Purification and characterization of malate dehydrogenase from sheep liver (Ovis aries): Application in AST assay diagnostic kit

© 2018 Doaa A. Darwish et al. This is an open access article distributed under the terms of the Creative Commons Attribution License -NonCommercialShareAlikeUnported License (http://creativecommons.org/licenses/by-nc-sa/3.0/). *Corresponding Author Hassan Mohamed M. Masoud, Molecular Biology Department, National Research Centre, Postal code: 12622, El-Tahrir st., Dokki, Giza, Egypt. E-mail: hssnmasoud @ yahoo.com Purification and characterization of malate dehydrogenase from sheep liver (Ovis aries): Application in AST assay diagnostic kit


INTRODUCTION
Malate dehydrogenase (MDH: EC 1.1.1.37) is widely distributed enzyme through living organisms that present in animals, plants and microorganisms and it is an essential enzyme in the central oxidative pathway. MDH is a catalyst in the oxidation process of malate to oxaloacetate and uses the reduced NAD + to NADH as a cofactor. MDH is one of the main enzymes in the tricarboxylic acid cycle (TCA) that performs significant metabolic part in aerobic energy producing pathways and in malate shuttle. In eukaryotes, MDH is present in two forms either mitochondrial or cytosolic according to their location (Fickenscher et al., 1987;Domenech et al., 1988;Minárik et al., 2002;Takahashi-Iñiguez et al., 2016). Malate dehydrogenases were also found in plants and some eukaryotes in different organelles as glyoxysomes, chloroplasts and peroxisomes (Gietl, 1992). In aerobic prokaryotic and eukaryotic organisms, the TCA cycle is the main oxidative pathway (Sidhu et al., 2011). A main reaction of TCA cycle is the transformation between malate and oxaloacetate stimulated by NAD + or NADP + dependent malate dehydrogenases. In addition to their important metabolic role in aerobic energy production, they participate in different metabolic actions comprising aspartate biosynthesis, the malate-aspartate shuttle, gluconeogenesis and lipogenesis (Labrou and Clonis, 1997). MDH has other tasks inside cells (Molenaar et al., 1998) as participation in the reductive TCA cycle for oxidative stress protection by binding the free radicals and in transfer of substrates throughout the metabolic pathways (Wu et al., 2007). Also, MDH with malic enzyme and pyruvate carboxylase are participated in an antioxidative pathway converting the pro-oxidant NADH into the antioxidant NADPH (Singh et al., 2008). In some microorganisms, MDH enzyme can interact with other TCA cycle enzymes to increase the cycle activity as in Bacillus subtilis that interacts with isocitrate dehydrogenase and citrate synthase forming a metabolon (Meyer et al., 2011;Bartholomae et al., 2014) and in E. coli, it interacts with the respiratory chain complex I for the immediate transport of NADH (Amarneh and Vik, 2005). MDH activity is vigorously blocked by oxaloacetate and NADH excess (Langelandsvik et al., 1997;Thompson et al., 1998;Madern and Zaccai, 2004;Mendoza et al., 2009). Lactate dehydrogenase (LDH) and MDH enzymes are major components in aspartate aminotransferase (AST) diagnostic kit. This kit is very sensitive in the detection of AST enzyme concentration in blood, serum and plasma which is mainly utilized as a liver function marker test in diagnosis, monitoring and treating of hepatic diseases. The kinetic assay of AST for diagnostic purposes uses a combined reaction of MDH, LDH and NADH (Henry, 1974;Tietz, 1976;Henry, 1984;Young, 1990). Therefore, the main aim of this study is purification, characterization and production of MDH enzyme from the sheep liver as locally available, safe and rich source for use in preparation of the AST diagnostic kit.

Liver tissues
Six fresh sheep liver samples were obtained from different male individuals in a local slaughter-house and stored at −40˚C.

Assay of malate dehydrogense activity
For assaying MDH activity, a reaction mixture contained 50 mM glycine-NaOH buffer (pH 9.6), 3 mM malate and 0.52 mM NAD was used. NADH increase at 340 nm was followed for 3 minutes and one MDH unit is defined as the amount of enzyme required to oxidize or reduce 1 μmol coenzyme min -1 (Nava et al., 2011).

