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Incorporation of Ammonium in Amino Acids by Trypanosoma cruzi Author(s): Ruy A. Caldas, Elza F. Araújo, Carlos R.

Felix, Isaac Roitman Source: The Journal of Parasitology, Vol. 66, No. 2 (Apr., 1980), pp. 213-216 Published by: The American Society of Parasitologists Stable URL: http://www.jstor.org/stable/3280806 Accessed: 17/11/2010 13:05
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1970. 1963) for of L-glutamate dehydrogenase (=GDH) culture forms of T. cruzi has been studied by Hampton and others (Hampton.J.3. cruzi has been purified.6. a major health prob. Caldas.. Carlos R. . was maintained in Caldas et al. EC 1. The ammonium produced pendent on its nitrogen metabolism because it has no known source of storage carbohy.2 and NAD oxidoreductase. 213-216 ?) American Society of Parasitologists 1980 INCORPORATION OF AMMONIUM IN AMINO ACIDS BY TRYPANOSOMACRUZI Ruy A. Universidade de Brasflia. we studied the comwere studied by Bash-Lewinson and Grossowicz (1957) and by Zeledon (1960a). has a high endogenous respiration.1.1. its Bone and Parent's medium) were harvested by centrifugation at 2. Araujo.1 M KHCO3 containing 5 x 10-3 M MgSO4 and 10-3 M 6f-mercaptoethanol (extraction buffer) using 1. but so far the pathways that T.000 g and washed twice with sterile saline solution. Disc electrophoresis showed the presence of cathodic bands of GDH activity.1. as folforms of T. cruzi (Zeledon. Caz. and ic compound. Proline low: also stimulated respiration in starved T. 1980. 43-55% of its dry weight is protein (Gutter. NADP-linked glutamate dehyCells at mid-log phase (120 hr after transfer to drogenase from T... GOGAT]. (1976) reported the presence LIT medium (Camargo.910-Bras[lia.skeletons is of great interest. cruzi 1) L-glutamate dehydrogenase (L-glutamate: (Sylvester and Krassner. genase and aspartate aminotransferase in the New Jersey). 1966.rpm) in a rotatoryshaker (Controlled Environment Incubator Shaker. producing other amino acids (Hampton. Elza F. New Brunswick Scientific Inc. cruzi are similar to those 1. cruzi is known to produce ammonium as the final lem in South and Central America. NADPH-dependent glutamate synthase. 1976). Goldberg et MATERIALS AND METHODS al. Felix.. EC 6. described for animals (Mancilla et al. Further studies The study of NH3 incorporation into carbon could lead to information useful in the treatment of Chagas' disease. 3) L-glutamine synthetase (L-glutamine: amThe transport of L-arginine and L-lysine by monia ligase. cruzi is not well understood. GDH).4.cruzi uses to accomplish the immobilization idge. 1964) and grown in Bone in and Parent's medium (Bone and Parent. Parasitol. T. 1978).53. The pH optima and Km'sfor the glutamate dehydrogenase system were determined.product of protein and amino acid catabolism (von Brand. and 1971a).may be immobilized in some less toxic organdrate. at 28 C with constant agitation (100 zulo et al. 2) L-glutamate synthase [glutamine (amide): 1967). cruzi and.amino groups of L-glutamate and L-glutamine partate stimulated the respiration of culture via three different enzymatic systems. 1971b. (1977) studied glutamate dehydro. L-glutamine synthetase and NADHdependent glutamate synthase. In the present paper. Some of the metabolic pathways involving L-glutamate: NADP oxidoreductase. because T. It also has been shown that the culture 2-oxoglutarate amino transferase oxidoreform of T. cruzi have not been established. The parative incorporation of ammonium into latter found that both L-glutamate and L-as. cruzi actively metabolizes L-serine ductase (NADP+). The pellet was resuspended in 0. and Isaac Roitman Departamento de Biologia Celular. 1960b). Instituto de Ciencias Biolo6gicas. Trypanosoma cruzi. which were highly dependent on NADP+. Trypanosoma cruzi seems to be highly de. 66(2). GSase). pp.1. and sonre of its The nitrogen metabolism of Trypamosoma properties studied (Juan et al. 1976).. 1976). Some transaminases of T.bulk growth. 1966).2.4. molecular weight determined. DF-Brasil ABSTRACT: Ammonium ions were incorporated into L-glutamate and a-ketoglutarate in epimastigote forms of Trypanosoma cruzi through the following enzymatic systems: NADPH and NADH-dependent glutamate dehydrogenase. EC amino acids in T.0 213 Received for publication 1 August 1978.4. EC 2. same system. recently. Y strain. in order of decreasing specific activity (/tmoles of product formed/min/mg protein). 70.

