CHAPTER SIX
Measurement of Enolase Activity in Cell Lysates Keigo Fukano, Kazuhiro Kimura1 Department of Biomedical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan 1 Corresponding author: e-mail address: [email protected]
Contents 1. Introduction 2. Assay of Enolase Activity 2.1 Sample preparation 2.2 Assay 3. DEAE-Cellulose Chromatography References
115 118 118 119 122 122
Abstract Enolase (EC 4.2.1.11) is a cytosolic metalloenzyme responsible for the conversion of 2-phosphoglycerate into phosphoenolpyruvate, the second to last step in glycolysis. In mammals, enolase is encoded by three homologous genes. These gene products not only possess distinct biochemical and immunological properties but also show different tissue distribution. Besides its glycolytic function, a-enolase plays a variety of roles in pathophysiological settings including oncogenesis, tumor progression, ischemia, and bacterial infection. The expression levels of a-enolase have been attributed diagnostic and prognostic value in a number of tumors. Furthermore, neuron-specific a-enolase is released into the cerebrospinal fluid as well as in the systemic circulation upon traumatic brain injury and ischemic episodes. Thus, the measurement of the enzymatic activity of enolase is relevant for diverse fields of investigation, including oncometabolism. Here, we described simple and rapid protocols to measure the activity of enolase in lysates from mammalian cells and tissues.
1. INTRODUCTION Enolase, also known as phosphopyruvate hydratase, is a key glycolytic enzyme in the cytoplasm of prokaryotic and eukaryotic cells. It is a metalloenzyme that requires the metal ion such as Mg2+ and catalyzes the dehydration of 2-phosphoglycerate (2-PG) to phosphoenolpyruvate (PEP), in Methods in Enzymology, Volume 542 ISSN 0076-6879 http://dx.doi.org/10.1016/B978-0-12-416618-9.00006-6
#
2014 Elsevier Inc. All rights reserved.
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Keigo Fukano and Kazuhiro Kimura
2-Phosphoglycerate O–
Phosphoenolpyruvate O– enolase
O H
O
P
O O
Mg2+ H2O
OH
P
H 2C
Figure 6.1 Conversion of 2-phosphoglycerate to phosphoenolpyruvate by enolase. Enolase, phosphopyruvate hydratase, catalyzes Mg2+-dependent conversion of 2-phosphoglycerate to phosphoenolpyruvate in the glycolytic pathway.
the catabolic glycolytic pathway (Fig. 6.1). It can also catalyze the reverse reaction in the process of gluconeogenesis, depending on environmental concentrations of substrates. In mammals, enolase is encoded by three homologous genes: ENO1 which is ubiquitously expressed, ENO2 which is expressed exclusively in neural tissues, and ENO3 which is expressed in muscle tissues. Thus, the enzyme occurs as three isoforms: a-enolase coded by ENO1 which is found in almost all tissues, b-enolase coded by ENO3 which is predominantly found in muscle tissues, and g-enolase coded by ENO2 which is only found in neuron and neuroendocrine tissues (Marangos, Parma, & Goodwin, 1978; Merkulova et al., 1997; Pancholi, 2001). Three isoforms are characterized by distinct biochemical and immunological properties, in addition to different tissue distributions. Functional enzyme is a dimer made up of two homologous and heterologous isoforms, and only five types (aa, ab, bb, ag, and gg) of enolase isozyme can be found due to selective localization of band g-enolases (Fletcher, Rider, & Taylor, 1976; Kato, Asai, Shimizu, Suzuki, & Ariyoshi, 1983; Royds, Parsons, Taylor, & Timperley, 1982). Interestingly, proportions of dimeric forms (aa, bb, and ab) of enolase in rat heart and skeletal muscle change during embryonic development. In both tissues, aa type is predominant in the fetus, although, as development progresses, aa type is replaced by ab and bb types in adult heart and by bb type in adult striated muscle (Merkulova et al., 1997). a-Enolase is considered to be a multifunctional protein, aside from its enzymatic function in the glycolytic pathway (Diaz-Ramos, Roig-Borrellas, Garcia-Melero, & Lopes-Alemany, 2012; Pancholi, 2001). Among its pleiotropic actions, a-enolase plays an important role in regulation of c-myc promoter activity in the form of an alternative translation product, c-myc promoter-binding protein 1 (MBP-1). It is localized in the nucleus and can bind to the c-myc P2 promoter and negatively regulates transcription of the
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proto-oncogene (Subramanian & Miller, 2001). In addition, a-enolase has been detected on the surface of hematopoietic cells such as monocytes, T cells and B cells, neuronal cells, and endothelial cells. Cell surface a-enolase serves as a receptor of plasminogen to enhance pericellular fibrinolytic activity and to lead extracellular matrix degradation (Godier & Hunt, 2012). a-Enolase has also been described as a neurotrophic factor, a heat-shock protein (HSP48), and a hypoxic stress protein. In the latter, it is suggested that upregulation of a-enolase contributes to hypoxia tolerance through nonglycolytic mechanisms (Aaronson, Graven, Tucci, McDonald, & Farber, 1995). Multifunctional a-enolase plays variety roles on pathophysiological situations (Butterfield & Bader Lange, 2009; Capello, Ferri-Borgogno, Cappello, & Novelli, 2011; Diaz-Ramos et al., 2012; Godier & Hunt, 2012; Pancholi, 2001; Terrier et al., 2007). During tumor formation and expansion, tumor cells must increase glucose