Maurer MS

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Kinetic Data of D-Glyceraldehyde-3-phosphate Dehydrogenase from HeLa Cells Juliana B.B. Maurer*1, Fernanda Bovo1, Elisa M. Gomes2, Helena M.S. Loureiro3, Fabíola R. Stevan4, Selma F. Zawadzki-Baggio1 and Momoyo Nakano1 1

Department of Biochemistry and Molecular Biology, Federal University of Paraná, P.O. Box 19046, 81531-990, Curitiba, Paraná, Brazil; 2Institute of Technology of Paraná - TECPAR, 81350-010, Curitiba, PR, Brazil; 3Department of Nutrition, Centers of Biology and Healthy Sciences, Catholic University of Paraná, 80215-901, Curitiba, PR, Brazil; 4 Centers of Biology and Healthy Sciences, Positivo University, 81280-330, Curitiba, PR, Brazil Abstract: D-Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the reversible oxidation of D-glyceraldehyde-3-phosphate (G3P) to 1,3-bis-phospho-D-glycerate, and as such participates in the glycolytic conversion of glucose to pyruvic acid in most living organisms. The glycolytic pathway plays an important role in tumor cells, but it is not clear whether the enzyme kinetics of GAPDH or their response to inhibitors, substrates, and cofactors differ between tumor cells and normal cells. To obtain a tumor-derived GAPDH sample, HeLa cells were chosen on the basis of their homogeneous differentiation pattern and ease of harvesting. We carried out experiments to investigate whether the enzyme kinetics of purified GAPDH from HeLa cells were altered in the presence of reagents containing sulfhydryl groups, divalent metal ions, and cellular metabolites such as nucleotides and coenzymes. The kinetic data were compared with data for GAPDH from normal tissue. GAPDH from HeLa cells was activated by 2-mercaptoethanol and dithioerythritol. The maximum activation was obtained at a 1 mM concentration of each reducing agent. Cupric and mercuric ions (1 mM), as well as p-chloro and phydroxymercuribenzoate (10 µM), fully inhibited enzymatic activity. Among the nucleotides tested, 3′–5′-cyclic AMP (cAMP) was the most effective inhibitor at 30 mM concentration, with a relative activity of 22.79 (±1.76), which was significantly different (p ≤ 0.05) from that of the control, which had 100% activity in the absence of adenine nucleotide. Enzyme inhibition by adenine nucleotides appeared to be via competition with NAD+. The apparent inhibition constants (Ki) for ADP, 5′-AMP, and cAMP were 2.1 mM, 1.0 mM, and 0.6 mM, respectively. GAPDH from HeLa cells was inactivated when incubated in the presence of G3P or NADH at 37 °C, and in both cases the presence of 2-mercaptoethanol protected the enzyme against inhibition. The presence of EDTA did not affect the inactivation of the enzyme by NADH, which suggested that the inactivation of HeLa GAPDH by NADH is not related to the presence of heavy metal ions. Our kinetic analysis showed that although the GAPDH of HeLa cells has a lower specific activity and stability compared with GAPDH from normal tissue, its kinetic characteristics were similar, reinforcing the key role of this enzyme in the metabolism of tumor cells.

Keywords: Adenine nucleotides, D-Glyceraldehyde-3-phosphate p-hydroxymecuribenzoate, sulfhydryl reagents. 1. INTRODUCTION The enzyme D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH, D-glyceraldehyde-3-phosphate: NAD + oxidoreductase, EC 1.2.1.12), one of the key enzymes in glycolysis, was first isolated from yeast by Warburg and Christian (1939) [cited by 1] and since then, it has been isolated and purified from various other sources [2-6]. GAPDH has been well characterized not only because of its central role in intermediary metabolism but also because of its abundance and ease of preparation. GAPDH enzymes isolated from various sources do not differ markedly with respect to their sedimentation coefficients [7] or their amino acid composition [7-9]. Because of the extensive homology *Address correspondence to this author at the Department of Biochemistry and Molecular Biology, Federal University of Paraná, P.O. Box 19046, 81531-990, Curitiba, Paraná, Brazil; Tel: + 55 41 3361 1576; Fax: + 55 41 3266 2042; E-mail: [email protected]; 1573-4080/15 $58.00+.00

