Q 2008 by The International Union of Biochemistry and Molecular Biology
BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION Vol. 36, No. 2, pp. 135–138, 2008
Laboratory Exercises Measuring Intracellular Enzyme Concentrations*hS ASSESSING THE EFFECT OF OXIDATIVE STRESS ON THE AMOUNT OF GLYOXALASE I Received for publication, June 27, 2007, and in revised form, December 5, 2007 Hugo Vicente Miranda‡§, Anto´nio E. N. Ferreira‡, Alexandre Quintas‡§, Carlos Cordeiro‡{, and Ana Ponces Freire‡ From the ‡Departamento de Quimica e Bioquı´mica, FCUL, 1749-016 Lisboa, Portugal and §Instituto Superior das Cieˆncias da Sau´de Egas Moniz, Laborato´rio de Patologia Molecular, 2829-511 Caparica, Portugal
Enzymology is one of the fundamental areas of biochemistry and involves the study of the structure, kinetics, and regulation of enzyme activity. Research in this area is often conducted with purified enzymes and extrapolated to in vivo conditions. The specificity constant, kS, is the ratio between kcat (the catalytic constant) and Km (Michaelis–Menten constant), and expresses the efficiency of an enzyme as a catalyst. This parameter is usually determined for purified enzymes, and in this work, we propose a classroom experiment for its determination in situ, in permeabilized yeast cells, based on a method of external enzyme addition, which was previously reported. Under these conditions, which resemble the in vivo state, enzyme concentrations and protein interactions are preserved. The students are presented with a novel approach in enzymology, based on the titration methods that allow the measurement of the enzyme amount, and thus the kcat and kS. The method will also be used to investigate the effect of exposure to oxidative stress conditions on yeast glyoxalase I. Keywords: Enzymes and catalysis, laboratory exercises, problem-based learning. Kinetic parameters known for the vast majority of enzymes have been obtained in vitro for the purified forms. Recently, enzymology is moving toward the understanding of enzyme kinetics in vivo [1]. One of the best approaches, however, is to study the enzymes in situ [2]. This can be achieved by selective membrane permeabilization of low molecular weight molecules, in which all the macromolecules, namely enzymes, remain in the cell at unchanged concentrations. Digitonin-based methods specifically complexify cholesterol (or other sterols), leading to membrane permeabilization [3]. This technique poses new insights in enzymology, given that the enzyme under study remains in its native environment. All the protein interactions and localizations are maintained, giving more accurate results on its kinetic behavior than those of purified enzymes. A fundamental kinetic parameter of an enzyme is its efficiency, kS, defined as kcat/Km [4]. The catalytic rate con-
& s This article contains supplementary material available via the Internet at http://www.interscience.wiley.com/jpages/14708175/suppmat. * This work is supported by: Fundac¸a˜o para a Cieˆncia e a Tecnologia, Ministe´rio da Cieˆncia e Tecnologia, Portugal (grant no. SFRH/BD/23035/2005). { To whom correspondence should be addressed. DQBFCUL, Lisboa 1749-016, Portugal. Tel: +351217500929; Fax: +351217500088; E-mail:
[email protected]. 1 The abbreviations used are: GSH, glutathione; MES, 2-(Nmorpholino) ethanesulfonic acid; YPD, yeast/peptone/dextrose; HTA, hemithioacetal. This paper is available on line at http://www.bambed.org
stant, kcat, is also known as the turnover number because it is a reciprocal time and defines the number of catalytic cycles the enzyme can undergo in a unit of time, or the number of molecules of substrate that one molecule of enzyme can convert into products in one unit of time [4]. It is given by the proportion or ratio between the limiting rate, V, and the total enzyme concentration. Enzyme concentration is a very difficult parameter to assay while studying the enzyme activity in a nonpurified form, like in vivo and in situ studies. The kS takes all the kinetic and substrate binding ability into consideration and is very useful to compare the enzymes or the competing substrates for the same enzyme. The aim of this work is to illustrate the application of a recently described method [5] to assay the changes in intracellular concentration of some enzymes upon exposure to oxidative stress. In this practical experiment, we propose to study the in situ kinetic properties of the enzyme glyoxalase I (S-Dlactoylglutathione:methylglyoxal lyase, EC 4.4.1.5), and also assay its intracellular concentration and kcat. We also suggest to study the effect of H2O2 on the homeostasis of the yeast Saccharomyces cerevisiae cells by evaluating the change in the intracellular enzyme concentration of glyoxalase I. Methylglyoxal, a reactive dicarbonyl compound, is an unavoidable product of the nonenzymatic b-elimination of dihydroxyacetone phosphate and D-glyceraldehyde-3phosphate during glycolysis [6]. In eukaryotic cells, the methylglyoxal main catalytic pathway is the glyoxalase
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DOI 10.1002/bmb.20166
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BAMBED, Vol. 36, No. 2, pp. 135–138, 2008 rithmic phase (5 hours of growth), one of them supplemented with hydrogen peroxide (1 mM). Saccharomyces cerevisiae strain BY4741 from Euroscarf collection was used, but any other yeast strain may be deployed.