Samples of dialysate for disc electrophoresis were treated with protamine sulfate (10 mg/ml of dialysate) and centrifuged (14. 1970).5 and that for the NADH is approximately 9. The basic procedure described by Davies (1964) was used for disc electrophoresis.02* 0. L-glutamate synthase (GOGAT). GDH is the most efficient way of immobilizing NH3 derived from protein catabolism in T. 1980 OF TABLE I. cruzi. x log S0: ca-ketoglutarate (4. using NAD+ or NADP+ as electron acceptors. See Material and = re= reaction with NADPH. rather than NADH. Enzyme GDH-NADPH GDH-NADH GDH-NADP+ GOGAT-NADH GOGAT-NADPH GSase * Average of five experiments.0. mate via the GDH-NADPH-dependent 5 x 10-4 M MgSO4and 10-4 M /3-mercaptoethanol). APRIL VOL.2 as running buffer for the specific detection of GDH on the gels. The hydroxamate assay as described by Elliot (1953) was used for measuring glutamine synthetase activity. *-* action with NADH. and L-glutamine synthetase (GSase) in T. Incorporation of ammonium ion into L-gluta- reaction was 19 times greater than that for the GOGAT-NADPH reaction and GSase was not a very effective way of immobilizing ammonium ions as compared with GDH.0 g of cell wet weight. or NADH 90. The pH optimum using phosphate buffer. for 2 x 15 sec with 90% setting.1 M buffers were used to determine the pH optima of the GDH-catalyzed reactions. we decided to further investigate this system. RESULTS then disrupted by sonification in a sonicator. the high specificity for NADPH.24 0. The catGAT-NADPH-dependent abolic activity of GDH-NADP+ was roughly one-seventh that of the NADPH-dependent GDH biosynthetic activity. The homogenate was cooled in an icebath during sonication.10 0. L-glutamine (5 mM). cruzi.07* 0. Micromoles of product/min/mg protein 4.002* 0. 66. Tris (hydroxymethyl) aminomethane 0. NADP+ (3 x 10-5 M).03* 0.0 30 4. is about 8.1 cm) eluted with the extractionbuffer diluted 1:10. NO.and GOreactions.214 THEJOURNAL PARASITOLOGY. the rate of oxidation of NADPH or NADH was recorded at 340 nm (Meers et al.009t V 1.044 + + ? + + + 0.2 x 10-4 M). Protein was determined using the microbiuret assay described by Goa (1953).5. The cells were The dialysis was done in a cold room (0-4 C) using dialysis tubing with pore diameter of 48 A. NADPH (0. we assumed that. FIGURE 1. We followed the concentrations given by Lee (1973) with 4. NADPH (1. The degradative assay for GDH was carried out by following the procedure described by Strecker (1955).55 2.7 x 10-4 M). The biosynthetic activity of L-glutamate dehydrogenase was determined following the procedure described by Ryan and Fottrell (1974).6 x 10-5 M). 0-4 C). Table I compares the enzymatic activity.KETOGLUTARATE 5.25% polyacrylamide and Tris-glycine 0. Saturation curve for a-ketoglutarate. using NADH or NADPH.01 M pH 8. after Polyacrylamide gel electrophoresis protamine sulfate treatment showed a region . The following apparent Km'swere obtained from the double reciprocal plots using concentrations of substrates in the linear region of the plot v(. 2. cruzi-culture epimastigotes. NH4C1 (4. Micromoles of productformed/min/mg of protein by L-glutamate dehydrogenase (GDH). for the GDH-NADPH-dependent reaction is about 8.0 (mM) 0a. The broken cells were centrifuged at 14.25 mM).65 1. Therefore.033 0. New York). model Biosonik (Bronwill Scientific. In Figures 1 and 2. is shown in both the a-ketoglutarate and NH4 Cl saturation curves. From the results shown in Table I.000 g for 20 min (0-4 C). 0-0 Methods.1 M and phosphate (potassium salt) 0..57 0. The supernatant was chromatographedin a Sephadex G-150 column (15 x 1.25 mM).25* 0. The pH optimum for the catabolic reactivity of the GDH-NADP+-dependent action. For the L-glutamate synthase activity the following concentrations were used: a-ketoglutarate(5 mM). of the systems studied.74 0.000 g for 20 min. t Average of three experiments. Velocity of the reaction (V) is expressed as A340nm/ min/mg of protein of T. and the supernatant dialysed overnight with two changes of buffer (10-2 M KHCO3.61 0.0 ml of buffer/1.