dehydrogenase,

GAPDH,

HeLa

cells,

in its amino acid sequence among different sources, GAPDH is also used for studying evolutionary relationships [10]. GAPDH is a critical enzyme in the carbohydrate metabolism of cells [11, 12]. GAPDH catalyzes the reversible oxidation of D-glyceraldehyde-3-phosphate (G3P) to 1,3-bisphospho-D-glycerate (1,3-BPG) (Fig. 1A), and as such participates in the glycolytic conversion of glucose to pyruvic acid in most living organisms [11]. The enzyme with 144 kDa consists of 4 identical subunits, as indicated by the letters O, P, Q, and R in (Fig. 1B). Each 330-amino acid subunit has 2 domains, 1 of which binds NAD+ (NAD+binding domain) [8, 10, 13, 14]. The NAD+-binding domain comprises amino acids 1–148 [10]. The other is the catalytic domain (residues 149–329), containing a cysteine residue (Cys-149) that is involved in the enzyme-coenzyme chargetransfer interactions [10, 15, 16]. In addition to its pivotal glycolytic function, a fraction of GAPDH is localized in the nucleus and can be involved in numerous processes, including regulation of the length of telomeres [17], DNA repair © 2015 Bentham Science Publishers

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Maurer et al.

Fig. (1). Overview of the reaction and the structure of HeLa GAPDH. (A) Schematic reaction of HeLa GAPDH: linkage of the substrate (G3P) to the thiolate group of the Cys residue (Cys-S-); formation of a thiohemiacetal intermediary (not shown) and oxidation of the intermediary by NAD+; NADH formed is changed by another NAD+; Pi participates in phosphorolysis (not shown), liberating 1,3-BPG [11]. (B) Schematic representation of the overall structure of the human liver GAPDH [modified from 10]. The identical subunits are indicated by the letters O, P, Q, and R. Each monomer is bound to an NAD+ molecule (the scale is not representative). Symmetry axes are shown as dashed arrows and the Q axis (not shown) is perpendicular to the plane of the paper.

[18], gene expression [19], and regulation of cyclin functions [20]. The glycolytic pathway plays an important role in tumor cells [21]. The key glycolytic enzymes, as well as other enzymes such as GAPDH, are crucial because they are involved in the production of energy and the formation of glycolytic intermediates, which in turn are important for supplying the pentose phosphate pathway [21]. The importance of GAPDH in tumor cells has previously been highlighted. It was reported that human lung cancer cells contained a protein of 37 kDa that had strong homology with GAPDH [22]. The cited authors concluded that the protein was GAPDH due to the finding of partial amino acid sequence homology between the 37 kDa peptide and GAPDH and they suggested a possible involvement of GAPDH itself or a GAPDH-related protein in lung tumorigenesis [22]. Considering that studies on the identification and kinetics of GAPDH in tumors may contribute to knowledge about metabolism in tumor cells, our group previously undertook a pilot study with human breast tumor tissue, in which we isolated and purified the enzyme, and performed basic kinetic assays (unpublished data). However, the interpretation of some kinetic results was hindered because different parts of the tumor tissue exhibited variable degrees of differentiation. Hence, in order to obtain a tumor-derived sample that has high homogeneous differentiation pattern and is easier

to harvest, HeLa cells (a human tumor cell line) were chosen. These cells are more amenable to analysis than are solid tumor samples from organs such as the uterus or breast, because the latter present several disadvantages, including different degrees of cell differentiation, tissue heterogeneity, and difficulty in obtaining the material. The purification of GAPDH from HeLa cells and some kinetics data were described in a previous work [23]. The basic kinetic rate constants and pH profile of GAPDH from HeLa cells [23] showed similarity with those of muscle enzymes isolated from different sources such as Caiman sp. (reptile) [5], Anas sp. (duck) [6], and Oryctolagus sp. (rabbit) [24]. However, both the yield and specific activity of HeLa GAPDH were lower than those of the animal muscle enzymes [23]. It was also observed that the GAPDH from HeLa cells exhibited a more rapid loss of activity than did GAPDH from other sources. Thus, we purified GAPDH from HeLa cells and carried out experiments in order to investigate if the enzyme source (a tumor cell lineage) affects its activity and stability in the presence of reagents containing sulfhydryl groups, divalent metal ions, and cellular metabolites such as nucleotides and coenzymes. The kinetic data were compared with kinetic data for GAPDH from normal tissue. Comparative analyses of GAPDH from different sources and different cell lines are also important for evolutionary studies of this enzyme, which have shown that its amino acid sequence is highly conserved, thus reinforcing the biochemical importance of this enzyme in different metabolic situations.

