Differential energetic metabolism during Trypanosoma cruzi ...

Molecular and CellularBiochemistry94: 71-82, 1990. © 1990KluwerAcademic Publishers. Printedin the Netherlands. Original Article

Differential energetic metabolism during Trypanosoma cruzi differentiation. II. Hexokinase, phosphofructokinase and pyruvate kinase

Francisco-Javier Adroher 1, Antonio Osuna 1 and Jos6 A. Lupififiez 2 1Departamento de Parasitologfa, Facultad de Farmacia, Universidad de Granada, 18071-Granada, Spain; 2Departamento de Bioqufmica y Biologta Molecular, Facultad de Ciencias, Universidad de Granada, 18001-Granada, Spain Received 13 December1988; accepted17 July 1989

Key words: Trypanosoma cruzi, epimastigotes, metacyclic trypomastigotes, hexokinase, phosphofructokinase, pyruvate kinase Summary

The activities of hexokinase (ATP:hexose-6-phosphate transferase, E.C. 2.7.1.1), phosphofructokinase (ATP:fructose-6-phosphate 1-phosphotransferase, E.C. 2.7.1.11) and pyruvate kinase (ATP:pyruvate transferase, E.C. 2.7.1.40), and their kinetic behaviour in two morphological forms of Trypanosoma cruzi (epimastigotes and metacyclic trypomastigotes) have been studied. The kinetic responses of the three enzymes to their respective substrates were normalized to hyperbolic forms on a velocity versus substrate concentration plots. Hexokinase and phosphofructokinase showed a higher activity in epimastigotes than in metacyclics, whereas pyruvate kinase had similar activity in both forms of the parasite. The specific activity of hexokinase from epimastigotes was 102.00 mUnits/mg of protein and the apparent Km value for glucose was 35.4/~M. Metacyclic forms showed a specific activity of 55.25 mUnits/mg and a Km value of 46.3/~M. The kinetic parameters (specific activity and Km for fructose 6-phosphate) of phosphofructokinase for epimastigotes were 42.60mUnits/mg and 0.31mM and for metacyclics 13.97mUnits/mg and 0.16raM, respectively. On the contrary, pyruvate kinase in both forms of T. cruzi did not show significant differences in its kinetic parameters. The specific activity in epimastigotes was 37.00 mUnits/mg and the Km for phosphoenolpyruvate was 0.47 mM, whereas in metacyclics these values were 42.94 mUnits/mg and 0.46 mM, respectively. The results presented in this work, clearly demonstrate a quantitative change in the glycolytic pathway of both culture forms of T. cruzi. Abbreviations: NNN - Novy-Nicolle-McNeal medium, Eagle's MEM - Eagle's Minimal Essential Medium with Earle's salts, IFCS - heat Inactivated Fetal Calf Serum (56 ° C, 30 rain), Tris - tris(hydroxymethyl) aminomethane, EDTA - Ethylenediaminetetraacetic Acid

Introduction

At least four morphologically well-characterized forms are found in the whole biological cycle of Trypanosoma cruzi, the causative agent of the

American trypanosomiasis, Chagas' disease. These forms are described as the intracellular amastigotes and bloodstream trypomastigotes present in the mammalian host tissues, and epimastigotes and metacyclic trypomastigotes found in the

72 midgut and faeces of the insect vector [1, 2]. One of the most important steps in the T. cruzi life cycle is the differentiation of epimastigotes into metacyclics, the natural infective form, a process known as metacyclogenesis. This occurs naturally in the triatomine insect vector digestive tract [1, 2]. The physiological conditions at the end of the gut of the insect are very similar to those in the culture system where epimastigote forms are differentiated into metacyclics. During the metacyclogenesis process several morphological changes take place in the parasite. These changes have been extensively studied [3, 4], but, on the contrary, there is a paucity of information regarding the biochemical modifications and molecular aspects of this process [5-71 . Epimastigote and metacyclic forms are both readily obtained in axenic cultures of T. cruzi [810]. Nevertheless, most of the biochemical studies have been made in epimastigotes and only a few metabolic works have been carried out in metacyclic forms, mainly due to the lack of uncontaminated amounts of these forms necessary for these studies [11-16]. Carbohydrate metabolism and respiration are two of the most investigated areas of T. cruzi in the last 30 years. Recently, these aspects of the metabolism of epimastigote forms have been reviewed [17, 18]. It is generally accepted that different cultured forms of this flagellate and other trypanosomatids preferentially catabolize glucose as principal energetic source by a process of incomplete oxidation instead of 'aerobic fermentation', as usually is named in the literature. Apart from CO2, as the main end product of glucose catabolism, these cells excrete into the medium some amounts of acetate, malate and succinate [19, 20]. All the enzymes of glycolysis from hexokinase to pyruvate kinase have been reported in cell-flee extracts (see 17 for references). The enzymes hexokinase, phosphofructokinase and pyruvate kinase have been shown to be present with similar activities in three forms of the flagellate (amastigotes, bloodstream trypomastigotes, and epimastigotes) which are all able to degrade glucose and produce succinate with a similar efficiency [21]. Opperdoes et al. [22, 23] were the first to report