See Material and Methods. studied by Cazzulo et al. however. those of Cazzulo et al. indicating that this protein has a lower molecular weight than the proteins of the for these differences between our results and cathodic region. (1977). However. Linda Styer Caldas for the English review. dent on NADP+) leads us to postulate that the The low apparent Km's of NH3 (4.-AMMONIUM INCORPORATIONT. One cannot exclude the possibility that the GDH enzyme also is used to degrade L-glutamate in T. . Velocity agreement with Cazzulo and co-workers (Cazof of the reaction (V) is expressed as A340nm/min/mg zulo et al. we were able to protein of T. Both regions showed activ(Cazzulo et al. We thank Dr. In both buffers (Tris and phosphate).ET IN CALDAS AL. Based on end-product inhibition studies Shalitov et al. CRUZI 215 the GOGAT system and to other biosynthetic reactions of the cells. ACKNOWLEDGMENTS This work was supported by the grant SIP08-072 from the Conselho Nacional de Desenvolvimento Cientifico e Tecnolo6gico of Brasil. cruzi as suggested by Cazzulo et al. therefore. cruzi strains used. The pH optimum for the reduction of a-ketoglutarate by the ity with NAD+ to lesser extent. might use the degradative pathway as they suggested. the pH optimum curve for the degradative GDH NH4 CI (mM) gives a higher activity around 8. Tulahuen strain. cruzi cultures. and 2) differences in extracDISCUSSION tion procedure. We also observed an optimal pH of 8. cruzi. However. whereas the T. L-glutamate is a key amino acid in the L-glutamate family. Under our experimental conditions.: 1) difference in T.B-mercaptoethanol was used in the The higher activity of the biosynthetic extraction buffer. therefore. which is dent GDH activity and a more anodic band higher than the value reported by Cazzulo with less activity. 1974). the flow of glutamine is too low to supply this substrate to both M) and a-ketoglutarate (4. FIGURE Saturationcurve for NH4C1. (1977). it is very advantageous for T. We can suggest two possible explanations G-150. this was found to be a crureaction in comGDH-NADPH-dependent cial factor in stabilizing the GDH-NAD+ and with the catabolic enzyme (depenparison NADH activities.. which was not detected by with NADH. . 1970). the specific activity of GSase (which synthesizes L-glutamine) is roughly one-fifth that of GOGAT (NADPH).0. The pH optimum for this reaction was 9. In our experimental conditions. Helio Peixoto for the T. (1975) suggested that in Chlorella..2 x 10-4 main function of the L-glutamate dehydrogenase system is to incorporate NH3 into organic compounds and not to produce ammonium. cruzi to have a high GDH biosynthetic activity for reincorporating NH3 into amino acids while it is metabolizing proteins. cruzi. the GDH-NADP+-dependent system has a synthetic function. the possibility remains that the system studied in our laboratorycould preferably use the GDH biosynthetic pathway.. the more anodic band was retained in Sephadex NADP-GDH system in the Tulahuen strain is 7.7 x 10-4 M) may have some biological significance. 0-0 *-* dent on NADH. them. which is in 2. detect a biosynthetic GDH activity depen= reaction = reaction with NADPH. 1977). However. Incorporation studies of 15NH3 are required to prove definitively the fate of ammonium in T.5 for near the cathode with strong NADP+-depenthe enzyme dependent on NADPH. cruzi. as suggested elsewhere (Miflin.5. and Dr.0. 1977). The GOGAT enzyme (NADPH) requires a source of L-glutamine and ketoglutarate to produce L-glutamate during growth under low ammonia levels (Tempest et al.