Cell Permeabilization After harvesting by centrifugation (5,500 3 g at 4 8C), cells were washed twice with distilled water and suspended in 0.1 M MES-NaOH (pH 6.5). Cells were permeabilized with 0.01% (w/v) digitonin by using a modification of a previously described method [3] for 15 minutes. After permeabilization, cells were washed twice with MES buffer and finally concentrated 10 times in assay buffer.
Protein Assay
FIG. 1. The glyoxalase pathway reactions.
system that comprises the enzymes glyoxalase I and glyoxalase II (hydroxyacylglutathione hydrolase, EC 3.1.2.6) in a glutathione- (GSH)1 dependent two-step pathway. First, glutathione reacts with methylglyoxal forming an hemithioacetal, and then glyoxalase I catalyses the formation of S-D-lactoylglutathione. Glyoxalase II catalyses the hydrolysis of S-D-lactoylglutathione to D-lactate and regenerates the glutathione [7, 8] (Fig. 1). The glyoxalase I reaction can be followed spectrophotometrically by the S-D-lactoylglutathione formation at 240 nm and e240nm ¼ 2.86 mM21 cm21 [9]. MATERIALS AND METHODS
Reagents and Equipment Absorbance determinations for protein assay and enzyme activity can be performed using various spectrophotometers, although the diode array spectrophotometers equipped with stirring and temperature control in the cuvette holder are particularly advised. Determinations were made in a Beckman DU7400 diode array spectrophotometer equipped with stirring and temperature control. Centrifugations were performed in a refrigerated Eppendorf 5804 R centrifuge (F34-6-38 Eppendorf rotor). Digitonin was purchased from CalBiochem. 2-(N-Morpholino)ethanesulfonic acid (MES), Coomassie Brilliant Blue G, glyoxalase I (S-D-lactoylglutathione:methylglyoxal lyase; EC 4.4.1.5), hydrogen peroxide, and methylglyoxal were from Sigma. Glutathione was purchased from Roche. Other reagents were of analytical grade and used without further purification. Deionised, double distilled water was used.
Microbiological Techniques Preculture was performed by growing cells in yeast/peptone/ dextrose- (YPD) rich medium (D-glucose (2%, w/v), yeast extract (0.5%, w/v), and peptone (1%, w/v)), and the cells were harvested by centrifugation (5,500 3 g at 4 8C) at the end of the logarithmic phase (17 hours of growth). Two populations were then grown in YPD-rich medium for a 2-hour period in the loga-
The proteins in the permeabilized cells were assayed using a modified Coomassie brilliant blue assay [10]. To a volume of 2 mL of dye reagent solution, 200 lL of sample was added and the absorbance measured at 600 and 460 nm. The absorbance difference at these two wavelengths varies proportionally with protein concentration. A standard calibration curve, with bovine serum albumin, was prepared (between 0.02 and 0.2 mg/mL). The dye reagent solution is prepared as follows: 0.01 g Coomassie brilliant blue, 5 mL of 95% (w/v) ethanol, 10 mL of 85% (w/v) phosphoric acid, and distilled water up to 100 mL of final volume.
