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
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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 epimastigote
forms of Trypanosoma cruzi through the following enzymatic systems: NADPH and NADH-dependent
glutamate dehydrogenase, NADPH-dependent glutamate synthase, L-glutamine synthetase and NADH-
dependent glutamate synthase, in order of decreasing specific activity (/tmoles of product formed/min/mg
protein). The pH optima and Km'sfor the glutamate dehydrogenase system were determined. Disc elec-
trophoresis showed the presence of cathodic bands of GDH activity, which were highly dependent on
NADP+.

The nitrogen metabolism of Trypamosoma molecular weight determined, and sonre of its
cruzi is not well understood. Further studies properties studied (Juan et al., 1978).
could lead to information useful in the treat- The study of NH3 incorporation into carbon
ment of Chagas' disease, a major health prob- skeletons is of great interest, because T. cruzi
lem in South and Central America. is known to produce ammonium as the final
Trypanosoma cruzi seems to be highly de- product of protein and amino acid catabolism
pendent on its nitrogen metabolism because (von Brand, 1966). The ammonium produced
it has no known source of storage carbohy- may be immobilized in some less toxic organ-
drate, has a high endogenous respiration, and ic compound, but so far the pathways that T.
43-55% of its dry weight is protein (Gutter- cruzi uses to accomplish the immobilization
idge, 1976). Some transaminases of T. cruzi have not been established.
were studied by Bash-Lewinson and Gros- In the present paper, we studied the com-
sowicz (1957) and by Zeledon (1960a). The parative incorporation of ammonium into
latter found that both L-glutamate and L-as- amino groups of L-glutamate and L-glutamine
partate stimulated the respiration of culture via three different enzymatic systems, as fol-
forms of T. cruzi (Zeledon, 1960b). Proline low:
also stimulated respiration in starved T. cruzi
1) L-glutamate dehydrogenase (L-glutamate:
(Sylvester and Krassner, 1976). NAD oxidoreductase, EC 1.4.1.2 and
Some of the metabolic pathways involving
L-glutamate: NADP oxidoreductase, EC
amino acids in T. cruzi are similar to those
1.4.1.4, GDH);
described for animals (Mancilla et al., 1966,
2) L-glutamate synthase [glutamine (amide):
1967). It also has been shown that the culture
2-oxoglutarate amino transferase oxidore-
form of T. cruzi actively metabolizes L-serine
ductase (NADP+), EC 2.6.1.53, GOGAT];
producing other amino acids (Hampton, and
1971a).
3) L-glutamine synthetase (L-glutamine: am-
The transport of L-arginine and L-lysine by
monia ligase, EC 6.3.1.2, GSase).
T. cruzi has been studied by Hampton and
others (Hampton, 1970, 1971b; Goldberg et MATERIALS AND METHODS
al., 1976).
Caldas et al. (1976) reported the presence Trypanosoma cruzi, Y strain, was maintained in
LIT medium (Camargo, 1964) and grown in Bone
of L-glutamate dehydrogenase (=GDH) in and Parent's medium (Bone and Parent, 1963) for
culture forms of T. cruzi and, recently, Caz- bulk growth, at 28 C with constant agitation (100
zulo et al. (1977) studied glutamate dehydro- rpm) in a rotatoryshaker (Controlled Environment
Incubator Shaker, New Brunswick Scientific Inc.,
genase and aspartate aminotransferase in the New
Jersey).
same system. NADP-linked glutamate dehy- Cells at mid-log phase (120 hr after transfer to
drogenase from T. cruzi has been purified, its Bone and Parent's medium) were harvested by 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
Received for publication 1 August 1978. M 6f-mercaptoethanol (extraction buffer) using 1.0
213
214 THEJOURNAL VOL.66, NO. 2, APRIL1980
OF PARASITOLOGY,
TABLE I. Micromoles of productformed/min/mg of
protein by L-glutamate dehydrogenase (GDH),
L-glutamate synthase (GOGAT), and L-glutamine
synthetase (GSase) in T. cruzi-culture epimasti-
gotes.