metabolism ( Jin, DiPaola, Mathew, & White, 2007). Hypoxia is a common feature of solid tumors. Consistent with this, several reports have shown an upregulation of a-enolase in several types of cancer (Capello et al., 2011; Chang et al., 2006; Katayama et al., 2006; Lo´pez-Pedrera et al., 2006), which may support anaerobic proliferation of tumor cells. In addition, a-enolase–plasminogen interaction on the cell surface is involved in promoting cell migration in tumor invasion and cancer metastasis (Capello et al., 2011; Godier & Hunt, 2012). Furthermore, the interaction mediates recruitment of monocytes to acutely inflamed tissue (Wygrecka et al., 2009). That is, it increases plasmin generation, promotes matrix degradation, and enhances monocyte migration. In rat heart ischemia–reperfusion model, a-enolase is induced in response to ischemic hypoxia and improves contractility of cardiomyocytes (Mizukami et al., 2004). The a-enolase–plasminogen interaction may also be involved in myogenesis and muscle regeneration (Diaz-Ramos et al., 2012). Diagnostic and prognostic value of ENO1 expression has been described in a number of tumors, as increased expression of a-enolase has been reported to correlate with progression of tumors (Capello et al., 2011; Diaz-Ramos et al., 2012). In addition, several posttranslational modifications such as acetylation, methylation, and phosphorylation have been found in a-enolase, and it is subjected to more acetylation, methylation, and phosphorylation in tumor cells than in normal tissues (Capello et al., 2011). Therefore, analysis of posttranslational modifications of a-enolase could also be of diagnostic and prognostic value in cancer, although it is uncertain whether such modifications affect the enzyme activity, localization, and
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stability. Furthermore, in many cancer patients, autoantibody against a-enolase has been detected. For example, in pancreatic ductal adenocarcinoma, a-enolase is upregulated and elicits a specific production of autoantibodies against isoforms phosphorylated on serine 419 (Tomaino et al., 2011). The presence of these autoantibodies is correlated with a significantly better clinical outcome in advanced patients treated with standard chemotherapy (Tomaino et al., 2011). Thus, levels of autoantibodies are also proposed as markers for diagnosis and prognosis of cancer. Autoantibodies to a-enolase have been found in patients with chronic autoimmune diseases and inflammatory disorders such as rheumatoid arthritis, systemic sclerosis, and Hashimoto’s encephalopathy (Diaz-Ramos et al., 2012; Terrier et al., 2007). In these diseases, the autoantibodies could induce endothelial injury through immune complex–complement activation, inhibit a-enolase–plasminogen interaction with perturbation of intravascular and pericellular fibrinolytic system, and induce apoptotic cell death. Autoantibodies to microbial a-enolase are seen in infectious disease and play a role in limiting microbial tissue invasion (Terrier et al., 2007). Thus, autoantibodies to a-enolase present in the sera of patients with autoimmune and infectious disease have potential diagnostic and prognostic value. g-Enolase is located in central and peripheral neurons and neuroendocrine cells and called neuron-specific enolase (NSE). This enzyme is released into the cerebrospinal fluid and blood when neural tissue is injured by traumatic brain injury and ischemic stroke (Ahmed, Wardlaw, & Whiteley, 2012; Meric, Gunduz, Turedi, Cakir, & Yandi, 2010). Tumors derived from neural or neuroendocrine tissue also release NSE into the blood, and therefore, NSE is a well-established tumor marker of small cell lung cancer and so on (Braga, Ferraro, Mozzi, Dolci, & Panteghini, 2013; Zhao & Luo, 2013). Therefore, measurement of enolase activity in biological fluids is of interest to investigators studying cancer cell metabolism. We here describe simple and rapid in vitro protocols to measure enolase activity in native lysates from mammalian cells and tissues.
2. ASSAY OF ENOLASE ACTIVITY 2.1. Sample preparation Enolase is localized in cytosol, nuclei, and plasma membrane, but majority of the enzyme is the most probably present in cytosol as a glycolytic enzyme. So preparation of cytosolic protein by any means may be able to use for total enolase activity assay. Two examples are shown as follows.
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Animal tissues are removed and frozen immediately with liquid nitrogen and stored at 80 C for several days. The tissues (0.1–0.3 g wet weight) are homogenized in 1 or 2 ml of 15 mM Tris–acetate (pH 6.5), 5 mM MgSO4, and 1 mM EDTA. The homogenate is centrifuged at 100,000 g for 1 h at 4 C, and the supernatant is used for the assay of enolase activity (Kato, Ishiguro, Suzuki, Ito, & Semba, 1982). Mammalian cells in culture are washed once with the buffer containing 250 mM sucrose, 20 mM HEPES (pH 7.5), 10 mM KCl, 1 mM MgCl2, 1 mM EGTA, 1 mM EDTA, 1 mM dithiothreitol, and 1 mM phenylmethanesulfonylfluoride and homogenized in 1 ml of the buffer. The homogenate is then centrifuged at 15,000 g for 15 min at 4 C, and the resultant supernatant is recovered for enzymatic assay of enolase (Ishii et al., 2012). RIPA buffer (25 mM Tris–HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) may be used, but the specific enolase activity is lower.
