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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 RoitmanSource: The Journal of Parasitology, Vol. 66, No. 2 (Apr., 1980), pp. 213-216Published by: The American Society of ParasitologistsStable URL: http://www.jstor.org/stable/3280806
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J. Parasitol.,66(2), 1980, pp. 213-216
?) American Society of Parasitologists 1980
INCORPORATION OF AMMONIUM IN AMINO ACIDS BY
TRYPANOSOMACRUZI
Ruy A. Caldas, Elza F. Araujo, Carlos R. Felix, and Isaac Roitman
Departamento de Biologia Celular, Instituto de Ciencias Biolo6gicas,Universidade de Brasflia, 70.910-Bras[lia, DF-Brasil
ABSTRACT: Ammonium ions were incorporated into L-glutamate and a-ketoglutarate in epimastigoteforms of Trypanosomacruzi through the following enzymatic systems: NADPH and NADH-dependentglutamate dehydrogenase, NADPH-dependent glutamatesynthase, L-glutamine synthetase and NADH-
dependent glutamatesynthase, in order of decreasing specific activity (/tmoles of product formed/min/mgprotein). The pH optima and Km's or the glutamatedehydrogenase system were determined. Disc elec-
trophoresis showed the presence of cathodic bands of GDH activity, which were highly dependent onNADP+.
The nitrogen metabolism of Trypamosoma
cruzi is not well understood. Further studies
could lead to information useful in the treat-
ment of Chagas' disease, a major health prob-
lem in South and Central America.
Trypanosoma cruzi seems to be highly de-
pendent on its nitrogen metabolism because
it has no known source of storage carbohy-
drate, has a high endogenous respiration, and
43-55% of its dry weight is protein (Gutter-
idge, 1976). Some transaminases of T. cruzi
were studied by Bash-Lewinson and Gros-
sowicz (1957) and by Zeledon (1960a). The
latter found that both L-glutamate and L-as-
partate stimulated the respiration of culture
forms of T. cruzi (Zeledon, 1960b). Proline
also stimulated respiration in starved T. cruzi
(Sylvester and Krassner, 1976).
Some of the metabolic pathways involving
amino acids in T. cruzi are similar to those
described for animals (Mancilla et al., 1966,
1967). It also has been shown that the culture
form of T. cruziactively
metabolizes L-serine
producing other amino acids (Hampton,
1971a).
The transport of L-arginine and L-lysine by
T. cruzi has been studied by Hampton and
others (Hampton, 1970, 1971b; Goldberg et
al., 1976).
Caldas et al. (1976) reported the presence
of L-glutamate dehydrogenase (=GDH) in
culture forms of T. cruzi and, recently, Caz-
zulo et al. (1977) studied glutamate dehydro-
genase and aspartate aminotransferasein the
same system. NADP-linked glutamate dehy-
drogenase from T. cruzi has been purified, its
Received forpublication 1 August 1978.
molecular weight determined, and sonre of its
properties studied (Juan et al., 1978).
The study of NH3 incorporation into carbon
skeletons is of great interest, because T. cruzi
is known to produce ammonium as the final
product of protein and amino acid catabolism
(von Brand, 1966). The ammonium produced
may be immobilized in some less toxic organ-
ic compound, but so far the pathways that T.
cruzi uses to accomplish the immobilization
have not been established.
In the present paper, we studied the com-
parative incorporation of ammonium into
amino groups of L-glutamate and L-glutamine
via three different enzymatic systems, as fol-
low:
1) L-glutamate dehydrogenase (L-glutamate:
NAD oxidoreductase, EC 1.4.1.2 and
L-glutamate: NADP oxidoreductase, EC
1.4.1.4, GDH);
2) L-glutamate synthase [glutamine (amide):
2-oxoglutarate amino transferase oxidore-
ductase (NADP+), EC 2.6.1.53, GOGAT];and
3) L-glutamine synthetase (L-glutamine: am-
monia ligase, EC 6.3.1.2, GSase).
MATERIALS AND METHODS
Trypanosoma cruzi, Y strain, was maintained in
LIT medium (Camargo, 1964) and grown in Bone
and Parent's medium (Bone and Parent, 1963) forbulk growth, at 28 C with constant agitation (100
rpm)in a rotatoryshaker(Controlled EnvironmentIncubator Shaker, New Brunswick Scientific Inc.,
New Jersey).Cells at mid-log phase (120 hr after transfer to
Bone and Parent'smedium) were harvestedby cen-trifugation at 2,000 g and washed twice with sterile
saline solution. The pellet was resuspended in 0.1
M KHCO3 containing 5 x 10-3 M MgSO4 and 10-3
M 6f-mercaptoethanol (extraction buffer) using 1.0
213
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214 THEJOURNAL FPARASITOLOGY,OL.66, NO.2, APRIL 980
TABLE I. Micromolesof productformed/min/mgofprotein by L-glutamate dehydrogenase (GDH),L-glutamate synthase (GOGAT),and L-glutaminesynthetase (GSase) in T. cruzi-culture epimasti-gotes.
