Introduction

The pentose phosphate pathway (PPP) represents an alternative route for glucose oxidation in cells. The major products of its oxidative phase, NADPH and ribose 5-phosphate, are essential for cell survival playing a variety of metabolic roles, e.g., lipid and nucleotide biosynthesis, as well as regulation of the redox state of the cell (Wood 1986). Glucose 6-phosphate dehydrogenase (β-d glucose 6-phosphate: NAD(P) oxidoreductase EC 1.1.1.49) catalyzes the first step of the PPP through the reaction:

$${\text{ $ \beta $ - D}}\;{\text{glucose }}6{\text{ - phospate}} + {\text{NAD}}\left( {\text{P}} \right)^ + \to 6{\text{ - phosphogluconolactone}} + {\text{NAD}}\left( {\text{P}} \right){\text{H}} + {\text{H}}^ + $$

Under in vivo conditions, the reaction is irreversible due to the hydrolysis of the lactone by a lactonase (Levy 1979). The activity of glucose 6-phosphate dehydrogenase (G6PDH) is the main control site for the flux of glucose 6-phosphate (Glc-6P) toward the PPP. In this sense, the concentration of NAD(P)+ is a primary factor governing such activity (Wood 1986). The enzyme shows a wide spectrum with regard to the specificity for the nicotinamide coenzyme, ranging from NADP-specific G6PDH to NAD-specific G6PDH (Levy 1979).

In parasitic flatworms, the presence of the PPP has been documented (Agosin and Aravena 1960; Agosin and Repetto 1961) and the activity of both G6PDH and 6-phosphogluconate dehydrogenase in crude extracts measured (Smith and Brown 1977; McManus and Smyth 1982). However, in spite of the importance of carbohydrate metabolism in these organisms, no detailed information on G6PDH is available. Up to 20% of glucose consumption in larval Echinococcus granulosus is due to the PPP (Agosin and Aravena 1960; Agosin and Repetto 1961). In addition to its critical involvement in determining the flux of glucose into the PPP, G6PDH activity is also essential for those NADPH-dependent processes. Therefore, knowledge of the properties of G6PDH of cestodes is essential in order to understand its role in cell metabolism. In the present work, we undertook the purification and characterization of such enzyme from the Taenia crassiceps metacestode (cysticercus).

Materials and methods

Reagents

d-Glucose 6-phosphate (sodium salt), 6-phosphogluconate (trisodium salt), NADP+, NADPH, NAD+, glucose, glucosamine 6-phosphate (free acid), and DEAE-Trisacryl were obtained from Sigma-Aldrich (St. Louis, MO, USA). All reagents needed for electrophoresis were purchased from Biorad. 2′5′-ADP-Sepharose 4B and gel strips for isoelectric focusing were obtained from Amersham-Pharmacia Biotech (Uppsala, Sweden). All chemicals were used without further purification.

Growth of T. crassiceps cysticerci

Female Balb/c mice were inoculated intraperitoneally with about 15 cysticerci of the T. crassiceps HYG strain, as described in Larralde et al. (1989). Six to 8 months later, mice were killed and the cysticerci recovered from the peritoneal cavity. Before use, larvae were washed thoroughly with phosphate-buffered saline solution.

Enzyme assays

G6PDH activity was determined following the increase in absorbance at 340 nm due to NADP+ reduction. Unless otherwise indicated, all enzyme assays were performed in 0.1 M Tris/HCl buffer (pH 7.8) 1 mM EDTA at 25°C in a final volume of 1 mL. NADP+ and Glc-6P were incubated in the buffer in a typical experiment, and the reaction was started by adding an enzyme aliquot. The assays were carried out at final concentrations of 160 μM NADP+ and 600 μM Glc-6P for the purification of the enzyme. One unit of enzyme activity is defined as the amount of protein catalyzing the reduction of 1 μmol NADP+ per minute at 25°C. 6-Phosphogluconate dehydrogenase activity was determined under the same conditions by replacing Glc-6P by 500 μM 6-phosphogluconate.