D-Glyceraldehyde-3-phosphate

dehydrogenase from HeLa Cells

2. MATERIALS AND METHODS 2.1. Reagents G3P, NAD+, NADH, adenine nucleotides, CDP, CMP, UDP, DEAE-Sephadex A-50 (Sigma Chemical Co.; St. Louis, MO, USA); minimum essential Eagle’s medium (MEM, Flow Laboratories; Irvine, CA, USA); trypsin (Difco; Detroit, MI, USA); and fetal bovine serum (Laborclin; Curitiba, Paraná, Brazil) were purchased as indicated. All other chemicals were of analytical grade. 2.2. Cell Line The human cervix adenocarcinoma cell line (HeLa) was obtained from TECPAR Institute (Curitiba, Paraná, Brazil). The HeLa cells were maintained in MEM supplemented with 10% fetal bovine serum at 37 °C in a humidified incubator containing 5% CO2 for 72 h. The cells were harvested by trypsinization and viable cells were counted by the trypan blue exclusion test using a phase-contrast microscope [25]. All cell preparations used for experiments had at least 95% cell viability. 2.3. Isolation and Purification of GAPDH from HeLa Cells GAPDH from HeLa cells was purified according to Ohkubo, Nakamura, Tokunaga, and Sakiyama [22] with some modifications. Solid ammonium sulfate was slowly added to the crude extract to 60% saturation. After 30 min, the precipitate was removed by centrifugation at 15,000 ×g for 30 min and discarded. The enzyme was precipitated by addition of solid ammonium sulfate (90% saturation) to the supernatant. The solution was left to stand for 30 min, centrifuged (15,000 ×g), and the precipitate was resuspended in 1 mL of 50 mM Tris-HC1 buffer (pH 8.2) containing 1 mM EDTA and 1 mM 2-mercaptoethanol. The enzyme was dialyzed overnight against the same buffer. The dialyzed enzyme was layered onto a DEAE-Sephadex® A-50 column (2.3 × 40 cm), equilibrated with 50 mM Tris-HC1 buffer (pH 8.2) containing 1 mM EDTA and 1 mM 2-mercaptoethanol. Elution was carried out with the same buffer at a flow rate of 30 mL/h, with the enzyme being eluted immediately after the column void volume. Fractions containing enzyme activity were collected and concentrated by ultrafiltration (GF-25, Amicon Corporation, Millipore; Bedford, MA, USA). Under these assay conditions, the specific activity for the purified enzyme was 32 units/mg of protein. 2.4. Substrate DL-Glyceraldehyde-3-phosphate was prepared as described by Vieira, Veiga, and Nakano [26]. The concentration of the D-isomer was determined spectrophotometrically, according to Dagher & Deal [27].

Current Enzyme Inhibition, 2015, Vol. 11, No. 2

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1 mM NAD+, 1 mM G3P, and 10 µg of protein. An enzyme unit was defined as the amount of enzyme capable of reducing 1 µmol of NAD+/min at 25 °C. The specific activity is expressed as enzyme units/mg of protein. 2.6. Protein Determination Protein quantity was estimated according to previously described methods [28, 29]. 2.7. Statistical Analysis The equilibrium constant inhibition, Ki, was calculated using non-linear regression analysis. Normality and homogeneity data were analyzed using an analysis of variance (ANOVA). Tukey’s test was used for determining the statistical significance (p ≤ 0.05). The results are expressed as relative activity (%) ± standard deviation. All experiments were performed at least in triplicate for each indicated condition and in at least 2 independent experiments. 3. RESULTS 3.1. Effect of 2-Mercaptoethanol and Dithioerythritol on Enzyme Activity Fig. (2) shows that HeLa cell GAPDH exhibited maximum activation in the presence of 2-mercaptoethanol or dithioerythritol at 1 mM concentration. In the absence of reducing agents, the enzyme had approximately 60% of its maximum activity.

Fig. (2). Effects of 2-mercaptoethanol and dithioerythritol on the activity of GAPDH from HeLa cells. The assay mixture contained the following in a final volume of 1 mL: 6.6 µg of enzyme, 50 mM Tris-HCl buffer (pH 8.2), containing 1 mM EDTA, 1 mM G3P, 50 mM arsenate, 1 mM NAD+, and varying amounts of 2mercaptoethanol (MSH) or dithioerythritol (DTT). Results not connected by the same letter were significantly different (p ≤ 0.05, Tukey’s test).