Micromoles ofEnzyme product/min/mg protein
GDH-NADPH 4.57 + 0.25*
GDH-NADH 0.74 + 0.03*
GDH-NADP+ 0.61 ? 0.07*
GOGAT-NADH 0.033 + 0.002*
GOGAT-NADPH 0.24 + 0.02*
GSase 0.044 + 0.009t
* Average of five experiments.
t Average of three experiments.
ml of buffer/1.0g of cell wet weight. The cells werethen disrupted by sonification in a sonicator, model
Biosonik (Bronwill Scientific, New York), for 2 x 15
sec with 90% setting. The homogenate was cooled
in an icebath during sonication.
The broken cells were centrifuged at 14,000 g for
20 min (0-4 C), and the supernatant dialysed over-
night with two changes of buffer (10-2 M KHCO3,5 x 10-4 M MgSO4and 10-4 M/3-mercaptoethanol).The dialysis was done in a cold room (0-4 C) usingdialysis tubing with pore diameter of 48 A.
Samplesof dialysatefor disc electrophoresisweretreated with
protaminesulfate
(10 mg/mlof
dialy-sate) and centrifuged (14,000 g for 20 min, 0-4 C).The supernatantwas chromatographed n a Seph-adex G-150 column (15 x 1.1 cm) eluted with theextraction buffer diluted 1:10.
The hydroxamateassay as described by Elliot
(1953) was used for measuring glutamine synthe-tase activity. The biosynthetic activity of L-gluta-mate dehydrogenasewas determined following theprocedure described by Ryan and Fottrell (1974),using NADH or NADPH. The degradative assay forGDH was carried out by following the proceduredescribed by Strecker (1955), using NAD+ orNADP+ as electron acceptors. For the L-glutamate
synthase activity the following concentrations wereused: a-ketoglutarate(5 mM), L-glutamine (5 mM),NADPH (0.25 mM), or NADH 90.25 mM);the rateof oxidation of NADPH or NADH was recorded at340 nm (Meerset al., 1970).Protein was determinedusing the microbiuret assay described by Goa(1953). The basic procedure described by Davies(1964) was used for disc electrophoresis. We fol-lowed the concentrationsgiven by Lee (1973) with4.25%polyacrylamideand Tris-glycine 0.01 M pH8.2 as running buffer for the specific detection ofGDH on the gels. Tris (hydroxymethyl) amino-methane 0.1 M and phosphate (potassium salt) 0.1
M buffers were used to determine the pH optimaof the GDH-catalyzed reactions.
RESULTS
Table I compares the enzymatic activity.
Incorporation of ammonium ion into L-gluta-
V
1.65
1.10
0.55
2.0 30 4.0
0a- KETOGLUTARATEmM)
5.0
FIGURE 1. Saturation curve for a-ketoglutarate.Velocity of the reaction (V) is expressed as A340nm/
min/mg of protein of T. cruzi. See Material andMethods. *-* = reaction with NADPH; 0-0 = re-
action with NADH.
mate via the GDH-NADPH-dependent reac-
tion was 19 times greater than that for the
GOGAT-NADPH reaction and GSase was not
a very effective way of immobilizing ammo-
nium ions as compared with GDH- and GO-GAT-NADPH-dependent reactions. The cat-
abolic activity of GDH-NADP+ was roughly
one-seventh that of the NADPH-dependent
GDH biosynthetic activity.
From the results shown in Table I, we as-
sumed that, of the systems studied, GDH is
the most efficient way of immobilizing NH3
derived from protein catabolism in T. cruzi.
Therefore, we decided to further investigate
this system. In Figures 1 and 2, the high spec-
ificity for NADPH, rather than NADH, isshown in both the a-ketoglutarate and NH4 Cl
saturation curves.
The pH optimum using phosphate buffer,
for the GDH-NADPH-dependent reaction is
about 8.5 and that for the NADH is approxi-
mately 9.0. The pH optimum for the catabolic
activity of the GDH-NADP+-dependent re-
action, is about 8.5.
The following apparent Km'swere obtained
from the double reciprocal plots using con-
centrations of substrates in the linear regionof the plot v(, x log S0: ca-ketoglutarate (4.7 x
10-4 M), NH4C1 (4.2 x 10-4 M), NADPH (1.6 x
10-5 M), NADP+ (3 x 10-5 M).