New York. J. Rev Brasil Biol 20: 409-414. 1974. M. Alguns enzimas do metabolismo nitrogenado de Trypanosoma cruzi. Disc electrophoresis. Biokhimiya 40: 1237-1245. R.. New York. V. J Biol Chem 201: 661-672. 1971b.. J Gen Microbiol 31: 261-266. 1976. G. 1955. 220225. KRASSNER. SEGURA. J.D. H. 266-269. Ann NY Acad Sci 121: 404-427.. Comparative physiological studies on four species of hemoflagellates in culture. AND G. A. E. A. P. L.. . PEREIRA. Biochemistry of parasites. Comp Biochem Physiol 18: 241-248. Comparative study of glutamate dehydrogenase of Chlorella. p. A. HAMPTON. CAMARGO.AND V. J. 1975. In vivo incorporation of leucine-1l-4C into the proteins of Trypanosoma cruzi. L. TEMPEST. C. DAVIES. LANAS. Glutamate dehydrogenase and aspartate aminotransferase in Trypanosoma cruzi. Effect of carbohydrates and related substances and some amino compounds on the respiration. 0. M. L-glutamic dehydrogenase from liver. VON BRAND. M. .. Growth and differentiation in Trypanosoma cruzi. Stearic acid. Comp Biochem Physiol 39B: 999-1003. Isolation of glutamine synfrom green thetase and glutamotransferase peas. E. 1970. RYAN. an essential growth factor for Trypanosoma cruzi. II. M.. MIFLIN. Comp Biochem Physiol 55B: 443448. GAZZINELLI. I. AND E. W. KASPAROVA. In S. The metabolic fate of DL-leucine-1l-4C in Trypanosoma cruzi. 1964. 1970. NAQUIRA.D. Transaminases of Trypanosoma cruzi. M. J Parasitol 46: 541-545. FOTTRELL. NO. R.. 1974. Comp Biochem Physiol 56B: 301-303. SHATILOV. Arginine transport in the culture form of Trypanosoma cruzi. Kaplan (eds. Bull Res Counc Isr Sect E 6: 91-92. GOA. J. 1967. Plant Physiol 54: 550-555. R. Purification and some properties of the NADP-linked glutamate dehydrogenase from Trypanosoma cruzi. isozymes in Ricinus communis seedlings. G. Inc. S. T. E. E. 1953. CALDAS. . an enzyme involved in the synthesis of glutamate by some bacteria. Academic Press. New York. II.. E. W. 1976.AND C. Biochemistry of Trypanosoma cruzi. AMBARTSUMYAN. ELLIOT. M. Lysine uptake in cultured Trypanosoma cruzi: Interaction of competitive inhibitors..J. S. SEGURA. E. J Gen Microbiol 64: 187-194. 66.R. 1970. MARES-GUIA. GUTTERIDGE. C. . BROWN.. Scand J Clin Lab Invest 5: 218-222. D. Int J Biochem 9: 395-400. Academic Press. KRETOVICH. AND J. p. 1973. Rev Inst Med Trop 6: 93-100. Colowick and N.W. thesis in Trypanosomatidae. W. J. SYLVESTER. S. L. Protein biosyn. L. CAZZULO. APRIL VOL. L. CERON. MANCILLA. In New approaches in American trypanosomiasis research. Serine metabolism in the culture form of Trypanosoma cruzi: Synthesis of other amino acids. Glutamine (amide): 2-oxoglutarate amino transferase oxidoreductase (NADP). J Protozool 17: 597-600. AND P. 1964. S.D. 1977. Vol. Glutamate dehydrogenase .AND C. FERNANDES. Protein biosynthesis in Trypanosomatidae. Pan Am Health Organ Publ 318: 255-262. 1960b. 2. Methods of enzymology. Comparative physiology of four species of hemoflagellates in culture. BONE. R. LITERATURE CITED BASH-LEWINSON. Cien Cult Sao Paulo 28: 483. II. CHIARI. Biochem J 117: 405-407. D. Phytochemistry 12: 2631-2634.AND C. 1978. Inc. BROWN. J. J Protozool 23: 186-193. R. Phytochemistry 12: 2647-2652. 1966.1957..R. AND S. TEMPEST. CAZZULO. J Protozool 18: 701-703. AND N. 1953. B. I. Origin of metacyclic trypanosomes in liquid media. J.. 1960a. Exp Parasitol 21: 154-159. J. GOLDBERG. A.216 1980 OF THEJOURNAL PARASITOLOGY. Proline metabolism in Trypanosoma cruzi epimastigotes. Determination of protein by the microbiuret method. AND G.. V. H. S. The location of nitrate reductase and other enzymes related to amino acid biosynthesis in the plastids of root and leaves. GROSSOWICZ. AND C. 1971a. and . Subcellular localization of enzymes involved in the assimilation of ammonia by soybean root nodules. Synthesis of glutamate in Aerobacter aerogenes by a hitherto unknown route. PARENT. F. STRECKER. P. 1976. F. New York. 1976. MEERS. J. MEERS. M. JUAN. FELIX. II. 1963. Method and application to human serum proteins.. JUAN. Transaminases. B.). V. 1966. Comparative kinetics of arginine and lysine transport by epimastigotes and trypomastigotes from two strains of Trypanosoma cruzi. LEE. ZELED6N. A. W.