2.5. GAPDH Activity Assay

3.2. Effect of Divalent Metal Ions on GAPDH Activity

Enzyme activity was determined by the method of Dagher & Deal [27]. The reaction mixture, in a final volume of 1.0 mL, contained 50 mM Tris-HCl buffer (pH 8.2) with 1 mM EDTA and 1 mM 2-mercaptoethanol, 50 mM arsenate,

The effect of various metal ions at 1 mM concentration on enzyme activity was studied using the standard assay system (Table 1). Potent inhibition by cupric and mercuric ions showed the presence of sulfhydryl groups at the active site.


Table 1.

Maurer et al.

Effect of divalent metal ions on the activity of GAPDH from HeLa cells.

Salt (1 mM)

Relative activity (%)

none

100 ± 0.57

MnCl2

98.6 ± 0.92

MgCl2

94.6 ± 2.51

CaCl2

71.3 ± 2.50

CdCl2

24.0 ± 3.00

CuCl2

0 ± 0.00

HgCl2

0 ± 0.00

3.3. Effect of Sulfhydryl Reagents on GAPDH Activity The effect of sulfhydryl reagents was studied using the standard assay system without 2-mercaptoethanol (Table 2). The effect of 2-mercaptoethanol on the reversal of inhibition by mercury was also shown. The most effective inhibitors were p-chloro (p-CMB) and p-hydroxymercuribenzoate (pHMB), at 10 µM concentration, but their inhibition was partially reversed by subsequent addition of excess 2mercaptoethanol (5 mM). The enzyme activity was 2% (± 1.00) and 52% (± 3.60) in the presence of p-HMB at concentrations of 5 µM and 0.005 µM, respectively. The addition of 2-mercaptoethanol (5 mM) partially reversed the inhibition by p-HMB at these concentrations.

The enzyme was incubated for 10 min at 25 °C in the presence of the metal ion and absence of G3P. The enzymatic activity was assayed in a final volume of 1 mL containing: 50 mM Tris-HCl buffer pH 8.2, containing 1 mM EDTA and 1 mM 2mercaptoethanol, 1 mM G3P, 50 mM arsenate, 1 mM NAD, and 13.2 µg of enzyme.

3.4. Inhibition of HeLa Cell GAPDH by Adenine Nucleotides

Similar results were observed for the GAPDH enzymes from Thermus thermophilus [30], Caiman sp. [5], and Anas sp. [6]. The cadmium ion inhibited 71% (± 2.51) of HeLa GAPDH activity at 1 mM concentration, whereas the enzyme from T. thermophilus [30] was completely inhibited under the same condition.

The inhibition of HeLa cell GAPDH by ATP, ADP, 5′AMP, and 3′–5′-cyclic AMP (cAMP) is shown in Fig. (3). The most effective inhibitor was found to be cAMP, which produced approximately 70% inhibition at 10 mM concentration. The inhibition of the enzyme by adenine nucleotides appeared to be via competition with NAD+. The apparent

Table 2.

Effect of sulfhydryl reagent on the activity of GAPDH from HeLa cells. Added compound (concentration µM)

Relative activity (%) a

Relative activity in the presence of 5 mM 2mercaptoethanol (%) b

None

100 ± 0.50

100 ± 0.00

Iodoacetamide (100 µM)

21 ± 1.51

23 ± 2.51

2-chloro4-aminobenzoate (100 µM)

22 ± 2.00

44 ± 2.50

Ethylmaleimine (100 µM)

11 ± 1.90

19 ± 2.31

p-chloromercuribenzoate (10 µM)

0 ± 0.00

50 ± 1.51

p-hydroxymercuribenzoate (50 µmM)

0 ± 0.00

50 ± 0.80

p-hydroxymercuribenzoate (10 µM)

0 ± 0.00

50 ± 0.90

p-hydroxymercuribenzoate (5 µM)

2 ± 1.00

50 ± 0.90

p-hydroxymercuribenzoate (0.005 µM)