2.2. Assay Enolase activity is measured by either direct or coupled methods. The direct method is to monitor PEP produced from 2-PG by enolase (Fig. 6.1; Ishii et al., 2012), while coupled method is to measure NADH decrease linked to conversion of PEP to lactate by a coupled enzyme system containing lactate dehydrogenase (LDH) and pyruvate kinase (PK) (Fig. 6.2; Hoorn, Flikweert, & Staal, 1974; Rider & Taylor, 1974). 2.2.1 Micro direct assay a. Prepare a reaction buffer consisting of 20 mM imidazole HCl (pH 7.0), 400 mM KCl, and 1 mM magnesium acetate. b. Put 50 ml of the reaction buffer into the tube. c. Put 25 ml of the sample after appropriate dilution or dilution buffer into the tube. d. Record absorbance of the solution at 240 nm at room temperature (25 C). e. Put 25 ml of 2-PG (1–2 mM as a final concentration) solution into the tube. f. Record an increase in absorbance of the solution at 240 nm for 0–30 min (when time dependency is tested) after the addition of 2-PG at room temperature. g. Calculate changes in the absorbance reflecting the conversion of 2-PG to PEP by enolase. Since molecular extinction coefficient for PEP is
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Enolase 2-Phosphoglycerate (2-PG)
Phosphoenolpyruvate (PEP) H2O ADP
Pyruvate kinase (PK)
ATP Lactate
Pyruvate NAD
NADH
Lactate dehydrogenase (LDH)
Activity (U/mg) =
(change in absorbance/min of sample – that of blank) ⫻ (dilution factor) 6.22 ⫻ (volume of sample used) ⫻ (sample concentration in mg/ml)
Figure 6.2 Measurement of enolase activity by a coupled enzyme system of lactate dehydrogenase and pyruvate kinase. Schema represents flow of the sequential conversion of substrates for enzymes involved, showing the last reaction is coupled with conversion of NADH to NAD. Thus, enolase activity can be monitored as a decrease in NADH absorbance at 340 nm and estimated by the equation.
1.4 103 M 1 cm 1, the change of one absorbance unit at 240 nm means increase of PEP concentration in the reaction mixture from 0 to 714 mM during the reaction time. Velocity of the reaction should be calculated from initial rates of absorbance increase. h. Do not forget to make an appropriate blank/control tube. Note a. Due to limitation of amount of 2-PG in terms of cost, we recommend this micro assay system, although a spectrophotometer measurable in small volume (1 ml) is necessary. b. Mg2+ should be included in the reaction buffer. Some other divalent cations such as Zn2+ and Mn2+ work as a cofactor of enolase with lower efficacy (Baranowski & Wolna, 1975). c. Inclusion of sodium fluoride (2 mM), phosphonoacetohydroxamate (2 mM), or ENOblock (10 mM) in the assay inhibits the enolase activity ( Jung et al., 2013; Wedekind, Poyner, Reed, & Rayment, 1994). So you can confirm that specific changes in the absorbance are dependent on the enzyme. d. Enolases from yeast and rabbit muscle as a positive control can be purchased from Oriental Yeast Co. (Tokyo, Japan) and Sigma-Aldrich (St. Louis, MO, USA), respectively. Alternatively mammalian blood
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which is diluted 1:1 with a hypotonic solution, sonicated, and centrifuged at 20,000 g can be used. e. We have experienced that inclusion of reduced glutathione (GSH, 5 mM) in the assay inhibits the enolase activity, due to acidification of the assay reagent by GSH. Be careful in pH in the reaction system if you include some additives. 2.2.2 Coupled assay a. Freshly prepare a reaction buffer containing 100 mM triethanolamine (pH 7.4), 20 mM MgCl2, 150 mM KCl, 0.6 mM b-NADH, 2 mM ADP, 20 units/ml PK, and 30 units/ml LDH. b. Put 50 ml of the reaction buffer into the tube. c. Put 25 ml of the sample after appropriate dilution or dilution buffer into the tube. d. Record absorbance of the solution at 340 nm at room temperature (25 C). e. Put 25 ml of 2-PG (1–2 mM as a final concentration) solution into the tube. f. Record absorbance of the solution at 340 nm for 5 min after the addition of 2-PG at room temperature. g. Calculate changes in the absorbance reflecting the sequential conversion of 2-PG to PEP by enolase, PEP to pyruvate by PK, and pyruvate to lactate by LDH. Since molecular extinction coefficient for NADH is 6.22 103 M 1 cm 1, the change of one absorbance unit at 340 nm means increase of lactate concentration in the reaction mixture from 0–161 mM during the reaction time. h. Ordinary, enolase activity can be calculated by the equation shown in Fig. 6.2. Note a. Changes in NADH concentration could be monitored fluorometrically (NADH excitation 360 nm, emission 460 nm). This method is suitable for evaluating relative abundance of enolase activity and can be applied for high-throughput assay (Muller, Aquilanti, & DePhinho, 2012). 2.2.3 Isozyme-specific assay Three isoforms of enolase have been identified: a-enolase, b-enolase, and g-enolase. a-Enolase has been detected on most tissues, whereas b-enolase is expressed predominantly in muscle tissue, and g-enolase is detected only
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in nervous tissue. These isoforms exist as both homodimers and heterodimers, and therefore, five types of dimeric enolase (isozymes) are found as aa, ab, bb, ag, and gg. Three isoforms can be distinguished immunologically, and five isozymes are separated by DEAE-cellulose chromatography (Rider & Taylor, 1974; Suzuki, Umeda, & Kato, 1980).