the existence in Trypanosoma brucei of cytosolic microbodies names glycosomes. These organelles contain the first seven enzymes of glycolysis (hexokinase, glucose-phosphate isomerase, phosphofructokinase, fructose-bisphosphate aldolase, triose-phosphate isomerase, glyceraldehyde-phosphate dehydrogenase and phosphoglycerate kinase) as well as two enzymes related to glycerol metabolism, glycerol kinase and glycerol-3-phosphate dehydrogenase (NAD+), [22-24]. Later, Gutteridge and his group [25] reported that glycosomes are also present in T. cruzi with similar characteristics to those found in other trypanosomatids. Recent studies on these organelles confirmed the additional presence of other related enzymes such as phosphoenolpyruvate carboxykinase and malate dehydrogenase [26, 27]. These microbodies constitute by far the most important functional difference between trypanosomatids and higher eukaryotic cells where those enzymes mentioned above are all present in the cytosol. Considering the especial features of the glycolytic enzymes compartmentation and the morphological changes that take place during the metacyclogenesis, our aim in this work was to investigate the behaviour of the glycolytic activity in both epimastigote and metacyclic forms. For this purpose, we have comparatively studied some metabolic changes during the growth and differentiation of T. cruzi as well as the kinetics and other parameters of the more important enzymes involved in the regulation of glycolysis. Hexokinase, phosphofructokinase and pyruvate kinase are universally considered as the three rate-controlling enzymes in the regulation of the glycolytic pathway and in the integration of this process with the tricarboxylic acid cycle in most organisms. The activities of these enzymes are continuously modified under the most important types of cellular regulation. Our results clearly indicate significant changes in the kinetics of the glycosomal enzymes in the two morphological forms of T. cruzi involved in the metacyclogenic process of this parasite.

73 Materials and methods

the supernatant fraction was used for the enzymatic activity assays.

Organism The strain of T. cruzi used in these studies was originally supplied from Maracay, Venezuela. Stock cultures were maintained in Novy-NicolleMcNeal (NNN) medium overlaid with Eagle's minimal essential medium (MEM) with Earle's salts plus 20% (v/v) fetal calf serum (FCS) heat inactivated at 56°C for 30 min (IFCS). Subcultures were made every 10-14 days by inoculating freshly prepared flasks with the previous subculture.

Growth conditions For routine experiments, epimastigotes were grown in a monophasic cell-free liquid medium from Bon6 and Parent [8]. Metacyclic forms were obtained in a Grace's insect medium [28] modified and prepared in our laboratory according to the method described previously [29]. The experimental procedure has been described elsewhere [10, 16]. Epimastigote forms were harvested on the 8th day of growth from the cultures that reached about 2.0 × 10 7 cells/ml. The parasites were counted in a Neubauer's haemocytometric chamber. Metacyclic trypomastigote forms were harvested on the 9th day of culture and the mean of the cultures selected had more than 86% of metacyclics, as examined by Giemsa stained preparation under light microscope.

Cell extracts For the preparation of cell extracts, parasites were removed by centrifugation at 3000 x g for 15 min at 4°C and washed three times with a large excess of 0.154 M NaC1. The trypanosomes were resuspended in a small volume of buffer, and homogenized in a MSE sonifier at 4 microns, four times of 30 sec each and 60 sec of resting in an ice bath. The homogenate was then centrifuged at 31000 x g for 30 rain, at 2 ° C. The cell pellet was discarded and

Glucose and ammonium determinations throughout T. cru~ development For the experiments related to determinations of glucose and ammonium levels in the culture medium throughout the parasite development was used Grace's insect medium supplemented with 100 mM of NaC1 and 10% (v/v) IFCS. This medium was inoculated with 1 x 106 epimastigotes/ml grown in NNN culture medium. Under these experimental conditions, also growth and differentiation were followed in the supplemented Grace's medium [15] instead of that culture media especially used for obtaining high amount of epimastigotes [8] and metacyclic forms [29]. This is due because in this later case the almost absence of glucose and the appearance of high amount of free ammonium during the experimental manipulations could make difficult the accurate glucose and ammonium measurements. For these reasons, the parasites transformed into metacyclic forms under these experimental conditions were always below (50-55%) that those obtained using the especial medium [29].

Enzyme assays Hexokinase activity was assayed by Joshi and Jagannathan's method [30] with a light modification, where the D-glucose 6-phosphate formation was coupled to the oxidation of this metabolite by NADP + in the presence of glucose 6-phosphate dehydrogenase (E.C. 1.1.1.49). The final concentrations in the reaction mixture were: 20 mM TrisHC1, pH7.5; 20raM MgC12; 0.01 mM EDTA, disodium salt; 0.13mM NADP, sodium salt; l mM ATP, magnesium salt; 0.22 Units/ml D-glucose 6phosphate dehydrogenase; about 0.1 mg of extract protein; and D-glucose in a range of 0.01-15 mM. The reduction of NADP + was followed spectrophotometrically at 340 rim. Phosphofructokinase activity was measured by a