Enzyme Assay Glyoxalase I activity was measured by assessing the S-D-lactoylglutathione formation at 240 nm in 0.1 M MES-NaOH (pH 6.5) buffer in the presence of 6 mM glutathione and 6.5 mM methylglyoxal [11].
Kinetic Study The apparent kinetic parameters of glyoxalase I were determined by varying the hemithioacetal concentration between 0.3 and 1.5 mM, which was obtained by the nonenzymatic reaction between GSH and methylglyoxal with an equilibrium constant of Keq ¼ 0.323 mM. The Michaelis constant, Km, and the limiting rate, V, were determined by the hyperbolic nonlinear regression and by the direct linear plot [11] using the computer program HYPERFIT [12]. Further calculations were made by using the direct linear plot results. The choice to include a parametric (nonlinear least-squares regression) as well as a ‘‘distributionfree’’ method (direct linear plot [11]) to estimate the Km and V stimulates the students to understand and explore the fundamental ideas that support each of the statistical types of data analysis, and become aware of their differences. Although a comparison of these two methods is beyond the scope of this paper, the target population of students should have taken the first- or second-year statistical courses in which this topic should have been addressed.
Assay of the Intracellular Enzyme Concentration First, after taking a suitable amount of permeabilized yeast cell suspension, the activity of the enzyme to be assayed was determined at a fixed substrate concentration. Then, the purified enzyme was added to the cell suspension in known increasing amounts. It is important to notice that the purified enzyme added must be from the same origin, that is, the one to be assayed (in this work, Saccharomyces cerevisiae). The amount of intracellular enzyme concentration in the cell suspension is given by the absolute value of the abscissa interception of the curve obtained from the representation of total enzyme activity versus added enzyme concentration [5].
137 STUDENT ACTIVITIES
At the beginning of the class, groups of two or three students are provided with the following information: 1) 2)
The composition of culture media and the cell growth conditions. Original papers on cell permeabilization [3], protein assay [10], glyoxalase pathway study [11], and intracellular enzyme concentration assay [5].
Our experience shows that the experiments can be carried out in three sessions of 3–4 hours in a 15-student class. In the first period, students prepare the solutions required, including buffers and enzymatic substrates (methylglyoxal and glutathione), and discuss and elaborate an experimental protocol (Supplementary material). In the following session, students are presented with the previously prepared cell cultures (logarithmic phase) [11] to determine the in situ glyoxalase I kinetic parameters, including kcat and its intracellular concentration. In the last class, the physiological response to oxidative stress due to hydrogen peroxide is evaluated. Students are presented with the previously prepared cell cultures. These are separated into two assays, one of them exposed to hydrogen peroxide for a 2-hour period in the beginning of the session. The cells are then permeabilized and glyoxalase I assayed in both the populations. At the end of the class, an extra hour is useful for group discussion. RESULTS AND DISCUSSION
In this work, students are provided with a practical experiment intended to assay an intracellular enzyme concentration and kcat. The activity of glyoxalase I was determined in the permeabilized yeast cells, to determine the kcat, Km, and intracellular concentration of the enzyme. First, the glyoxalase I kinetic parameters were determined in situ. Using the software HYPERFIT [13], which implements the direct linear plot, the limiting rate and Km can be assayed. Nevertheless, to determine the enzyme kcat, the intracellular enzyme concentration was determined. The results are shown in Table I and Fig. 2b and 2c. To study the physiological response to oxidative stress due to hydrogen peroxide, yeast cells were subjected to different H2O2 concentrations. The intracellular glyoxalase I concentration was assayed by performing in situ studies, which showed clear effects of