3. DEAE-CELLULOSE CHROMATOGRAPHY a. Tissue extract from brain or muscle is dialyzed against 15 mM Tris–HCl (pH 7.9), 5 mM MgCl2, and 0.1 mM EDTA. b. The dialysate is applied on DEAE-cellulose column preequilibrated with the same buffer. c. The aa type of enolase is recovered from flow-through (unbound) fraction. d. The bb, ab, and ag types of enolase are eluted with the same buffer containing 0.1 M NaCl after successful washing. e. The gg type of enolase is eluted with the same buffer containing 0.3 M NaCl.
REFERENCES Aaronson, R. M., Graven, K. K., Tucci, M., McDonald, R. J., & Farber, H. W. (1995). Non-neuronal enolase is an endotherial hypoxic stress protein. The Journal of Biological Chemistry, 270, 27752–27757. Ahmed, O., Wardlaw, J., & Whiteley, W. N. (2012). Correlation of levels of neuronal and glial markers with radiological measures of infarct volume in ischaemic stroke: A systematic review. Cerebrovascular Diseases, 33, 47–54. Baranowski, T., & Wolna, F. (1975). Enolase from human muscle. Methods in Enzymology, 42, 335–338. Braga, F., Ferraro, S., Mozzi, R., Dolci, A., & Panteghini, M. (2013). Biological variation of neuroendocrine tumor markers chromogranin A and neuron-specific enoolase. Clinical Biochemistry, 46, 148–151. Butterfield, D. A., & Bader Lange, M. L. (2009). Multifunctional roles of enolase in Alzheimer’s disease brain: Beyond altered glucose metabolism. Journal of Neurochemistry, 111, 915–933. Capello, M., Ferri-Borgogno, S., Cappello, P., & Novelli, F. (2011). a-Enolase: A promising therapeutic and diagnostic tumor target. FEBS Journal, 278, 1064–1074. Chang, G. C., Liu, K. J., Hsieh, C. L., Hu, T. S., Charoenfuprasert, S., Liu, H. K., et al. (2006). Identification of a-enolase as an autoantigen in lung cancer: Its overexpression is associated with clinical outcomes. Clinical Cancer Research, 12, 5746–5754. Diaz-Ramos, A., Roig-Borrellas, A., Garcia-Melero, A., & Lopes-Alemany, R. (2012). a-Enolase, a multifunctional protein: Its role on pathophysiological situations. Journal of Biomedicine and Biotechnology, 2012, 156795. http://dx.doi.org/10.1155/2012/156795. Fletcher, L., Rider, C. C., & Taylor, C. B. (1976). Enolase isoenzymes. III. Chromatographic and immunological characteristics of rat brain enolase. Biochimica et Biophysica Acta, 452, 245–252.
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Godier, A., & Hunt, B. J. (2012). Plasminogen receptors and their role in the pathogenesis of inflammatory, autoimmune and malignant disease. Journal of Thrombosis and Haemostasis, 11, 26–34. Hoorn, R. K. J., Flikweert, J. P., & Staal, G. E. J. (1974). Purification and properties of enolase of human erythrocytes. International Journal of Biochemistry, 55, 845–852. Ishii, T., Fukano, K., Shimada, K., Kamikawa, A., Okamatsu-Ogura, Y., Terao, A., et al. (2012). Proinsulin C-peptide activates a-enolase activity: Implications for C-peptide– cell membrane interaction. Journal of Biochemistry, 152, 53–62. Jin, S., DiPaola, R. S., Mathew, R., & White, E. (2007). Metabolic catastrophe as a means to cancer cell death. Journal of Cell Science, 120, 379–383. Jung, D.-W., Kim, W.-H., Park, S.-H., Lee, J., Kim, J., Su, D., et al. (2013). A unique small molecule inhibitor of enolase clarifies its role in fundamental biological processes. ACS Chemical Biology, 8, 1271–1282. Katayama, M., Nakano, H., Ishiuchi, A., Wu, W., Oshima, R., Sakurai, J., et al. (2006). Protein pattern difference in the colon cancer cell lines examined by two-dimensional differential in-gel electrophoresis and mass spectrometry. Surgery Today, 36, 1085–1093. Kato, K., Asai, R., Shimizu, A., Suzuki, F., & Ariyoshi, Y. (1983). Immunoassay of three enolase isozymes in human serum and in blood cells. Clinica Chimica Acta, 127, 353–363. Kato, K., Ishiguro, Y., Suzuki, F., Ito, A., & Semba, R. (1982). Distribution of nervous system-specific forms of enolase in peripheral tissues. Brain Research, 237, 441–448. Lo´pez-Pedrera, C., Villalba, J. M., Siendones, E., Barbarroja, N., Go´mez-Dı´az, C., Rodrı´guez-Ariza, A., et al. (2006). Proteomic analysis of acute myeloid leukemia: Identification of potential early biomarkers and therapeutic targets. Proteomics, 6(Suppl. 