74 modification of the assay described by Ling et al. [31]. This method utilizes a coupled assay system in the presence of glucose 6-phosphate isomerase (E.C. 5.3.1.9), aldolase (E.C. 4.1.2.13), triosephosphate isomerase (E.C. 5.3.1.1), glycerol-3phosphate dehydrogenase (E.C. 1.2.1.12) and NADH. The final concentrations in the reaction mixture were: 30mM Tris-HC1, pH8.0; 5mM MgSO4; 50mM KC1; l m M dithiothreitol; 2mM ATP, magnesium salt; 2raM AMP, sodium salt; 25/xg/ml aldolase; 17/xg/ml triose-phosphate isomerase plus glycerol-3-phosphate dehydrogenase; 1 Unit./ml glucose 6-phosphate isomerase; 0.25 mM NADH, sodium salt; about 0.1 mg of protein from cell extract, and substrate. The substrate used was glucose 6-phosphate and fructose 6-phosphate in a 3 : 1 ratio. The concentration of fructose 6-phosphate increased from 0.01 mM to 2.0 mM. The oxidation of N A D H was monitored spectrophotometrically at 340 nm. Pyruvate kinase activity was assayed by the method of Valentine and Tanaka [32]. The formation of pyruvate was coupled to its reduction by N A D H in the presence of lactate dehydrogenase (E.C. 1.1.1.27). The final concentrations in the assay mixture were: 8.33mM triethanolamineHC1, pH7.5; 75mM KCI; 8raM MgSO4; 0.4raM ADP, sodium salt; 0.2mM NADH, sodium salt; 6 Units/ml lactate dehydrogenase; about 0.05 mg of extract protein; and phosphoenolpyruvate in a range of concentration of 0.1-10 raM. The reaction was initiated by the addition of substrate and the oxidation of N A D H was followed spectrophotometrically at 340 urn. All spectrophotometric determinations were carried out at 37° C. The enzyme activities are expressed as enzyme units. One unit of hexokinase was defined as that amount catalyzing the reduction of one micromole of NADP + per minute. One unit of phosphofructokinase and pyruvate kinase were defined as those amounts catalyzing the oxidation of two and one micromole of N A D H per minute, respectively.

Kinetic parameters Since the double-reciprocal plot tends to emphasize the data points obtained at low concentrations of substrate, where the degree of error is likely to be greatest [33], the data from the experiments presented in this work were analyzed by the linear Eadie-Hofstee plot. As an additional check, for comparative purposes, the kinetic parameters were also determined from a simple least-squares fit of the untransformed data to a rectangular hyperbola [34] described by the equation: V = Vmax. IS]/ (Kin + [S]). This non-linear plot was constructed with the aid of a computer program designed by us in this laboratory. The activity ratio is defined as the relationship between the enzyme activity at subsaturating substrate concentration (Vss) and maximum velocity (Vmax). Catalytic efficiency, defined as the ratio between enzyme activity and Km, was determined at two substrate concentrations: Vss/Km, which indicates the relationship between the amount of enzyme-substrate complex [ES] at S substrate concentration and the affinity of the enzyme; and Vmax/Km, which relates the total enzyme concentration [Et] with the interaction between the enzyme and the substrate.

Analysis of data Results are expressed as means + S.E.M. Statistical comparisons between epimastigotes and metacyclic trypomastigotes of T. cruzi were done using the Student's t distribution.

Other procedures Protein was determined in the supernatant of the cell extracts according to Lowry et al. [35] and Bradford [36], using crystalline bovine serum albumin as standard. Glucose was estimated by the method of Bergmeyer and Berut [37]. Ammonium was determined by a micromodification of a procedure recommended by Sigma Chemical Co. for its 170-UV kit.

75 ul O100

tl.3

11.2

6.0

5.5

5.2

5.8

6.5

30

O m

i'r20

,-,

t'i'-

O Z 10 \ I"

0

2

4

DAYS

6

OF

II

10

12

CULTURE

Fig. 1. Opposite changes in the levels of glucose and ammonium in the medium during growth and differentiation of Trypanosoma cruzi. The culture conditions for these experiments are d e s ~ b e d in the Materials and Methods section. The variations of glucose ([]) and ammonium (W) in the medium along the culture time are expressed as percentage of initial (day O) and final (day 12) values, respectively. The data at these time were, respectively, 5.9 mM and 1.9 mM for glucose and 0.04 mM and 7.80 mM for ammonium. The growth of T. cruzi ( ) are expressed as (cells/ml) × 10 -6 and metacyclic forms (MI) as percentage of total number of organisms. Epimastigote cells correspond to the difference from 100 with respect to metacyclics.

Chemicals

Results

Chemicals were purchased from Riedel de Ha6n (Seelze, Hannover, FRG). Biochemicals were obtained from Sigma Chemical Co. (St. Louis, MO., USA). Auxiliar enzymes were supplied by Boehringer Mannheim (FRG). Eagle's MEM, FCS and Grace's insect medium were obtained from Gibco (Middlesex, UK). Ammonium determinations were carried out by following the instructions of the 170-UV kit from Sigma Chemical Co. All other chemicals used were analytical reagents of the highest purity available.

Metacyclogenesis and variations of glucose and ammonium in the medium throughout T. cruzi development The growth of the organisms, metacyclics differentiation as well as the variations of the extracellular levels of glucose and ammonium in the presence of oxygen, have been followed and the results are shown in Fig. 1. In our experimental conditions, the growth of the organisms was clearly associated with a decrease in the levels of glucose in the medium. Under these conditions, the process of cellular differentiation of T. cruzi took place with a continuous increase in metacyclic forms. The exponential phase of metacyclogenesis coincided with the stationary phase of the growth. At the end of growth

76

V o

o

100

a6o

200

5

v S

rn Units / m M

o

10

v

mUnits / r a M

B

l

10

o'.5

i

I1

'

[Gtucos~]

g mM

1'0

1 S

Fig. 2. Effect of glucose concentration on hexokinase activities in epimastigote and metacyclic forms of Trypanosoma cruzi. Panel A: Eadie-Hofstee plots with respect to substrate. The Vmax and Km obtained from these plots were 98.8mU/mg of protein and 25/aM, respectively, for epimastigotes, with a regression coefficient of r = 0.961; and 53.7 mU/mg and 35/~M for metacyclics (r = 0.972). Panel B: Initial velocities are plotted against substrate concentrations. Data are the mean _+S.E.M. of at least three experiments in trip]icate. V represents enzyme activity (mU) and S glucose concentration. Epimastigote forms (0). Metacyclic forms (0).