52 ± 3.60

73 ± 2.00

The enzyme (6.6 µg) was incubated for 10 min at 25 °C in the presence of the reagents in 50 mM Tris-HCl buffer pH 8.2, containing 1 mM EDTA. The remaining activity was assayed by adding 1 mM G3P, 50 mM arsenate, and 1 mM NAD; b The incubation mixture was the same as described in (a), except that 5 mM 2-mercaptoethanol was added immediately before the assay for determining the remaining activity of the enzyme. a

Fig. (3). Effect of adenine nucleotides on the activity of GAPDH from HeLa cells. The assay mixture contained the following in a final volume of 1 ml: 6.6 µg of enzyme, 50 mM Tris-HCl buffer pH 8.2, containing 1 mM EDTA, 1 mM 2-mercaptoethanol, 1 mM G3P, 50 mM arsenate, 1 mM NAD+, and varying amounts of the inhibitor. All results of the experiments with adenine nucleotides (0.3–30 mM concentration) were statistically different (p ≤ 0.05, Tukey’s test) from those of the control experiment, which corresponds to 100% of relative activity.



inhibition constants, Ki, for the adenine nucleotides were KiADP = 2.1 mM, Ki5′-AMP = 1.0 mM, and KicAMP = 0.6 mM. 3.5. Comparison of the Effect of Nucleotides on HeLa Cell GAPDH Activity Fig. (4) shows the effect of the nucleotides CDP, UDP, and CMP on HeLa cell GAPDH activity. These nucleotides inhibited approximately 30% of the GAPDH activity at 30


5

mM concentration. On the basis of their effectiveness in inhibiting GAPDH activity, these nucleotides were divided into 3 classes [31]: (a) the strong inhibitor class (>50% inhibition), consisting only of cAMP, which produced 70% inhibition at 10 mM concentration; (b) the moderate inhibitor class (22–50% inhibition), including CDP, ADP, 5′-AMP, and CMP, which produced 22–50% inhibition at a 10 mM concentration; and (c) the weak inhibitor class (0–21% inhibition), including ATP, CDP, and UDP, which produced ≤21% inhibition at 10 mM concentration. These results were similar to those described for yeast GAPDH [31]. 3.6. Effect of Glyceraldehyde-3-phosphate or NADH on GAPDH Stability at 37 °C Fig. (5) shows that HeLa cell GAPDH at 2.5 µM concentration was inactivated at 37 °C in the presence of G3P (0.05 and 0.5 mM concentration). NAD+ did not protect the enzyme from inactivation by G3P, but 2-mercaptoethanol provided protection mainly at a 0.05 mM concentration of G3P.

Fig. (4). Effects of diphosphate and monophosphate nucleotides on the activity of GAPDH from HeLa cells. The assay mixture contained the following in a final volume of 1 mL: 12 µg of enzyme, 50 mM Tris-HCl buffer (pH 8.2), containing 1 mM EDTA, 1 mM 2-mercaptoethanol, 1 mM G3P, 50 mM arsenate, 1 mM NAD+, and varying amounts of the nucleotide. All results of the experiments with the adenine nucleotides (0.3–30 mM concentration) were statistically different (p ≤ 0.05, Tukey’s test) in relation to the control experiment without addition of adenine nucleotide, which corresponds to 100% of relative activity.

Fig. (6) shows that HeLa cell GAPDH at 2.5 µM concentration was inactivated at 37 °C in the presence of NADH. Furthermore, when NAD+ was present in the assay mixture, it protected the enzyme from inactivation by NADH, but only at a concentration of 0.05 mM of reduced coenzyme (Fig. 6A). The protective effect of 2-mercaptoethanol was also evident (Figs. 6A and 6B). Maximum protection was observed when the enzyme was incubated in the presence of both NAD+ and 2-mercaptoethanol. The possibility that the inactivation of GAPDH by NADH was in some manner related to the effect of heavy

Fig. (5). Effect of G3P on the activity of GAPDH from HeLa cells. The enzyme at 2.5 µM concentration was incubated at 37 °C with 50 mM Tris-HCl buffer (pH 8.2), containing 1 mM EDTA and 0.05 mM G3P (A) or 0.5 mM G3P (B). The enzymatic activity was assayed in a final volume of 1 mL containing: 50 mM Tris-HCl buffer (pH 8.2), containing 1 mM EDTA, 1 mM 2-mercaptoethanol (2-MSH), 1 mM G3P, 50 mM arsenate, 1 mM NAD+, and 0.04 µmol of enzyme. Results not connected by the same letter were significantly different (p ≤ 0.05, Tukey’s test).


metals was investigated. Table 3 shows that 1 mM EDTA did not protect the enzyme from inactivation either with or without NADH addition. The enzyme, in the presence of 0.5 mM NADH, was approximately 95% inactivated in the presence and absence of EDTA, suggesting that there is no correlation between the inactivation of GAPDH by NADH and the presence of heavy metals. Similar results were observed for the enzymes from rabbit muscle [32] and Caiman sp. [26]. 4. DISCUSSION The results of this study regarding the effect of reducing agents on enzyme activity were similar to those previously Table 3.

