Quantificação de glutamato synthase

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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



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



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.



216 THEJOURNAL FPARASITOLOGY,OL.66, NO.2, APRIL 980

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Quantificação de glutamato synthase