2 [F6P]

mM

tl

5 S

Fig. 3. Effect of fructose 6-phosphate concentration on phosphofructokinase activities in epimastigote and metacyclic forms of Trypanosoma cruzi. Panel A: Eadie-Hofstee plots with respect to substrate. The Vmax and Km obtained from these plots were 40.3mU/mg of protein and 0.29raM, respectively, for epimastigotes, with a regression coefficient of r = 0.969; and 13.9 mU/mg and 0.15 mM for metacyclics (r = 0.997). Panel B: Initial velocities are plotted against substrate concentrations. Data are the mean ± S.E.M. of at least three experiments in triplicate. V represents enzyme activity (mU) and S fructose 6-phosphate concentration. Epimastigote forms (O). Metacy-

clic forms (O). more than 50% of metacyclic forms were differentiated in these media. The consumption of glucose and the growth of epimastigote cells followed a similar pattern. The highest value of glucose consumption was obtained with the largest number of epimastigote cells. When these flagellate forms began to decrease, something similar occurred with the values of glucose consumption, even when an equal total number of parasite forms (epimasfigotes plus meta-

cyclics) were present, which is indicative of a reduced glucose catabolism by the metacyclic trypomastigote forms. At the same time, there was an increase of ammonium production of about 200fold and these high values coincided with the maximum number of metacyclic forms of the parasite. It is interesting to point out that the highest concentrations of ammonium produced were parallel to the exponential phase of metacyclics differentiation. This increase was maximum when the pop-

77 ulation of epimastigotes diminished, indicating that metacyclic forms induced a stimulation of the oxidative metabolism of amino acids, quantitatively m o r e important than that carried out by epimastigote cells.

Glycolytic enzymes in epimastigote and metacyclic trypomastigote forms of Y. cruzi The three enzymes universally considered as the key regulatory enzymes in the glycolytic pathway, hexokinase, phosphofructokinase and pyruvate kinase in both, epimastigote and metacyclic forms of T. cruzi, were studied.

Hexokinase. The effect of glucose concentration on enzyme activity is depicted in Fig. 2. Results show that in both forms of the parasite, hexokinase activity display simple Michaelis-Menten kinetics. In epimastigotes, the enzyme activity was found to be higher than in the metacyclic forms. At all concentrations of glucose used (0.01 to 15.00raM) the reaction rate in epimastigotes, measured as initial velocity, was always double (in a range of 1.85 to 2.35 fold). The apparent Michaelis constant (Kin), the maximal velocity (Vmax) and the activity ratio measured as the relationship between the initial velocity at subsaturating substrate concentration

and V m a x ( V o J V m a x ) are shown in Table 1. With regard to the substrate, the enzyme in both T. cruzi forms exhibited a high and similar affinity (35 and 46/xM) whereas specific activity was almost 2-fold higher in epimastigotes than in metacyclic trypomastigote forms. On the other hand, V0.0jVmax was the same in both forms of the flagellate. The catalytic efficiency of the enzyme (Vmax/Km) was almost 2.5 times higher in epimastigotes [2.9 x 106nrnol/(mg p r o t e i n . m i n . M ) ] than in metacyclics [1.2 x 106 nmol/(mg protein, m i n . M)].

Phosphofructokinase. The dependence of the reaction rate of phosphofructokinase in both forms of the parasite on fructose 6-phosphate concentration is shown in Fig. 3. Hyperbolic kinetic plots of enzyme velocity against concentration of fructose 6phosphate were obtained without evidence of sigmoidicity. This was confirmed by the Hill's plots of the data (not shown) which gave a values for interaction coefficient (n) of 1.37 and 1.41 for epimastigotes and metacyclics, respectively. The respective plots showed regression coefficients (r) of 0.994 and 0.980. The kinetic p a r a m e t e r s are given in Table 1. Significant differences can be observed in all parameters studied (Kin, Vmax and V0.U Vmax). It is interesting to note a significant increase in the apparent K m for the epimastigote forms, which probably indicates a m o d e r a t e inhib-

Table1. Changes in kinetic parameters of the glycosomalhexokinase (HK) and phosphofructokinase (PFK) during Trypanosomacruzi differentiation at insect vector stages Enzyme

Parasite forms

Kin (~M)

Vmax (mU/mg)

Activityratio Vss/Vmax

Catalyticefficiency Vss/Km

HK PFK

Epimastigotes Metacyclics Epimastigotes Metacyclics

35.4+ 2.6 46.3+ 6.4 310_+ 50 160-+ 10"