Maurer et al.

observed for rabbit muscle GAPDH [2, 24, 32], yeast GAPDH [1], Caiman sp. muscle GAPDH [5], and Anas sp. muscle GAPDH [6]. HeLa cell GAPDH was inhibited by sulfhydryl reagents and among the reagents tested, p-CMB and p-HMB were the most potent inhibitors. The inhibitory effects of these reagents were partially reversible by the subsequent addition of excess 2-mercaptoethanol, which differs from the described results for GAPDHs from rabbit muscle [2], Caiman sp. [5], and Anas sp. [6], in which cases the inhibition was completely reversible under similar experimental conditions. This suggests that p-chloro- and p-hydroxymercuribenzoate, besides reacting with the important sulfhydryl groups necessary for enzyme activity, could also react with other func-

Effect of EDTA on enzyme stability at 37 ºC, during 6 h, in the presence of NADH. Addition

Treatment 1

Treatment 2

Relative activity (%) a

Relative activity (%)

none

76 ± 1.10

78 ± 2.00

0.5 mM NADH

5 ± 2.00

4 ± 1.52

0.5mM NADH + 0.5 mM NAD +

10 ± 1.50

7 ± 1.80

0.5mM NADH + 1 mM MSH

70 ± 2.10

73 ± 1.20

a

Relative activity (%) was considered in relation to zero time of incubation. Treatment 1: The samples contained in a 1 mL final volume: 0.2 µmol of enzyme, 50 mM Tris-HCl buffer pH 8.2, containing 1 mM EDTA plus the indicated additional reagents. Treatment 2: The assay mixture was the same as described for Treatment 1, but EDTA was omitted. MSH: 2-mercaptoethanol.

Fig. (6). Effect of NADH on the activity of GAPDH from HeLa cells. The enzyme at 2.5 µM concentration was incubated at 37 °C with 50 mM Tris-HCl buffer (pH 8.2), containing 1 mM EDTA and 0.05 mM NADH (A) or 0.5 mM NADH (B). The enzymatic activity was assayed in a final volume of 1 mL containing: 50 mM Tris-HCl buffer (pH 8.2), containing 1 mM EDTA, 1 mM 2-mercaptoethanol (2-MSH), 1 mM G3P, 50 mM arsenate, 1 mM NAD+, and 0.04 µmol of enzyme. Results not connected by the same letter were significantly different (p ≤ 0.05, Tukey’s test).




7

tional groups normally involved in the maintenance of the enzyme’s 3-dimensional conformation. Kinetic studies with reducing agents, divalent metal ions, and sulfhydryl reagents showed the importance of sulfhydryl groups in maintaining the activity of HeLa cell GAPDH. Highly similar results have been shown for GAPDH enzymes obtained from other sources [2, 5, 6, 30]. The high conservation of the primary structure of HeLa cell GAPDH is possibly related to the importance of the enzyme in carbohydrate metabolism.

cal intracellular concentrations of the metabolites that can influence GAPDH activity.

The HeLa cell enzyme, like GAPDH from other sources, was also inhibited by triphosphate, diphosphate, and monophosphate nucleotides. Among the tested nucleotides, cAMP was the most effective inhibitor, causing 70% inhibition at 10 mM concentration. In yeast GAPDH [31], cAMP caused 100% inhibition at the same concentration, while GAPDHs from Caiman sp. [5] and Anas sp. [6] were inhibited by approximately 50% under similar conditions. The physiological significance of the inhibition caused by nucleotides in normal cells is a controversial subject, since the intracellular levels of the nucleotides are not generally thought to be high enough to cause inhibition [13, 31, 33]. However, it has been suggested that ATP could be present in high enough concentrations to cause appreciable inhibition in vivo [13, 31].