The effect of temperature on G6PDH activity was determined in the range of 15°C to 50°C in Tris/HCl buffers adjusted to pH 7.8 at the corresponding temperature. An enzyme aliquot was incubated for 2 min in the presence of NADP+ at the indicated temperature, and the reaction was started by the addition of Glc-6P.

Kinetic data analysis

Initial velocity data were obtained for the determination of the kinetic parameters by varying the concentration of both substrates in a 5 × 5 data matrix. K m values were obtained from the global fit of the data to the following rate equation:

$$v = \frac{{{\text{Vm}}{\left[ {\text{A}} \right]}{\left[ {\text{B}} \right]}}}{{K{\text{i}}_{{\text{A}}} K_{{\text{B}}} + K_{{\text{A}}} {\left[ {\text{B}} \right]} + K_{{\text{B}}} {\left[ {\text{A}} \right]} + {\left[ {\text{A}} \right]}{\left[ {\text{B}} \right]}}}$$
(1)

where A and B represent NADP+ and Glc-6P, respectively. KiA is the dissociation constant of substrate A from the EA complex, and K A and K B are the K m for substrates A and B, respectively. Initial velocity experiments involving an inhibitor were carried out at a saturating concentration of the fixed substrate, and data were analyzed with the corresponding velocity equation (Segel 1975). All kinetic data represent the average of five independent determinations.

Protein determination

The dye-binding technique (Bradford 1976) was used with bovine serum albumin as standard.

Electrophoresis and isoelectric focusing

Polyacrylamide gel electrophoresis under denaturing conditions was carried out essentially as described in Laemmli (1970). Gels were stained by conventional procedures. The relative mobility of the enzyme was compared with that of known protein standards for subunit molecular weight determination. The isoelectric point was determined by using a 3–10-pH-immobilized gradient gel strip. Immobiline dry strips were rehydrated and the focusing performed according to Berkelmen and Stenstedt (1998). A mixture of nine protein markers with isoelectric points ranging from 4.45 to 9.6 was used.

HPLC gel filtration

Size-exclusion chromatography was carried out in a SEC 3000 HPLC column (0.75 cm × 30 cm). The buffer used for pre-equilibration and elution was degassed and filtered through a Millipore 0.22-μm filter. The elution profiles were monitored recording the absorbance at 280 nm. The relative molecular weight and the Stokes radius were determined from the elution volumes data as described in Hagel (1998).

Purification of enzyme

All purification steps were performed at 4°C. The soluble fraction of T. crassiceps cysticerci was obtained as described by del Arenal et al. (2005). The high-speed supernatant was brought to 30% saturation by adding solid ammonium sulfate under continuous stirring. The resultant precipitate was discarded by centrifugation at 12,000×g for 20 min, and the supernatant was saturated up to 60% with ammonium sulfate. The protein pellet containing the G6PDH activity was recovered by centrifugation as described above, resuspended in 50 mM Tris/HCl buffer (pH = 8.5) containing 1 mM EDTA, and dialyzed overnight. The active fraction was adsorbed onto a DEAE-Trisacryl column (2.6 cm × 10 cm) previously equilibrated in the Tris/HCl buffer at pH 8.5. After washing the non-adsorbed protein, the enzyme was eluted by a linear NaCl concentration gradient (0–0.3 M) prepared in the same buffer. A single peak of activity was eluted at 0.05 M NaCl. Fractions containing the G6PDH activity were pooled and dialyzed overnight against 0.1 M Tris/HCl buffer (pH 7.8) containing 1 mM EDTA. The retentate was applied onto a 2′5′-ADP-Sepharose 4B column (2.6 cm × 2 cm) previously equilibrated at pH 7.8 in the Tris/HCl buffer. After washing the column, the enzyme was eluted by a linear NADP+ concentration gradient (0–200 μM) prepared in the same buffer. Under these conditions, G6PDH eluted as a symmetrical activity peak at about 100 μM NADP+. Active fractions containing the dehydrogenase activity were pooled, dialyzed overnight, and concentrated to a minimal volume in Amicon Ultra tubes (Millipore). The enzyme was stored frozen at −10°C until use.