TM

102.0+ 4.1 55.2_+ 2.5*** 42.60_+ 2.02 13.97+ 0.57"**

0.58 + 0.03 0.56_+ 0.05~' 0.14+ 0.01 0.33+ 0.02**

1.67 + 0.08 0.67_+ 0.05*** 19.2_+ 1.3 28.8_+ 1.4"*

Vmax/Km 2.89 + 0.06 1.20_+ 0.07*** 139.1+ 9.3 87.4_+ 1.1"*

Epimastigotes and metacyclictrypomastigotes of T. cruziwere isolated, grown and harvested as indicated in the Materials and Methods section. The kinetic parameters (Vmax and Km) were determined from a simple least-squares fit of the untransformed data and constructed with a computer program. Vss in both, the activityratio and catalytic efficiencyfor hexokinase (HK) and phosphoffuctoldnase (PFK), represents the specificactivities of these enzymes at 50 kLMof glucose and 50 izM of fructose 6-phosphate, respectively.The units of catalytic efficiencyare mU/(mg - M) for HK, and mU/(mg - mM) for PFK. Data are the means _+S.E.M. of 3 to 5 experiments in triplicate. P values refer to significance of difference between epimastigotes and metacyclic trypomastigotes: (*) P < 0.05; (**) P < 0.01; (***) P < 0.001; (ns) not siLsnificant.

78 Tab/e 2. Kinetic parameters of the cytosolic pyruvate kinase from epimastigote and metacyclic forms of Trypanosoma cruzi.

Km (raM) Vmax (mUnits/mg) Vss/Vmax Vss/Km (U/rag.M) Vmax/Km (U/mg.M)

2 mUnlts / m M

1

5 [P E P] mM

Epimastigotes

Metacyclics

0.47 + 0.04 37.00 + 3 . 4 6 0.28 + 0.02 22.0 + 2.3 78.7 _+ 8.0

0.46_+ 0.04 42.94_+ 2.87 0.33_+ 0.03 30.8 + 3.3 93.3 + 9.1

The kinetic parameters (Km and Vmax) were determined from a simple least-squares fit of untransformed data to a rectangular hyperbola. This non-linear plot was constructed with the aid of a computer program designed by us. Vss in both, the activity ratio and catalytic efficiency for pyruvate kinase, represents the specific activity of this enzyme of 20/zM of phosphoenolpyruvate. Data are the mean + S.E.M. of 3 experiments in triplicate. The difference of the data between epimastigotes and metacyclic trypomasfigotes was not significant (Student's t test).

4 V

10 S

Fig. 4. Effect of phosphoenolpyruvate concentration on pyruvate kinase activities in epimastigote and metacyclic forms of Trypanosorna cruzi. Panel A: Eadie-Hofstee plots with respect to substrate. The Vmax and Km obtained from these plots were 36.2 mU/mg of protein and 0.47 raM, respectively, for epimastigotes, with a regression coefficient of r = 0.930; and 43.2mU/ mg and 0.49raM for metacyclics (r =0.975). Panel B: Initial velocities are plotted against substrate concentrations. Data are the mean + S.E.M. of at least three experiments in triplicate. V represents enzyme activity (mU) and S phosphoenolpyruvate concentration. Epimastigote forms (O). Metacyctic forms (0). ition of the activity of this e n z y m e at subsaturating substrate concentrations in accordance with the results of U r b i n a and Crespo [38]. H o w e v e r , the catalytic efficiency of phosphofxuctokinase in epimastigotes was almost double than that in metacyclics [1.4 x 10 5 n m o l / ( m g p r o t e i n - rain. M) and 0.8 x 105 n m o l / ( m g p r o t e i n - m i n - M ) , respective-

lyl. Pyruvate kinase. T h e kinetic d e v e l o p m e n t s of this

e n z y m e for the two morphological forms of T. cruzi plotted as reaction rates against substrate concentrations are shown in Fig. 4. Similarly to the hexokinase and p h o s p h o f r u c t o k i n a s e kinetics, p y r u v a t e kinase displays in both forms of the parasite a typical Michaelis-Menten kinetics. N o evidence of sigmoidicity was found. W h e n these data were treated by Hill's equation, the interaction coefficients (n) for epimastigotes and metacyclics were 1.08 and 1.00, respectively (the values of the regression coefficients were 0.992 and 0.998, respectively). T h e values of Km, V m a x and V002/Vmax of pyruvate kinase in respect to p h o s p h o e n o l p y r u v a t e are shown in Table 2. In contrast to the other e n z y m e s assayed, there was no significant difference in any of the kinetic parameters of p y r u v a t e kinase in b o t h forms of T. cruzi. These findings indicate a different behaviour of p y r u v a t e kinase c o m p a r e d to hexokinase and phosphofructokinase, which is particularly interesting in the metacyclic forms.