Micromoles of
Enzyme 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/1.0 g of cell wet weight. The cells were
then 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, mate via the GDH-NADPH-dependent
5 x 10-4 M MgSO4and 10-4 M /3-mercaptoethanol).
The dialysis was done in a cold room (0-4 C) using
dialysis tubing with pore diameter of 48 A.
Samples of dialysate for disc electrophoresis were
treated with protamine sulfate (10 mg/ml of dialy-
sate) and centrifuged (14,000 g for 20 min, 0-4 C).
The supernatant was chromatographedin a Seph-
adex G-150 column (15 x 1.1 cm) eluted with the
extractionbuffer diluted 1:10.
The hydroxamate assay as described by Elliot
(1953) was used for measuring glutamine synthe-
tase activity. The biosynthetic activity of L-gluta-
mate dehydrogenase was determined following the
procedure described by Ryan and Fottrell (1974),
using NADH or NADPH. The degradative assay for
GDH was carried out by following the procedure
described by Strecker (1955), using NAD+ or
NADP+ as electron acceptors. For the L-glutamate
synthase activity the following concentrations were
used: a-ketoglutarate(5 mM), L-glutamine (5 mM),
NADPH (0.25 mM), or NADH 90.25 mM); the rate
of oxidation of NADPH or NADH was recorded at
340 nm (Meers et al., 1970). Protein was determined
using the microbiuret assay described by Goa
(1953). The basic procedure described by Davies
(1964) was used for disc electrophoresis. We fol-
lowed the concentrations given by Lee (1973) with
4.25% polyacrylamide and Tris-glycine 0.01 M pH
8.2 as running buffer for the specific detection of
GDH on the gels. Tris (hydroxymethyl) amino-
methane 0.1 M and phosphate (potassium salt) 0.1
M buffers were used to determine the pH optima
of 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 5.0
0a- KETOGLUTARATE
(mM)
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 and
Methods. *-* = reaction with NADPH; 0-0 = re-
action with NADH.
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, is
shown 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 region
of 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
 CALDASETAL.-AMMONIUM
NH4 CI (mM)
FIGURE2. Saturationcurve for NH4C1. Velocity agreement with Cazzulo and co-workers (Caz-
of the reaction (V) is expressed as A340nm/min/mg
protein of T. cruzi. See Material and Methods. detect a biosynthetic GDH activity depen-
*-* = 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 the
cathodic 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
15NH3 are 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
IN T. CRUZI
INCORPORATION 215
the GOGAT system and to other biosynthetic
reactions of the cells.
One cannot exclude the possibility that the
GDH enzyme also is used to degrade L-glu-
tamate in T. cruzi as suggested by Cazzulo et
al. (1977). However, L-glutamate is a key
amino acid in the L-glutamate family; there-
fore, it is very advantageous for T. cruzi to
have a high GDH biosynthetic activity for
reincorporating NH3 into amino acids while
it is metabolizing proteins.
In both buffers (Tris and phosphate), the
pH optimum curve for the degradative GDH
gives a higher activity around 8.5, which is in
of zulo et al., 1977). However, we were able to
dent on NADH, which was not detected by
them. The pH optimum for this reaction was
9.0. We also observed an optimal pH of 8.5 for
the enzyme dependent on NADPH, which is
higher than the value reported by Cazzulo
(Cazzulo et al., 1977). The pH optimum for
the reduction of a-ketoglutarate by the
NADP-GDH system in the Tulahuen strain is
7.0. We can suggest two possible explanations
for these differences between our results and
those 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 the
extraction buffer; this was found to be a cru-
cial factor in stabilizing the GDH-NAD+ and
NADH activities.
The low apparent Km's of NH3 (4.2 x 10-4
M) and a-ketoglutarate (4.7 x 10-4 M) may
have some biological significance, as suggest-
ed elsewhere (Miflin, 1974); however, the
possibility remains that the system studied in
our 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 grant SIP-
08-072 from the Conselho Nacional de Des-
envolvimento Cientifico e Tecnolo6gico of
Brasil. We thank Dr. Linda Styer Caldas for
the English review, and Dr. Helio Peixoto for
the T. cruzi cultures.
216 VOL.66, NO. 2, APRIL1980
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