Polyacrylamide gel electrophoresis after
protamine sulfate treatment showed a region
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CALDAS TAL.-AMMONIUMNCORPORATIONN T.CRUZI 215
NH4CI(mM)
FIGURE. Saturationcurve for NH4C1. Velocity
of the reaction (V) is expressed as A340nm/min/mgfprotein of T. cruzi. See Material and Methods.*-* = reaction with NADPH; 0-0 = reaction
with NADH.
near the cathode with strong NADP+-depen-
dent GDH activity and a more anodic band
with less activity. Both regions showed activ-
ity with NAD+ to lesser extent. However, the
more anodic band was retained in Sephadex
G-150, indicating that this protein has a lower
molecular weight than the proteins of thecathodic region.
DISCUSSION
The higher activity of the biosynthetic
GDH-NADPH-dependent reaction in com-
parison with the catabolic enzyme (depen-
dent on NADP+) leads us to postulate that the
main function of the L-glutamate dehydroge-nase system is to incorporate NH3 into organic
compounds and not to produce ammonium.
Based on end-product inhibition studies Sha-
litov et al. (1975) suggested that in Chlorella,
the GDH-NADP+-dependent system has a
synthetic function. Incorporation studies of
15NH3are required to prove definitively the
fate of ammonium in T. cruzi.
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., 1970).
Under our experimental conditions, the spe-cific activity of GSase (which synthesizes
L-glutamine) is roughly one-fifth that of GO-
GAT (NADPH); therefore, the flow of gluta-
mine is too low to supply this substrate to both
the GOGATsystem and to other biosyntheticreactions of the cells.
One cannot exclude the possibility that theGDH enzyme also is used to degrade L-glu-
tamatein T. cruzi as suggested by Cazzulo etal. (1977). However, L-glutamate is a keyamino acid in the L-glutamate family; there-
fore, it is very advantageous for T. cruzi tohave a high GDH biosynthetic activity for
reincorporatingNH3 into amino acids whileit is metabolizing proteins.
In both buffers (Tris and phosphate), the
pH optimum curve for the degradative GDH
gives a higher activity around8.5, which is in
agreement with Cazzulo and co-workers(Caz-
zulo et al., 1977). However, we were able todetect a biosynthetic GDH activity depen-dent on NADH, which was not detected bythem. The pH optimum for this reaction was9.0. We also observed an optimalpH of 8.5 forthe enzyme dependent on NADPH, which is
higher than the value reported by Cazzulo
(Cazzulo et al., 1977). The pH optimum forthe reduction of a-ketoglutarate by theNADP-GDH system in the Tulahuen strainis
7.0. We can suggest two possible explanations
for these differences between our results andthose of Cazzulo et al.: 1)difference in T. cru-zi strains used; and 2) differences in extrac-tion procedure. In our experimental condi-
tions, ,B-mercaptoethanol was used in theextractionbuffer; this was found to be a cru-cial factor in stabilizing the GDH-NAD+ andNADH activities.
The low apparent Km's of NH3 (4.2 x 10-4
M) and a-ketoglutarate (4.7 x 10-4 M) mayhave some biological significance, as suggest-
ed elsewhere (Miflin, 1974); however, thepossibility remains that the system studied inour laboratorycould preferably use the GDH
biosynthetic pathway, whereas the T. cruzi,Tulahuen strain, studied by Cazzulo et al.
(1977), might use the degradative pathway as
they suggested.
ACKNOWLEDGMENTS
This work was supported by the grantSIP-08-072 from the Conselho Nacional de Des-
envolvimento Cientifico e Tecnolo6gico ofBrasil. We thank Dr. Linda Styer Caldas forthe English review, and Dr. Helio Peixoto forthe T. cruzi cultures.
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216 THEJOURNAL FPARASITOLOGY,OL.66, NO.2, APRIL 980
LITERATURECITED
BASH-LEWINSON,D., AND N. GROSSOWICZ.1957.
Transaminases of Trypanosoma cruzi. Bull Res
Counc Isr Sect E 6: 91-92.
BONE, G. J., AND G. PARENT. 1963. Stearic acid, an
essential growth factor for Trypanosoma cruzi.J Gen Microbiol 31: 261-266.
CALDAS,R. A., C. R. FELIX, E. A. FERNANDES,AND
C. R. CERON. 1976. Alguns enzimas do meta-
bolismo nitrogenado de Trypanosoma cruzi.
Cien Cult Sao Paulo 28: 483.
CAMARGO, E. P. 1964. Growth and differentiation
in Trypanosoma cruzi. I. Origin of metacyclic
trypanosomes in liquid media. Rev Inst Med
Trop 6: 93-100.
