) and Physarum polycephalum (Teague & Henney. 1973). Little inhibition for soluble malate dehydrogenase with increasing concentration of substrates was observed for Drosophila melanogaster. Only 20-30!~i; inhibition with 10 mM oxalacetate was reported. How-

Cytosolic malate dehydrogenase in T. canis

151

Table 2. Comparison of the kinetic constants of the cytosolic malate dehydrogenase of T. canis muscle and that of other organisms

Organism Toxocara canis Ascaris lumbricoides Ascaris suum Fasciola hepatica Hvmenolepis diminuta Schistosoma mansoni

pH

Km(OOA) (/~M)

K,,(NADH) (/tM)

7.5 7.5 7.8 8.5 7.4 7.8

54 38 29 52 22 40

25 . -117 -80

ever, the enzyme was strongly inhibited by high concentrations of N A D H (Hay & Armstrong, 1976). Inhibition of the reverse reaction by the substrate L-malate was also observed for the enzyme of mammals (Englard & Breiger, 1962) if concentrations of L-malate above 39 x 1 0 - 3 M were used. This again seems to be a general property of malate dehydrogenases and has also been reported for different organisms such as Fasciola hepatica (Probert & Lwin, 1977), chicken heart (Kitto & Kaplan, 1966). However, activation of malate dehydrogenase by concentrations of malate above 1.3 × 1 0 - 4 M have been reported for the enzyme of Pseudomonas testosteroni (You & Kaplan, 1975). In Table 2, the kinetic constants determined for the enzyme from T. canis are shown in comparison with the data reported for other organisms. The apparent gin'S for oxalacetate reduction as well as for L-malate oxidation found for the enzyme of T. canis are similar to those found for the malate dehydrogenase of other organisms. The largest differences observed were between the values of the present investigation and the data reported for the K,, of N A D H oxidation in Fasciola hepatica (Probert & Lwin, 1977) and for N A D + reduction in Drosophila melanogaster (Hay & Armstrong, 1976). The low values found for the K,, of oxalacetate (54/iM) and N A D H (25/~M) as compared with the K,, values found for L-malate (1.8 mM) and N A D ÷ (0.11 mM) support the proposed mechanism by which the reaction of cytosolic malate dehydrogenase of T. canis occurs with the production of large quantities of malate, that is, quite opposite from the mechanism of the enzyme of mammals. The inhibition of malate dehydrogenase by silver ions is also observed for the enzyme from bovine brain which is strongly inhibited by silver ions (Higashida et al., 1975), 1000~ inhibition being found at 33~tM AgNO3, as in the present investigation. Complete loss of activity by 1 m M silver acetate was also reported for the enzyme of Drosophila melanoyaster (Hay & Armstrong, 1976). The non competitive type of inhibition found in our experiments was also reported for the malate dehydrogenase of Pseudomonas testosteroni (You & Kaplan, 1975). However, slight competitive inhibition was obtained for the reverse reaction with I~(+ )malate. Our investigation has yielded information on the malate dehydrogenase content and properties in muscle extracts of T. canis but the whole mechanism of the enzyme action and function require further investigations. Nevertheless, the data presented here strongly support the fact that malate dehydrogenase

pH 8.8 .

. 7.8 10 -10

K,,(MAL) (mM)

K,,(NAD +) (mM)

1.8 . 1.7 2.0 -0.3

0.11 -0.45 -0.025

References

Present investigation Barret & Fairbairn (1971) Zee & Zinkham (1968) Probert & Lwin (1977) Moon et al. (1977) Rotmans (1978)

of T. canis is very similar and most probably has the same functions of the enzyme of the model helminth A . lumbricoides. Acknowledgements--The present work was supported by grants from Conselho Nacional de Desenvolvimento Cientifico e Tecnol6gico (CNPq), from Conselho de Ensino para Graduados (CEPG/UFRJ) and from Funda~o Universit6ria Jos6 Bonifftcio (FUJB/UFRJ).

REFERENCES

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PEAK M. J., PEAK J. G. & TING I. P. (1972) lsoenzyme of malate dehydrogenase and their regulation in Euglena gracilis Z. Biochim. biophys. Acta 284, 1-15. PROBERT A. J. & LWlN T. (1977) Fasciola hepatica: the subcellular distribution and kinetic and electrophoretic properties of malate dehydrogenase. Expl. Parasit. 41, 89 94. REW R. & SAZ H, J. (1974) Enzyme localization in the anaerobic mitochondria of Ascaris lumbricoides. J. Cell Biol. 63, 125-135. RHODES M. B., MARSH C. L. & KELLEY JR, G. W. (1963) Trypsin and chymotrypsin inhibitors from Ascaris suum. Expl. Parasit. 13, 266-272. RHODES M. B., MARSH C. L. & KELLEY JR, G. W. (1964) Studies on helminth enzymology. III. Malic dehydrogenases of Ascaris suum. Expl. Parasit. 15, 403-409. RIBEIRO L. P., FERREZRAM. F. A. & ANDRADE C. M. (1981) Compartmentalization and one-step separation of enzymes of malate metabolism in muscle extracts of Toxocara canis. Comp. Biochem. Physiol. 69B, 859-864. ROTMANS J. P. (1978) Schistosoma mansoni: Purification and characterization of malate dehydrogenase. Expl. Parasit. 46, 31-48. RYAN L. D. & VESTLING C. S. (1974) Rapid purification of lactate dehydrogenase from rat liver and hepatoma: A new approach. Archs Biochem. Biophys. 160, 279-284. SAZ H. J. (1971) Facultative anaerobiosis in the invertebrates: Pathways and control systems. Am. Zool. 11, 125-135.

SAZ H. J. (1972) Comparative biochemistry of carbohydrates in nematodes and cestodes. In Comparative Biochemistry of Parasites (Edited by VAN DEY BOSSCHE H.), pp. 33-47. Academic Press, New York. SAZ H. J. & BUEDING E. (1966) Relationships between anthelmintic effects and biochemical and physiological mechanisms. Pharmac. Rev. 18, 871 894. SAZ H. J. & HtmBARO J. A. (1957) The oxidative decarboxylation of malate in Ascaris lumbricoides. J. biol. Chem. 225, 921-933. SAZ H. J. & LESCURE O. (1969) The function of phosphoenol pyruvate carboxykinase and malic enzyme in the anaerobid formation of succinate by Ascaris lumbricoides. Comp. Biochem. Physiol. 30, 49-60. TEAGUE W. M. & HENNEY JR, H. R. (1973) Purification and properties of cytoplasmic and mitochondrial malate dehydrogenase of Physarum polvcephalum. J. Bact. !16, 673-684. You K. S. & KAPLAN N. O. (1975) Purification and properties of malate dehydrogenase from Pseudomonas testosteroni. J. Bact. 123, 704-716. YOSHIDA A. (1965) Enzymic properties of malate dehydrogenase of Bacillus subtilis. J. biol. Chem. 240, 1118-1124. YOSHXDA A. (1969) L-Malate dehydrogenase from Bacillus subtilis. Meth. Enzym. 13, 141-145. ZEE D. S. & ZINKr~AMW. H. (1968) Malate dehydrogenase in Ascaris suum. Characterization, otongeny and genetic control, Archs Biochem. Biophys. 126, 574-584.