Discussion Despite the large v o l u m e of available data on the c a r b o h y d r a t e catabolism in several forms of the biological cycle of T. cruzi [17-21], there is very little information on the characteristics and role of this metabolic p a t h w a y in metacyclic trypomasti-

79 gote forms [11, 15], probably due to the difficulty in obtaining high populations and pure samples of these flagellate forms in vitro. For these reasons, the purpose on this investigation was to make a comparative study of the glucose catabolism in the two differentiated forms of T. cruzi at the insect vector stages, epimastigotes and metacyclic trypomastigotes. Accordingly, the activities and some kinetic parameters of the three enzymes carrying out the catalysis of those reactions generally considered as the key regulatory steps of this metabolic pathway in numerous organisms (hexokinase, phosphofructokinase, and pyruvate kinase) were studied. From the results presented in this work, it is clearly demonstrated that the glycolytic activity in epimastigote forms of the parasite is significantly higher than in metacyclic forms. In our assay systems, the activities of hexokinase (Fig. 2) and phosphofructokinase (Fig. 3) were always 2-3 times higher in epimastigotes than in metacyclic forms. Furthermore, the higher catalytic efficiency of those enzymes in epimastigote forms, especially at cellular substrate concentrations, also explain these results. However, no changes in the activities of pyruvate kinase in both forms of T. cruzi were found. The enzymes hexokinase and phosphofructokinase in T. cruzi are inside the glycosomes [25], an especial membrane-bound microbody-like organelle which contain a number of glycolytic enzymes involved in the conversion of glucose and glycerol into 3-phosphoglycerate [22-24]. For this reason, in Trypanosomatidae these microbodies play an important role in energy metabolism. Glucose is converted to two molecules of 3-phosphoglycerate by a mechanism of aerobic glycolysis and this metabolite constitutes one of the end-products of glycosomal metabolism. In T. brucei, 20-30% of the glycolytic intermediates are found in glycosomes and they equilibrate slowly with the cytosolic pool

[391. On the other hand, it is generally accepted that epimastigotes are the main forms of T. cruzi in which cellular division take place whereas metacyclic forms have lost this capacity. An increased cell division requires high availability of energy, in

terms of ATP, and epimastigote forms could obtain it by a high activity of glycolysis, as indicated by the augmented activities of the glycosomal enzymes. Furthermore, we could observe that metacyclogenesis was stimulated when the level of glucose in the culture medium diminished (Fig. 1 and Ref. 10 and 15). Under this nutritional situation, metacyclic forms have a limited capacity to utilize glucose as energetic source through the glycolytic pathway. The activities of hexokinase and phosphofructokinase in metacyclic forms support this assumption. The differences observed in the activities of glycosomal enzymes, but not in the pyruvate kinase activity in both forms of the parasite, could be due to differences in the number of glycosomes between epimastigote and metacyclic forms. In this sense, several authors have shown that the number of glycosomes per cell may vary greatly from one species to another and that, even within different stages of the life cycle of the same species, considerable variations can occur. So, in parasites highly glucose-dependents, such as T. brucei bloodstream trypomastigotes, it has been estimated that between 200 and 300 glycosomes are present per cell [40]. In the other genera of the family Trypanosomatidae, these organelles may not be as abundant as in T. brucei, since the amastigote stage of Leishmania mexicana has recently been reported to conrain as few as ten glycosomes per cell [41]. These variations in the number of glycosomes are probably reflecting changes in the relative metabolic importance of the organelle according to its capacity of glucose utilization. Results here reported appear to point out the possible existence of a coordinate repression mechanism by which the levels of glycosomal protein are modulated. By this mechanism, the number of glycosomes could vary between the different stages of the parasite biological cycle in agreement with its especial metabolic features. On the other hand, several authors [38, 42-44] reported that hexokinase and phosphofructokinase from T. cruzi epimastigotes present a small capacity of regulation by the cell energy charge and oxidative activity, and this fact suggests that the glycolytic flux is poorly regulated in this organism. However, a repression

80 mechanism during metacyclogenesis could explain the capacity of these cells to regulate glucose utilization. In addition, the unchanged activity of pyruvate kinase in both forms of the trypanosome give suport to this hypothesis. In a preceding paper [16], we have shown an important and significant mitochondrial metabolic shift during metacyclogenesis. The activities of mitochondrial enzymes, citrate synthase, NADPlinked isocitrate dehydrogenase and succinate dehydrogenase, in metacyclic trypomastigotes were higher than in epimastigotes. As a result of this, the mitochondrial activity in metacyclics was higher than in epimastigotes, which preferably use the carbon skeleton of carbohydrate as energy source [45]. Our results are also in agreement with Cannata and Cazzulo [27] who provide excellent evidence for the coordinated participation of the glycosomes and mitochondrion in the partial catabolism of carbohydrate in the epimastigotes. During epimastigote stage, the non-infective forms, T. cruzi have an active carbohydrate metabolism using mainly saccharides such as glucose, fructose, and other derivatives [46], however, its mitochondrial metabolic activity is reduced [16]. When the trypanosomes are differentiated from non-infective stage into infective (metacyclic) stage, a reduction of glycosomal metabolism is produced with a noted increase of the activities of, at least, some mitochondrial enzymes, which allow the utilization of amino acids as energetic source. These amino acids could constitute a metabolic reserve, accumulated as proteins, synthesized and stored in the epimastigote stage [6, 15, 16]. These changes in the metabolic activity throughout T. cruzi metacyclogenesis could be related to a phenomenon of enzyme induction-repression in different subcellular organelles dependent with the environmental conditions. The glycosomal enzymes could undergo an induction in the epimastigote stage, while the mitochondrial enzymes could suffer a catabolic repression, which could explain the incomplete mitochondrial oxidation of glucose to yield mono- and dicarboxylic acids by the coordinated participation of the specific enzymes of the glycosome and mitochondrion [20, 27]. On the contrary, a coordinated repression of the glycosomal