CAZZULO, J. J., S. M. JUAN, AND E. L. SEGURA.
1977. Glutamate dehydrogenase and aspartateaminotransferase in Trypanosoma cruzi. Comp
Biochem Physiol 56B: 301-303.DAVIES,B. J. 1964. Disc electrophoresis. II. Meth-
od and application to human serum proteins.Ann NY Acad Sci 121: 404-427.
ELLIOT, W. H. 1953. Isolation of glutamine syn-thetase and glutamotransferase from green
peas. J Biol Chem 201: 661-672.
GOA,J. 1953. Determination of protein by the mi-
crobiuret method. Scand J Clin Lab Invest 5:
218-222.
GOLDBERG, S. S., A. A. S. PEREIRA, E. CHIARI, M.
MARES-GUIA, AND G. GAZZINELLI. 1976.
Comparative kinetics of arginine and lysine
transport by epimastigotesand
trypomastigotesfrom two strains of Trypanosoma cruzi. J Pro-
tozool 23: 186-193.
GUTTERIDGE,W. E. 1976. Biochemistry of Try-panosoma cruzi. In New approaches in Amer-
ican trypanosomiasis research. Pan Am Health
Organ Publ 318: 255-262.
HAMPTON,J. R. 1970. Lysine uptake in cultured
Trypanosoma cruzi: Interaction of competitiveinhibitors. JProtozool 17: 597-600.
. 1971a. Serine metabolism in the culture
form of Trypanosoma cruzi: Synthesis of other
amino acids. Comp Biochem Physiol 39B:
999-1003.
. 1971b. Arginine transport in the cultureform of Trypanosoma cruzi. J Protozool 18:
701-703.
JUAN, S. M., E. L. SEGURA,AND J. J. CAZZULO.
1978. Purification and some properties of the
NADP-linked glutamate dehydrogenase from
Trypanosoma cruzi. Int J Biochem 9: 395-400.
LEE, D. W. 1973. Glutamate dehydrogenase iso-
zymes in Ricinus communis seedlings. Phyto-
chemistry 12: 2631-2634.
MANCILLA,R., C. NAQUIRA,AND C. LANAS. 1966.Protein biosynthesis in Trypanosomatidae. I.
In vivo incorporation of leucine-1l-4C into theproteins of Trypanosoma cruzi. Comp Bio-
chem Physiol 18: 241-248.
, , and . 1967. Protein biosyn-thesis in Trypanosomatidae. II. The metabolic
fate of DL-leucine-1l-4C in Trypanosoma cruzi.
Exp Parasitol 21: 154-159.
MEERS,J. L., D. W. TEMPEST,AND C. M. BROWN.
1970. Glutamine (amide): 2-oxoglutarate amino
transferase oxidoreductase (NADP), an enzymeinvolved in the synthesis of glutamate by some
bacteria. J Gen Microbiol 64: 187-194.
MIFLIN, B. F. 1974. The location of nitrate reduc-
tase and other enzymes related to amino acid
biosynthesis in the plastids of root and leaves.Plant Physiol 54: 550-555.
RYAN, E., ANDP. F. FOTTRELL. 1974. Subcellularlocalization of enzymes involved in the assim-
ilation of ammonia by soybean root nodules.
Phytochemistry 12: 2647-2652.
SHATILOV,V. R., M. A. KASPAROVA, . G. AMBART-
SUMYAN,AND V. L. KRETOVICH.1975. Com-parative study of glutamate dehydrogenase of
Chlorella. Biokhimiya 40: 1237-1245.
STRECKER,H. J. 1955. L-glutamic dehydrogenasefrom liver. In S. P. Colowick and N. 0. Kaplan
(eds.), Methods of enzymology. Vol. II. Aca-
demic Press, Inc., New York, New York, p. 220-225.
SYLVESTER,D., AND S. M. KRASSNER. 1976. Pro-
line metabolism in Trypanosoma cruzi epi-
mastigotes. Comp Biochem Physiol 55B: 443-
448.
TEMPEST,D. W., J. L. MEERS,AND C. M. BROWN.
1970. Synthesis of glutamate in Aerobacter
aerogenes by a hitherto unknown route. Bio-
chem J 117: 405-407.VON BRAND, T. 1966. Biochemistry of parasites.
Academic Press, Inc., New York, New York, p.266-269.
ZELED6N,R. 1960a.
Comparative physiologicalstudies on four species of hemoflagellates inculture. V. Transaminases. Rev Brasil Biol 20:409-414.
. 1960b. Comparative physiology of four
species of hemoflagellates in culture. II. Effectof carbohydrates and related substances andsome amino compounds on the respiration. JParasitol 46: 541-545.