1), S293–S299. Marangos, P. J., Parma, A. M., & Goodwin, F. K. (1978). Functional properties of neuronal and glial isoenzymes of brain enolase. Journal of Neurochemistry, 31, 727–732. Meric, E., Gunduz, A., Turedi, S., Cakir, E., & Yandi, M. (2010). The prognostic value of neuron-specific enolase in head trauma patients. Journal of Emergency Medicine, 38, 297–301. Merkulova, T., Lucas, M., Jabet, C., Lamande´, N., Rouzeau, J. D., Gros, F., et al. (1997). Biochemical characterization of the mouse muscle-specific enolase: Developmental changes in electrophoretic variants and selective binding to other proteins. Biochemical Journal, 323, 791–800. Mizukami, Y., Iwamatsu, A., Aki, T., Kimura, M., Nakamura, K., Nao, T., et al. (2004). ERK1/2 regulates intracellular ATP levels through a-enolase expression in cardiomyocytes exposed to ischemic hypoxia and reoxygenation. The Journal of Biological Chemistry, 279, 50120–50131. Muller, F., Aquilanti, E., & DePhinho, R. (2012). In vitro enzymatic activity assay for enolase in mammalian cells in culture. Protocol Exchange. http://dx.doi.org/10.1038/ protex.2012.040. Pancholi, V. (2001). Multifunctional a-enolase: Its role in diseases. Cellular and Molecular Life Sciences, 58, 902–920. Rider, C. C., & Taylor, C. B. (1974). Enolase isozymes in rat tissue: Electrophoretic, chromatographic, immunological and kinetic properties. Biochimica et Biophysica Acta, 365, 285–300. Royds, J. A., Parsons, M. A., Taylor, C. B., & Timperley, W. R. (1982). Enolase isoenzyme distribution in the human brain and its tumours. Journal of Pathology, 137, 37–49. Subramanian, A., & Miller, D. M. (2001). Structural analysis of a-enolase. The Journal of Biological Chemistry, 275, 5958–5965. Suzuki, F., Umeda, Y., & Kato, K. (1980). Rat brain enolase isozymes: Purification of three forms of enolase. Journal of Biochemistry, 87, 1587–1594. Terrier, B., Degand, N., Guilpain, P., Servettaz, A., Guillevin, L., & Mouthon, L. (2007). Alpha-enolase: A target of antibodies in infectious and autoimmune disease. Autoimmunity Reviews, 6, 176–182.
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Tomaino, B., Cappello, P., Capello, M., Fredolini, C., Sperduti, I., Migliorini, P., et al. (2011). Circulating autoantibodies to phosphorylated alpha-enolase are a hallmark of pancreatic cancer. Journal of Proteome Research, 1, 105–112. Wedekind, J. E., Poyner, R. R., Reed, G. H., & Rayment, I. (1994). Chelation of serine 39 to Mg2+ latches a gate at the active site of enolase: Structure of the bis(Mg2+) complex of yeast enolase and the intermediate analogue phosphonoacetohydroxamate at 2.1-A resolution. Biochemistry, 33, 9333–9342. Wygrecka, M., Marsh, L. M., Morty, R. E., Henneke, I., Guenther, A., Lohmeyer, J., et al. (2009). Enolase-1 promotes plasminogen-mediated recruitment of monocytes to the acutely inflamed lung. Blood, 113, 5588–5598. Zhao, W.-X., & Luo, J. (2013). Serum neuron-specific enolase levels were associated with the prognosis of small cell lung cancer: Meta-analysis. Tumor Biology, 34, 3245–3248.
Measurement of Enolase Activity in Cell Lysates Keigo Fukano, Kazuhiro Kimura1 Department of Biomedical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan 1 Corresponding author: e-mail address: [email protected]
Contents 1. Introduction 2. Assay of Enolase Activity 2.1 Sample preparation 2.2 Assay 3. DEAE-Cellulose Chromatography References
115 118 118 119 122 122
Abstract Enolase (EC 4.2.1.11) is a cytosolic metalloenzyme responsible for the conversion of 2-phosphoglycerate into phosphoenolpyruvate, the second to last step in glycolysis. In mammals, enolase is encoded by three homologous genes. These gene products not only possess distinct biochemical and immunological properties but also show different tissue distribution. Besides its glycolytic function, a-enolase plays a variety of roles in pathophysiological settings including oncogenesis, tumor progression, ischemia, and bacterial infection. The expression levels of a-enolase have been attributed diagnostic and prognostic value in a number of tumors. Furthermore, neuron-specific a-enolase is released into the cerebrospinal fluid as well as in the systemic circulation upon traumatic brain injury and ischemic episodes. Thus, the measurement of the enzymatic activity of enolase is relevant for diverse fields of investigation, including oncometabolism. Here, we described simple and rapid protocols to measure the activity of enolase in lysates from mammalian cells and tissues.