Results

Activity of G6PDH and 6-phosphogluconate dehydrogenase in crude extracts

Glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities were detected in the soluble fraction of T. crassiceps cysticerci. However, the specific activity of G6PDH was ten times higher (24 ± 3.1 mU mg−1) than the activity of 6-phosphogluconate dehydrogenase (2.2 ± 0.4 mU mg−1).

Purification of G6PDH

Table 1 shows the results of a typical purification. The enzyme was purified over 1,400-fold with a total yield of 32%. The preparation has a specific activity of 33.8 U mg−1 at pH 7.8 and 25°C. Such activity increased to 67.6 ± 3.9 U mg−1 when the enzyme assays were carried out at 39°C. Both denaturing PAGE and isoelectric focusing patterns revealed a homogeneous preparation (Fig. 1b and c).

Fig. 1
figure 1

Electrophoretic patterns of purified G6PDH from T. crassiceps cysticerci. Lines A and B represent polyacrylamide gel electrophoresis profile obtained under denaturing and reducing conditions. Before run, an enzyme sample was heated in the presence of SDS and β-mercaptoethanol at final concentrations of 2% and 0.7 M, respectively. A Molecular weight markers, B purified G6PDH, C isoelectric focusing pattern obtained in the presence of urea 8 M

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Table 1 Purification of glucose 6-phosphate dehydrogenase of T. crassiceps
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Physical properties

A Mr of 61,000 ± 1,700 was obtained from the electrophoretic mobility of the enzyme under denaturing conditions for the enzyme subunit (Fig. 1b). On the other hand, gel filtration experiments performed under native conditions gave values of 134,000 ± 17,200 and 44.2 ± 3.3 Å, respectively, for both Mr and Stokes radius. Hence, G6PDH from T. crassiceps exists as a dimeric protein. An isoelectric point of 4.5 was estimated for the enzyme.

General catalytic properties and substrate specificity

The enzyme showed maximal activity between pH 6.7 and 7.8 (data not shown). d-Glucose 6-phosphate and NADP+ were the preferred substrates of the enzyme. K m values of 14 ± 1.7 μM and 1.3 ± 0.45 μM for Glc-6P and NADP+, respectively, were obtained from the global fitting of the initial velocity data to the rate equation at pH 7.8 and 25°C. A turnover number of 34 s−1 was calculated at 25°C, resulting in a catalytic efficiency of 26.1 × 106 M−1 s−1 with NADP+ as substrate. Glucose, 2-deoxyglucose, and glucosamine 6-phosphate were not substrates at all. NAD+ was accepted as a substrate; however, concentrations of the nucleotide in the mM range were needed in order to observe activity. Table 2 summarizes the kinetic parameters of G6PDH from T. crassiceps. The enzyme showed no essential requirement for divalent cations.

Table 2 Kinetic parameters of G6PDH from Taenia crassiceps
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Activation energy

The activity of G6PDH was measured in the temperature range of 15°C to 50°C. The Arrhenius plot showed a curvature at the highest temperatures tested (Fig. 2), and such behavior was not due to protein denaturation, as revealed by experiments in which the enzyme was preincubated at various times at a high temperature (50°C) before addition of the second substrate. After 4-min incubation, no loss in activity was observed (data not shown). In order to obtain precise values for activation energies, a statistical analysis of data was performed (Puterman et al. 1988). The results of such analysis revealed activation energies of 9.91 ± 0.51 and 7.94 ± 0.5 kcal mol−1 with a transition temperature at 40.5°C.

Fig. 2
figure 2

Arrhenius plot shows the dependence of G6PDH activity on temperature. Enzyme samples were incubated at the indicated temperature as described under “Materials and methods section” in the presence of 75 μM NADP+. Reaction was started by adding glucose 6-phosphate at a final concentration of 300 μM. From the analysis of the biphasic plot, the activation energies, as well as the transition temperature, were calculated