LIST OF ABBREVIATIONS

HeLa cell GAPDH was also inactivated by incubation with G3P, in agreement with the results of Carr, Amelunxen, Curran, and Grisolia [34]; G3P decreases the stability of the enzyme and also enhances the effect of NADH. GAPDH from HeLa cells was inactivated in the presence of NADH under aerobic conditions. The enzyme was approximately 95% inactivated after 6 h of incubation at 37 °C. GAPDH enzymes from rabbit muscle [32], yeast [35], Caiman sp. [26], and Anas sp. [36] were also inhibited by NADH. According to Amelunxen and Grisolia [37], and Carr, Amelunxen, and Grisolia [38] the mechanism of GAPDH inactivation by NADH occurs by the following steps: (a) replacement of bound NAD+ by NADH, (b) exposure of sulfhydryl groups to oxidation with H2O2 formation and, (c) destruction of enzyme activity by H2O2. 5. CONCLUSION In relation to the kinetic studies, it can be concluded that the GAPDH of HeLa cells is a sulfhydryl-dependent enzyme since its activity was only maximally activated in the presence of 2-mercaptoethanol or dithioerythritol and was inhibited by divalent metallic ions, reagents such as p-CMB and p-HMB, and other inhibitors of sulfhydryl groups as iodoacetamide, ethylmaleimide, and 2-chloro-4aminobenzoate. Of the nucleotides tested, cAMP was the most effective inhibitor of GAPDH from HeLa cells. The enzyme was inhibited when incubated in the presence of G3P or NADH, and in both cases the presence of 2mercaptoethanol protected the enzyme against inhibition. The presence of EDTA did not affect the inactivation of the HeLa GAPDH by NADH, suggesting that the inactivation of NADH is not related to the presence of heavy metal ions. The investigation of the activity and stability of the enzyme in vitro in the presence of substrate or coenzymes, as well in presence of nucleotides could elucidate the physiological importance of these factors. However, it is necessary to consider the cellular metabolic environment and the physiologi-

Our kinetic analysis showed that while GAPDH of HeLa cells (a tumor cell line) has a lower specific activity and stability compared with enzymes from normal tissue, its kinetic characteristics were similar to those of GAPDH from normal tissue, which reinforces the key biochemical role of this enzyme in cellular metabolism.

ADP

=

adenosine diphosphate

5′-AMP

=

adenosine 5′-monophosphate

cAMP

=

cyclic adenosine 3′–5′monophosphate

ATP

=

adenosine triphosphate

CDP

=

cytidine diphosphate

CMP

=

cytidine monophosphate

EDTA

=

ethylenediaminetetraacetic acid

G3P

=

D-glyceraldehyde-3-phosphate

GAPDH

=

D-glyceraldehyde-3-phosphate

dehydrogenase p-HMB

=

+

p-hydroxymercuribenzoate

NAD (or NAD) =

nicotinamide adenine dinucleotide

NADH

=

reduced nicotinamide adenine dinucleotide

Tris

=

tri-hydroxymethylaminomethane

UDP

=

uridine diphosphate

CONFLICT OF INTEREST The authors confirm that this article content has no conflict of interest. ACKNOWLEDGEMENTS The authors thank Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for financial support. We are grateful to Maria Benigna Martinelli de Oliveira and Shigehiro Funayama, both in memoriam, for all contributions. REFERENCES [1] [2] [3] [4]

[5]

Krebs, E.G.; Rafter, G.W.; Jungle, J. Yeast D-glyceraldehyde-3phosphate dehydrogenase. II Yeast protein. J. Biol. Chem., 1953, 200, 479-492. Velick, S.F. Coenzyme binding and the thiol groups of dehydrogenase. J. Biol. Chem., 1953, 203, 563-573. Suzuki, K.; Harris, J.I. Glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus. FEBS Lett., 1971, 13, 217-220. Duggleby, R.G.; Dennis, D.T. Nicotinamide adenine dinucleotidespecific glyceraldehyde-3-phosphate dehydrogenase from Pisum sativum assay and steady state kinetics. J. Biol. Chem., 1974, 249, 167-174. Vieira, M.M.; Veiga, L.A.; Nakano, M. Muscle D-glyceraldehyde3-phosphate dehydrogenase from Caiman sp. I. Purification and


[6] [7] [8] [9] [10] [11] [12]

[13] [14] [15] [16] [17]

[18]

[19]

[20]

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Accepted: June 30, 2015