enzymes along with an induction mechanism of the mitochondrial enzymes could take place in the metacyclic stage, which could allow a significant increase in the functionality of the tricarboxylic acid cycle, necessary for the amino acids utilization [45]. Nevertheless, the mechanisms of metabolic adaptation during T. cruzi differentiation remain to be elucidated and are now under investigation. In conclusion, these results related to others previously reported [16] clearly demonstrate the existence of an extraordinary difference between both glycosomal and mitochondrial metabolism of the two different morphological forms of T. cruzi at the insect vector stage, namely the epimasfigote and the infective metacyclic forms. Undoubtedly, the knowledge of the metabolic differences not only between host and parasite but also between the two forms of the latter present in the life cycle of the organism, may provide targets for rational drug design programmes. Several potential targets for chemotherapic exploitation have been identified. The glycosome is one of them [47]. Therefore, the study of glycosomal glycolyfic enzymes could lead to a first approach to the rational drug design. In addition, and according to previous studies [16], the mitochondrial metabolism in T. cruzi is very important at least at the metacyclic stage, thus potentiating the study of mitochondrial enzyme inhibitors as other potential targets for new trypanocidal drugs.

Acknowledgements The authors are gratefully indebted to Dr. L.A. del Rfo Legazpi for the critical reading of this manuscript and helpful advice. We wish to thank to Dr. R. Benttez Rodrfguez and Dr. G. Ortega Tortes for advice and collaboration. F.J.A. was recipient of a long term fellowship from the Spanish Plan de Formaci6n de Personal Investigador (PFPI). This work was supported by the Spanish Comisi6n Asesora de Investigaci6n Cientffica y T6cnica (CAICYT) grants no. 3786/79 and 1067/82.

81 References 1. Brener Z: Biology of Trypanosoma cruzi. Annu Rev Microbiol 27: 34%382, 1973 2. De Souza W: Cell biology of Trypanosoma cruzi. Int Rev Cytol 86: 197-283, 1984 3. Hoare CA: The trypanosomes of mammals. BlackweU Scientific Publications, Oxford and Cambridge, 1972 4. Brener Z, Alvarenga N J: Life cycle of T. cruzi in the vector. In American Trypanosomiasis Research. Sci Publ No 318, PAHO, Washington DC, 1976, pp 83-86 5. Contreras VT, Morel CM, Goldenberg S: Stage specific gene expression precedes morphological changes during Trypanosoma cruzi metacyclogenesis. Mol Biochem Parasitol 14: 83-96, 1985 6. Contreras VT, Salles JM, Thomas N, Morel CM, Goldenberg S: In vitro differentiation of Trypanosoma cruziunder chemically defined conditions. Mol Biochem Parasitol 16: 315-327, 1985 7. Nagakura K, Tachibana H, Kaneda Y: Alteration of the cell surface acid phosphatase concomitant with the morphological transformation in Trypanosoma cruzi. Comp Biochem Physiol 81B: 815-817, 1985 8. Bon6 GJ, Parent G: Stearic acid, an essential growth factor for Trypanosoma cruzi. J Gen Microbio131: 261-266, 1963 9. Brun R, Jenni L: Cultivation of African and South American trypanosomes of medical or veterinary importance. Br Med Bull 41: 122-129, 1985 10. Adroher FJ, Lupi~ifiez JA, Osuna A: Influence of saccharides and sodium chloride on growth and differentiation of Trypanosoma cruzi. Cell Differ 22: 165-170, 1988 11. Funayama S, Funayama S, Ito I, Veiga LA: Trypanosoma cruzi: Kinetic properties of glucose 6-phosphate dehydrogenase. Exp Parasitol 43: 376-381, 1977 12. Wood DE: Trypanosoma cruzi: Fatty acid metabolism in vitro. Exp Parasitol 37: ~ , 1975 13. Wood DE, Schiller EL: Trypanosoma cruzi: Comparative fatty acid metabolism of the epimastigotes and trypomastigotes in vitro. Exp Parasitol 38: 202-207, 1975 14. Adroher FJ, Osuna A, Lupigifiez JA: Fructose 1,6-bisphosphatase activity in two Trypanosoma cruzi morphological forms. J Parasitol 73: 438-441, 1987 15. Lupig~fiez JA, Adroher FJ, Vargas AM, Osuna A: Differential behaviour of glucose 6-phosphate dehydrogenase in two morphological forms of Trypanosoma cruzi. Int J Biochem. 19: 1085-1089, 1987 16. Adroher FJ, Osuna A, Lupi~ifiez JA: Differential energetic metabolism during Trypanosoma cruzi differentiation. I. Citrate synthase, NADP-isocitrate dehydrogenase and succinate dehydrogenase. Arch Biochem Biophys 267: 252261, 1988 17. Gutteridge WE: Trypanosoma cruzi: Recent biochemical advances. Trans R Soc Trop Med Hyg 75: 484-492, 1981 18. Cannata JJB, Cazzulo JJ: The aerobic fermentation of glucose by Trypanosoma cruzi. Comp Biochem Physiol 79B: 297-308, 1984