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Kinetic mechanism

The initial velocity patterns with either Glc-6P or NADP+ as variable substrates shows that T. crassiceps G6PDH follows a sequential mechanism (Fig. 3). However, no clear distinction between a rapid equilibrium random bi bi system or a steady-state ordered bi bi mechanism can be made based solely on the initial velocity patterns (Segel 1975). Hence, inhibition studies were performed to discriminate between both options. Since the low stability of the phosphogluconolactone produced by the enzyme (Horecker and Smyrniotis 1953) prevents its use as an inhibitor, we selected NADPH, glucosamine 6-phosphate, and 6-phosphogluconate as inhibitors. As expected, NADPH behaves as a competitive inhibitor regarding NADP+, while 6-phosphogluconate acted similarly with respect to Glc-6P (data not shown). A K i value of 1.95 ± 0.4 μM was calculated from the fitting of initial velocity data to the corresponding equation for NADPH. On the other hand, NADPH was a noncompetitive inhibitor with Glc-6P as the variable substrate (data not shown). When glucosamine 6-phosphate was used as an inhibitor with NADP+ as the variable substrate, a nonintersecting pattern in the double reciprocal plots was obtained, suggesting a parabolic inhibition (data not shown). Table 3 summarizes the results of the inhibition studies. Taking initial velocity data and inhibition studies in conjunction lead us to assume a random bi bi sequential kinetic mechanism in rapid equilibrium for G6PDH from T. crassiceps.

Fig. 3
figure 3

Initial velocity patterns of G6PDH from T. crassiceps. a Dependence of enzyme activity on glucose 6-phosphate concentration at the following fixed NADP+ concentrations: filled circle 2.3 μM, empty circle 3.5 μM, inverted filled triangle 6 μM, filled square 11 μM, empty triangle 18 μM. b Dependence of enzyme activity on NADP+ concentration at the following fixed glucose 6-phosphate concentrations: filled circle 4 μM, empty circle 5.5 μM, inverted filled triangle 8.2 μM, empty triangle 14 μM, filled square 22 μM. Lines were traced by adjustment of initial velocity data to Eq. 1 described under “Materials and methods section”

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Table 3 Inhibition patterns and inhibition constants of T. crassiceps G6PDH
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Discussion

T. crassiceps cysticerci constitutes an interesting and practical model for the study of metabolism in the larval stages of cyclophyllidean cestodes. Its unusual ability to proliferate asexually inside the peritoneal cavity of mouse allows us to obtain a large amount of biomass under laboratory conditions (Freeman 1962). Furthermore, as the larval stage lacks both male and female reproductive systems, its molecular complexity must be lesser as compared with the adult worm. Thus, the purification of any protein is greatly simplified.

The detection of both G6PDH and 6-phosphogluconate dehydrogenase activities in the soluble fraction of larval T. crassiceps suggests the presence of an active PPP in this cestode. The specific activity obtained in the present work is similar to data available from protoscoleces of E. granulosus and Echinococcus multilocularis (McManus and Smyth 1982). G6PDH is the most active enzyme in both Echinococcus species as well as in T. crassiceps for the production of NADPH during the oxidative phase of PPP. An identical conclusion can be drawn from the data obtained in crude extracts of the digenean trematodes Schistosoma mansoni and Schistosoma japonicum (Smith and Brown 1977). With regard to the relative importance of T. crassiceps PPP in the production of cytosolic NADPH, the active NADP-dependent malic enzyme found in the larval stage of this species must be taken into consideration (Zenka and Prokopic 1987). Its specific activity in crude extracts of the cytoplasmic fraction is about half the value obtained in the present work for the G6PDH. Based on these data, it can be concluded that G6PDH plays an important role in cytosolic NADPH production in T. crassiceps larvae.

The specific activity of 33.8 U mg−1 found for T. crassiceps G6PDH is low when compared with the enzyme from animal sources. However, such activity was doubled at 39°C (67.6 ± 3.9 U mg−1). This latter value represents a more physiological condition in the mammalian intermediary host. Although a low dehydrogenase activity could be due to the absence of an activator in the enzyme assays, no increase in the enzyme activity was observed when either Mg2+ or Ca2+ was added.