1. INTRODUCTION Enolase, also known as phosphopyruvate hydratase, is a key glycolytic enzyme in the cytoplasm of prokaryotic and eukaryotic cells. It is a metalloenzyme that requires the metal ion such as Mg2+ and catalyzes the dehydration of 2-phosphoglycerate (2-PG) to phosphoenolpyruvate (PEP), in Methods in Enzymology, Volume 542 ISSN 0076-6879 http://dx.doi.org/10.1016/B978-0-12-416618-9.00006-6
#
2014 Elsevier Inc. All rights reserved.
115
116
Keigo Fukano and Kazuhiro Kimura
2-Phosphoglycerate O–
Phosphoenolpyruvate O– enolase
O H
O
P
O O
Mg2+ H2O
OH
P
H 2C
Figure 6.1 Conversion of 2-phosphoglycerate to phosphoenolpyruvate by enolase. Enolase, phosphopyruvate hydratase, catalyzes Mg2+-dependent conversion of 2-phosphoglycerate to phosphoenolpyruvate in the glycolytic pathway.
the catabolic glycolytic pathway (Fig. 6.1). It can also catalyze the reverse reaction in the process of gluconeogenesis, depending on environmental concentrations of substrates. In mammals, enolase is encoded by three homologous genes: ENO1 which is ubiquitously expressed, ENO2 which is expressed exclusively in neural tissues, and ENO3 which is expressed in muscle tissues. Thus, the enzyme occurs as three isoforms: a-enolase coded by ENO1 which is found in almost all tissues, b-enolase coded by ENO3 which is predominantly found in muscle tissues, and g-enolase coded by ENO2 which is only found in neuron and neuroendocrine tissues (Marangos, Parma, & Goodwin, 1978; Merkulova et al., 1997; Pancholi, 2001). Three isoforms are characterized by distinct biochemical and immunological properties, in addition to different tissue distributions. Functional enzyme is a dimer made up of two homologous and heterologous isoforms, and only five types (aa, ab, bb, ag, and gg) of enolase isozyme can be found due to selective localization of band g-enolases (Fletcher, Rider, & Taylor, 1976; Kato, Asai, Shimizu, Suzuki, & Ariyoshi, 1983; Royds, Parsons, Taylor, & Timperley, 1982). Interestingly, proportions of dimeric forms (aa, bb, and ab) of enolase in rat heart and skeletal muscle change during embryonic development. In both tissues, aa type is predominant in the fetus, although, as development progresses, aa type is replaced by ab and bb types in adult heart and by bb type in adult striated muscle (Merkulova et al., 1997). a-Enolase is considered to be a multifunctional protein, aside from its enzymatic function in the glycolytic pathway (Diaz-Ramos, Roig-Borrellas, Garcia-Melero, & Lopes-Alemany, 2012; Pancholi, 2001). Among its pleiotropic actions, a-enolase plays an important role in regulation of c-myc promoter activity in the form of an alternative translation product, c-myc promoter-binding protein 1 (MBP-1). It is localized in the nucleus and can bind to the c-myc P2 promoter and negatively regulates transcription of the
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proto-oncogene (Subramanian & Miller, 2001). In addition, a-enolase has been detected on the surface of hematopoietic cells such as monocytes, T cells and B cells, neuronal cells, and endothelial cells. Cell surface a-enolase serves as a receptor of plasminogen to enhance pericellular fibrinolytic activity and to lead extracellular matrix degradation (Godier & Hunt, 2012). a-Enolase has also been described as a neurotrophic factor, a heat-shock protein (HSP48), and a hypoxic stress protein. In the latter, it is suggested that upregulation of a-enolase contributes to hypoxia tolerance through nonglycolytic mechanisms (Aaronson, Graven, Tucci, McDonald, & Farber, 1995). Multifunctional a-enolase plays variety roles on pathophysiological situations (Butterfield & Bader Lange, 2009; Capello, Ferri-Borgogno, Cappello, & Novelli, 2011; Diaz-Ramos et al., 2012; Godier & Hunt, 2012; Pancholi, 2001; Terrier et al., 2007). During tumor formation and expansion, tumor cells must increase glucose metabolism ( Jin, DiPaola, Mathew, & White, 2007). Hypoxia is a common feature of solid tumors. Consistent with this, several reports have shown an upregulation of a-enolase in several types of cancer (Capello et al., 2011; Chang et al., 2006; Katayama et al., 2006; Lo´pez-Pedrera et al., 2006), which may support anaerobic proliferation of tumor cells. In addition, a-enolase–plasminogen interaction on the cell surface is involved in promoting cell migration in tumor invasion and cancer metastasis (Capello et al., 2011; Godier & Hunt, 2012). Furthermore, the interaction mediates recruitment of monocytes to acutely inflamed tissue (Wygrecka et al., 2009). That is, it increases plasmin generation, promotes matrix degradation, and enhances monocyte