19. Bowman IBR, Tobie El, Von Brand T: CO2 fixation studies with the culture form of Trypanosoma cruzi. Comp Biochem Physiol 9: 105-114, 1963 20. Cazzulo JJ, Franke de Cazzulo BM, Engel JC, Cannata JJB: End products and enzyme levels of aerobic glucose fermentation in trypanosomafids. Mol Biochem Parasitol 16: 32%343, 1985 21. Rogerson GW, Guttefidge WE: Catabolic metabolism in Trypanosoma cruzi. Int J Parasitol 10: 131-135, 1980 22. Opperdoes FR, Borst P: Local~ation of nine glycolytic enzymes in a microbody-like organelle in Trypanosoma brucei: the glycosome. FEBS Lett 80: 360-364, 1977 23. Opperdoes FR, Borst P, Bakker S, Leene W: Localization of glycerol-3-phosphate oxidase in the mitochondfion and NAD+-linked glycerol-3-phosphate dehydrogenase in the microbodies of the bloodstream form of Trypanosoma brucei. Eur J Biochem 76: 2%39, 1977 24. Opperdoes FR: Compartmentafion of carbohydrate metabolism in trypanosomes. Annu Rev Microbiol 41: 127152, 1987 25. Taylor MB, Berghausen H, Heyworth P, Messenger N, Rees LJ, Gutteridge WE: Subcellular localization of some glycolytic enzymes in parasitic flagellated protozoa. Int J Biochem 11: 11%120, 1980 26. Cannata JJB, Valle E, Docampo R, Cazzulo JJ: Subcellular l~alJzation of pbosphoenolpyruvate carboxykinase in the trypanosomatids Trypanosoma cruzi and Crithidia fascicu/ata. Mol Biochem Parasitol 6: 151-160, 1982 27. Cannata JJB, Cazzulo JJ: Glycosomal and mitochondrial malate dehydrogenases in epimastigotes of Trypanosoma cruzi. Mol Biochem Parasitol 11: 37-49, 1984 28. Grace TDC: Establishment of four strains of cells from insect tissues grown in vitro. Nature 195: 788-789, 1962 29. Osuna A, Jim6nez-Ortiz A, Lozano J: Medios de cultivo para la obtenci6n de formas metaciclicas de Trypanosoma cruzi. Rev Ib6r Parasitol 39: 12%133, 1979 30. Joshi MD, Jagannathan V: Hexokinase. I. Brain. Methods Enzymol 9: 371-375, 1966 31. Ling KH, Paetkan V, Marcus F, Lardy HA: Phosphofructokinase. I. Skeletal muscle. Methods Enzymol 9: 4LSM29, 1966 32. Valentine WN, Tanaka KR: Pyruvate kinase: clinical aspects. Methods Enzymol 9: 468~73, 1966 33. Fersht A: Enzyme structure and mechanism. 2nd edn. WH Freeman & Co., Reading and San Francisco, 1985 34. Atkins GL, Nimmo IA: A comparison of seven methods for fitting the Michaelis-Menten equation. Biochem J 149: 775777, 1975 35. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265-275, 1951 36. Bradford MM: A rapid and sensitive method for the quantitation of microgram quantifies of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254, 1976 37. Bergmeyer HU, Bernt E: D-glucose: Determination with

82

38.

39. 40.

41.

42.

43.

glucose oxidase and peroxidase. In: HU Bergmeyer (ed). Methods of enzymatic analysis. Verlag-Chemie Academic Press, New York, 1974, pp 1205-1215 Urbina JA, Crespo A: Regulation of energy metabolism in Trypanosoma (Schizotrypanurn) cruzi. I. Hexokinase and phosphofructokinase. Mol Biochem Parasitol 11: 22.5-239, 1984 Visser N, Opperdoes FR: Glycolysis in Trypanosorna brucei. Eur J Biochem 103: 6 ~ 2 , 1980 Opperdoes FR, Baudhuin P, Coppens I, De Roe C, Edwards SW, Weijers PJ, Misset O: Purification, morphometric analysis, and characterization of the glycosomes (microbodies ) of the protozoan hemoflagellate Trypanosoma brucei. J Cell Biol 98: 1178-1184, 1984 Tetley L, Coombs GH, Vickerman K: The distribution of cell organelles in Leishmania amastigotes as shown by three-dimensional reconstruction. Parasitology 87: xxxvi, 1983 Racagni GE, Machado de Domenech EE: Characterization of Trypanosoma cruzi hexokinase. Mol Biochem Parasitol 9: 181-188, 1983 Aguilar Z, Urbina JA: The phosphofructokinase of Trypa-

44.

45.

46.

47.

nosoma (Schizotrypanurn) cruzi: purification and kinetic mechanism. Mol Biochem Parasitol 21: 103-111, 1986 Taylor M, Gutteridge WE: The regulation ofphosphofructokinase in epimastigote Trypanosoma cruzi. FEBS Lett 201: 262-266, 1986 CAceres O, Fermindes JF: Glucose metabolism, growth and differentiation of Trypanosorna cruzi. Rev Brasil Biol 36: 397-410, 1976 LehmannDL: Comparative utilization of carbohydrates by culture forms of Trypanosoma (Schizotrypanum) cruzi and T. ranarum. Ann Trop Med Parasitol 57: 232-234, 1963 Michels PAM: Compartmentation ofglycolysis in trypanosomes: a potential target for new trypanocidal drugs. Bin Cell 64: 157-164, 1988

Address for offprints: J.A. Lupi(~fiez, Departamento de Bioqulmica y Biologta Molecular, Facultad de Ciencias, Avenida Fuentenueva s/n, Universidad de Granada, 18001-Granada, Spain