oxidative stress on this protein expression. Upon exposure to hydrogen peroxide, the enzyme amount increases 36% over its reference amount. Detailed results are shown in Table I. It is well known that in the presence of hydrogen peroxide, cells undergo a defense response that involves enzymatic and nonenzymatic removal of radicals formed by oxidative species. Catalase, superoxide dismutase, glutathione reductase, and glucose 6-phosphate dehydrogenase are examples of enzymes that undergo adaptive responses [14, 15]. It was then expected that glyoxalase I concentration would increase. Additional experiments may be performed with this system. For instance, any of the enzymes mentioned ear-
TABLE I Determination of the enzyme concentration, catalytic constant, and specificity constant for Saccharomyces cerevisiae glyoxalase I upon different oxidative stress conditions Enzyme kinetic analysis Total protein (lg) Number of cells (3106) Enzyme assay (lg) Molecular weight Total enzyme concentration (lM) Enzyme concentration per cell (310220 mol cell21) Cell volume (310214 dm3) Michaelis constant—direct linear plot (mM) Limiting rate—direct linear plot (mM min21) Michaelis constant—hyperbolic regression (mM) Limiting rate—hyperbolic regression (mM min21) kcat (3104 min21) kS (31010 mM21 min21)
14.00 13.32 0.082 37,209 5.74 16.64 2.90 1.03 89.14 1.03 89.19 1.55 1.51
Hydrogen peroxide glyoxalase I effect
Control
H2O2 exposure
Total protein (lg) Number of cells (3106) Enzyme assay (ng) Total enzyme concentration (lM) Enzyme concentration per cell (310220 mol cell21)
4.78 6.88 8.29 1.12
3.05 3.93 6.44 1.52
3.24
4.40
lier can be used to evaluate the oxidative stress effects, like glucose 6-phosphate dehydrogenase, whose activity can be spectrophotometrically followed by NADPH formation (340 nm), by reacting NADPþ with glucose 6phosphate [5]. Other stress conditions, such as heat shock and osmotic stress, can also be evaluated.
CONCLUDING COMMENTS
The kinetic characterization of enzymes by the technique referred to in this work can be considered to be a novel approach for the study of these molecules in their native environment [5]. It should be pointed out that the procedure includes the determination of enzyme concentration, kcat, and kS. These parameters are much more difficult to measure than the Km or the enzyme activity. This work is currently being performed by students of the biochemistry undergraduate program at the ‘‘Faculdade de Cieˆncias of Universidade de Lisboa,’’ Portugal, in a module of two laboratory classes of 3 hours each. It is our belief that students, after attending these classes, successfully improve their skills on enzyme kinetics and their understanding of the enzyme function and its response to changes in the cellular environment. Furthermore, the experimental procedure could be integrated with more elaborate assignments, adequate to the graduate level. As a suggestion, the titration method that was outlined in this paper could be complemented with transcriptomics experiments to study the enzyme adaptation to pathological situations at different levels of metabolic regulation.
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FIG. 2. Hyperbolic and direct linear plots representation of the Michaelis–Menten enzyme kinetics. (a) Activities were assayed upon variation of hemithioacetal (HTA) concentration between 0.3 and 1.5 mM in 0.1 M MES-NaOH (pH 6.5) in a 1.5 mL volume of 13.32 3 106 permeabilized cells containing 14.0 lg total protein. Measurement of glyoxalase I amount in situ in Saccharomyces cerevisiae upon no H2O2 exposure (b) and 1 mM H2O2 exposure (c). Activity was measured in the permeabilized cells ((b) 6.88 3 106 cells containing 4.78 lg total protein; (c) 3.93 3 106 cells containing 3.05 lg total protein). The assay was performed in the presence of 6 mM glutathione and 6.5 mM methylglyoxal in 0.1 M MES-NaOH (pH 6.5) in a 1.5 mL volume. Insets in (b) and (c): magnification of the plot between 20.01 and 0.01 lg, showing the abscissa’s interception. Acknowledgment— The authors thank Dr. Ricardo Gomes (Universidade de Lisboa, Departamento de Quimica e Bioquı´mica, Portugal) for fruitful discussion and revision of the manuscript.
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