migration. In rat heart ischemia–reperfusion model, a-enolase is induced in response to ischemic hypoxia and improves contractility of cardiomyocytes (Mizukami et al., 2004). The a-enolase–plasminogen interaction may also be involved in myogenesis and muscle regeneration (Diaz-Ramos et al., 2012). Diagnostic and prognostic value of ENO1 expression has been described in a number of tumors, as increased expression of a-enolase has been reported to correlate with progression of tumors (Capello et al., 2011; Diaz-Ramos et al., 2012). In addition, several posttranslational modifications such as acetylation, methylation, and phosphorylation have been found in a-enolase, and it is subjected to more acetylation, methylation, and phosphorylation in tumor cells than in normal tissues (Capello et al., 2011). Therefore, analysis of posttranslational modifications of a-enolase could also be of diagnostic and prognostic value in cancer, although it is uncertain whether such modifications affect the enzyme activity, localization, and
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stability. Furthermore, in many cancer patients, autoantibody against a-enolase has been detected. For example, in pancreatic ductal adenocarcinoma, a-enolase is upregulated and elicits a specific production of autoantibodies against isoforms phosphorylated on serine 419 (Tomaino et al., 2011). The presence of these autoantibodies is correlated with a significantly better clinical outcome in advanced patients treated with standard chemotherapy (Tomaino et al., 2011). Thus, levels of autoantibodies are also proposed as markers for diagnosis and prognosis of cancer. Autoantibodies to a-enolase have been found in patients with chronic autoimmune diseases and inflammatory disorders such as rheumatoid arthritis, systemic sclerosis, and Hashimoto’s encephalopathy (Diaz-Ramos et al., 2012; Terrier et al., 2007). In these diseases, the autoantibodies could induce endothelial injury through immune complex–complement activation, inhibit a-enolase–plasminogen interaction with perturbation of intravascular and pericellular fibrinolytic system, and induce apoptotic cell death. Autoantibodies to microbial a-enolase are seen in infectious disease and play a role in limiting microbial tissue invasion (Terrier et al., 2007). Thus, autoantibodies to a-enolase present in the sera of patients with autoimmune and infectious disease have potential diagnostic and prognostic value. g-Enolase is located in central and peripheral neurons and neuroendocrine cells and called neuron-specific enolase (NSE). This enzyme is released into the cerebrospinal fluid and blood when neural tissue is injured by traumatic brain injury and ischemic stroke (Ahmed, Wardlaw, & Whiteley, 2012; Meric, Gunduz, Turedi, Cakir, & Yandi, 2010). Tumors derived from neural or neuroendocrine tissue also release NSE into the blood, and therefore, NSE is a well-established tumor marker of small cell lung cancer and so on (Braga, Ferraro, Mozzi, Dolci, & Panteghini, 2013; Zhao & Luo, 2013). Therefore, measurement of enolase activity in biological fluids is of interest to investigators studying cancer cell metabolism. We here describe simple and rapid in vitro protocols to measure enolase activity in native lysates from mammalian cells and tissues.
2. ASSAY OF ENOLASE ACTIVITY 2.1. Sample preparation Enolase is localized in cytosol, nuclei, and plasma membrane, but majority of the enzyme is the most probably present in cytosol as a glycolytic enzyme. So preparation of cytosolic protein by any means may be able to use for total enolase activity assay. Two examples are shown as follows.
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Animal tissues are removed and frozen immediately with liquid nitrogen and stored at 80 C for several days. The tissues (0.1–0.3 g wet weight) are homogenized in 1 or 2 ml of 15 mM Tris–acetate (pH 6.5), 5 mM MgSO4, and 1 mM EDTA. The homogenate is centrifuged at 100,000 g for 1 h at 4 C, and the supernatant is used for the assay of enolase activity (Kato, Ishiguro, Suzuki, Ito, & Semba, 1982). Mammalian cells in culture are washed once with the buffer containing 250 mM sucrose, 20 mM HEPES (pH 7.5), 10 mM KCl, 1 mM MgCl2, 1 mM EGTA, 1 mM EDTA, 1 mM dithiothreitol, and 1 mM phenylmethanesulfonylfluoride and homogenized in 1 ml of the buffer. The homogenate is then centrifuged at 15,000 g for 15 min at 4 C, and the resultant supernatant is recovered for enzymatic assay of enolase (Ishii et al., 2012). RIPA buffer (25 mM Tris–HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) may be used, but the specific enolase activity is lower.