It is worth noting the low K m values for both NADP+ and Glc-6P found in this work for G6PDH. These values were obtained from the initial velocity patterns and thus represent authentic K m values. The K m of 1.3 μM for NADP+ is comparable with values found among some mammalian G6PDH (Shreve and Levy 1980; Özer et al. 2002). Thus, although the catalytic constant of the enzyme is relatively low, the affinity toward its substrates is high, resulting in a catalytic efficiency in the expected range for a G6PDH. As regards to substrate specificity, the enzyme of T. crassiceps showed absolute specificity for Glc-6P. From the viewpoint of coenzyme specificity, G6PDH from T. crassiceps must be included within the group of G6PDH that use NADP+ as the preferred coenzyme but with the ability to use NAD+ under forced conditions (Levy 1979).

The results of molecular weight determination under both native and denaturing conditions strongly suggest G6PDH from T. crassiceps larvae exists as a dimeric protein. Moreover, the fact that a single band was revealed in the electrofocusing experiment, which was performed in the presence of 8 M urea, indicates that both subunits are identical in amino acid composition. As regards to the isoelectric point, the value of 4.5 is comparable with that reported for a variety of G6PDH (Olive and Levy 1971; Levy 1979; Ulusu et al. 1999).

The finding of a biphasic Arrhenius plot was unexpected. In those cases where an Arrhenius plot is showed, a linear dependence was observed (Glaser and Brown 1955; Ulusu et al. 1999). The change in the rate-limiting step at high temperatures is the most plausible explanation for a biphasic Arrhenius plot (Segel 1975).

As regards to the kinetic mechanism followed by T. crassiceps G6PDH, it must be noted that, depending on the source of the enzyme, a variety of kinetic mechanisms have been reported (Kanji et al. 1976; Özer et al. 2001; Shreve and Levy 1980; Özer et al. 2002; Wang et al. 2002). In the case of G6PDH from T. crassiceps, all kinetic data were consistent with a random bi bi sequential mechanism in rapid equilibrium. This conclusion was based in the following experimental evidence: First, the intersecting patterns observed with the initial velocity data clearly exclude a ping-pong mechanism; secondly, the symmetrical character of the double reciprocal plots with either Glc-6P or NADP+ as the variable substrate lead us to discard an ordered sequential mechanism in rapid equilibrium; and third, the results obtained with NADPH and glucosamine 6-phosphate as inhibitors (Table 3). The inclusion of the aminosugar in the enzyme assays with NADP+ as the variable substrate for an ordered mechanism would result in uncompetitive inhibition. Instead, a general noncompetitive pattern was observed. An identical pattern was obtained when the product NADPH was tested with Glc-6P as the variable substrate. Thus, the results of the kinetic experiments lead us to conclude that the kinetic behavior of G6PDH from T. crassiceps cysticerci is best described by a random bi bi sequential kinetic mechanism in rapid equilibrium.

Finally, the low K i value found in this work for NADPH suggests a potential role as a regulator of G6PDH activity in T. crassiceps. Assuming that the total pool of NADP+ + NADPH remains essentially constant, a notable dependence of G6PDH activity on the “reducing charge” was found (Fig. 4), suggesting that in larval T. crassiceps, the flux of glucose 6-phosphate toward the PPP can be modulated by the reduction level of its nicotinamide coenzyme. This observation may establish a functional connection between G6PDH and the multifunctional NADPH-dependent thioredoxin-glutathione reductase (Rendón et al. 2004) through the redox status of the NADP pool.

Fig. 4
figure 4

Effect of the reducing charge on G6PDH activity. Each point was obtained by incubation of the enzyme in the presence of a variable NADP+/NADPH ratio such that [NADP+] + [NADPH] = 50 μM. Reaction was started by adding glucose 6-phosphate at a final concentration of 100 μM. The activity obtained at 50 μM NADP+ in the absence of NADPH was taken as 100% relative activity

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Notice

All efforts were made to minimize the number of animals used and their suffering. All procedures were performed in strict accordance with the Institute for Laboratory Animal Research (ILAR) Guide for Care and Use of Laboratory Animals as well as the guidelines and requirements of the Ethical Committee of the Faculty of Medicine at Universidad Nacional Autónoma de México (UNAM).