2.2. Assay Enolase activity is measured by either direct or coupled methods. The direct method is to monitor PEP produced from 2-PG by enolase (Fig. 6.1; Ishii et al., 2012), while coupled method is to measure NADH decrease linked to conversion of PEP to lactate by a coupled enzyme system containing lactate dehydrogenase (LDH) and pyruvate kinase (PK) (Fig. 6.2; Hoorn, Flikweert, & Staal, 1974; Rider & Taylor, 1974). 2.2.1 Micro direct assay a. Prepare a reaction buffer consisting of 20 mM imidazole HCl (pH 7.0), 400 mM KCl, and 1 mM magnesium acetate. b. Put 50 ml of the reaction buffer into the tube. c. Put 25 ml of the sample after appropriate dilution or dilution buffer into the tube. d. Record absorbance of the solution at 240 nm at room temperature (25 C). e. Put 25 ml of 2-PG (1–2 mM as a final concentration) solution into the tube. f. Record an increase in absorbance of the solution at 240 nm for 0–30 min (when time dependency is tested) after the addition of 2-PG at room temperature. g. Calculate changes in the absorbance reflecting the conversion of 2-PG to PEP by enolase. Since molecular extinction coefficient for PEP is
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Enolase 2-Phosphoglycerate (2-PG)
Phosphoenolpyruvate (PEP) H2O ADP
Pyruvate kinase (PK)
ATP Lactate
Pyruvate NAD
NADH
Lactate dehydrogenase (LDH)
Activity (U/mg) =
(change in absorbance/min of sample – that of blank) ⫻ (dilution factor) 6.22 ⫻ (volume of sample used) ⫻ (sample concentration in mg/ml)
Figure 6.2 Measurement of enolase activity by a coupled enzyme system of lactate dehydrogenase and pyruvate kinase. Schema represents flow of the sequential conversion of substrates for enzymes involved, showing the last reaction is coupled with conversion of NADH to NAD. Thus, enolase activity can be monitored as a decrease in NADH absorbance at 340 nm and estimated by the equation.
1.4 103 M 1 cm 1, the change of one absorbance unit at 240 nm means increase of PEP concentration in the reaction mixture from 0 to 714 mM during the reaction time. Velocity of the reaction should be calculated from initial rates of absorbance increase. h. Do not forget to make an appropriate blank/control tube. Note a. Due to limitation of amount of 2-PG in terms of cost, we recommend this micro assay system, although a spectrophotometer measurable in small volume (1 ml) is necessary. b. Mg2+ should be included in the reaction buffer. Some other divalent cations such as Zn2+ and Mn2+ work as a cofactor of enolase with lower efficacy (Baranowski & Wolna, 1975). c. Inclusion of sodium fluoride (2 mM), phosphonoacetohydroxamate (2 mM), or ENOblock (10 mM) in the assay inhibits the enolase activity ( Jung et al., 2013; Wedekind, Poyner, Reed, & Rayment, 1994). So you can confirm that specific changes in the absorbance are dependent on the enzyme. d. Enolases from yeast and rabbit muscle as a positive control can be purchased from Oriental Yeast Co. (Tokyo, Japan) and Sigma-Aldrich (St. Louis, MO, USA), respectively. Alternatively mammalian blood
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which is diluted 1:1 with a hypotonic solution, sonicated, and centrifuged at 20,000 g can be used. e. We have experienced that inclusion of reduced glutathione (GSH, 5 mM) in the assay inhibits the enolase activity, due to acidification of the assay reagent by GSH. Be careful in pH in the reaction system if you include some additives. 2.2.2 Coupled assay a. Freshly prepare a reaction buffer containing 100 mM triethanolamine (pH 7.4), 20 mM MgCl2, 150 mM KCl, 0.6 mM b-NADH, 2 mM ADP, 20 units/ml PK, and 30 units/ml LDH. b. Put 50 ml of the reaction buffer into the tube. c. Put 25 ml of the sample after appropriate dilution or dilution buffer into the tube. d. Record absorbance of the solution at 340 nm at room temperature (25 C). e. Put 25 ml of 2-PG (1–2 mM as a final concentration) solution into the tube. f. Record absorbance of the solution at 340 nm for 5 min after the addition of 2-PG at room temperature. g. Calculate changes in the absorbance reflecting the sequential conversion of 2-PG to PEP by enolase, PEP to pyruvate by PK, and pyruvate to lactate by LDH. Since molecular extinction coefficient for NADH is 6.22 103 M 1 cm 1, the change of one absorbance unit at 340 nm means increase of lactate concentration in the reaction mixture from 0–161 mM during the reaction time. h. Ordinary, enolase activity can be calculated by the equation shown in Fig. 6.2. Note a. Changes in NADH concentration could be monitored fluorometrically (NADH excitation 360 nm, emission 460 nm). This method is suitable for evaluating relative abundance of enolase activity and can be applied for high-throughput assay (Muller, Aquilanti, & DePhinho, 2012). 2.2.3 Isozyme-specific assay Three isoforms of enolase have been identified: a-enolase, b-enolase, and g-enolase. a-Enolase has been detected on most tissues, whereas b-enolase is expressed predominantly in muscle tissue, and g-enolase is detected only
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in nervous tissue. These isoforms exist as both homodimers and heterodimers, and therefore, five types of dimeric enolase (isozymes) are found as aa, ab, bb, ag, and gg. Three isoforms can be distinguished immunologically, and five isozymes are separated by DEAE-cellulose chromatography (Rider & Taylor, 1974; Suzuki, Umeda, & Kato, 1980).
3. DEAE-CELLULOSE CHROMATOGRAPHY a. Tissue extract from brain or muscle is dialyzed against 15 mM Tris–HCl (pH 7.9), 5 mM MgCl2, and 0.1 mM EDTA. b. The dialysate is applied on DEAE-cellulose column preequilibrated with the same buffer. c. The aa type of enolase is recovered from flow-through (unbound) fraction. d. The bb, ab, and ag types of enolase are eluted with the same buffer containing 0.1 M NaCl after successful washing. e. The gg type of enolase is eluted with the same buffer containing 0.3 M NaCl.
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