Malate dehydrogenase activity staining on polyacrylamide gels
After electrophoresis, the gels were submerged in 100 ml 0.1M Tris-HCl, pH 8 containing 250 mg malate sodium salt, 30 mg NAD, 25 mg NBT and 2 mg PMS. The gel was incubated in the staining solution in dark at 37˚C until dark blue bands appear (Richardson et al., 1986).

Preparation of crude extract
All of the procedures were performed at 4˚C unless stated otherwise. 10 grams of sheep liver were homogenized in 0.05 M sodium phosphate buffer pH 7.4 containing 10 mM EDTA and 1 mM MgCl 2 using Omni mixer homogenizer. Cell debris and insoluble materials were removed by centrifugation at 5000 xg for 20 min and the supernatant was saved and designated as crude extract.

Ammonium sulfate precipitation
The crude extract was brought to 40% saturation by gradually adding solid (NH 4 ) 2 SO 4 , stirred for 30 min at 4°C and centrifuged at 8000 xg for 20 min. The pellet of this step was discarded and the filtrate was brought to 80% (NH 4 ) 2 SO 4 saturation and the pellet was obtained by centrifugation at 12000 xg for 30 min, dissolved in 0.02 M sodium phosphate buffer pH 7.4 and dialyzed against the same buffer (3 × 2L buffer).

DEAE-cellulose column chromatography
The ammonium sulfate fraction was chromatographed on DEAE-cellulose column previously equilibrated with 0.02 M sodium Phosphate buffer pH 7.4. The proteins were eluted with stepwise NaCl gradient ranging from 0 to 1 M prepared in the equilibration buffer. Fractions of 5 ml were collected at a flow rate of 60 ml/hour. Fractions containing MDH activity were pooled and concentrated by lyophilization.

Sephacryl S-300 column chromatography
The concentrated solution containing the MDH activity was applied onto a Sephacryl S-300 column. The column was equilibrated and developed with 0.02 M sodium Phosphate buffer pH 7.4 at a flow rate of 30 ml/hour and 2 ml fractions were collected.

Electrophoretic analysis
Native gel electrophoresis was carried out with 7% PAGE (Smith, 1969). SDS-PAGE was performed with 12% polyacrylamide gel (Laemmli, 1970). The subunit molecular weight of the purified MDH enzyme was determined by SDS-PAGE (Weber and Osborn, 1969). Electrofocusing was performed according to O'Farrell (1975) and the isoelectric point (pI) value was calculated from a calibration curve as described by Ubuka et al. (1987). Proteins were stained with 0.25% Coomassie Brilliant Blue R-250.

Protein determination
Protein content was determined by the dye binding assay method using bovine serum albumin (BSA) as a standard protein (Bradford, 1976).

Construction of aspartate aminotransferase (AST) diagnostic kit
The purified sheep liver MDH is used in the construction of AST diagnostic kit. The kit is composed of two reagents: (R1: 80 mM Tris-HCl buffer pH 7.8, 240 mM L-aspartate, 900 IU/L LDH and 600 IU/L MDH) and (R2: 12 mM 2-oxoglutarate and 0.18 mM NADH) (Thomas, 1998).

Purification of sheep liver malate dehydrogenase (SLMDH)
Malate dehydrogenase is a major component in aspartate aminotransferase (AST) diagnostic kit. This kit is very sensitive in the measurement of AST enzyme concentration in blood, serum and plasma which is mainly used as a liver function marker test in diagnosis, monitoring and treatment of liver diseases (Henry, 1974;Tietz, 1976;Henry, 1984;Young, 1990). The main aim of this study is purification, characterization and production of MDH enzyme from the sheep liver as locally available, safe and rich source for using in preparation of the AST diagnostic kit. The purification procedure of MDH from the sheep liver is very convenient since it involved ammonium sulfate fractionation, anion-exchanger DEAEcellulose resin chromatography and size-exclusion chromatography on Sephacryl S-300 resin. Different MDH purification methods were stated from rat liver by ion-exchange chromatography with affinity elution by NADH (Wiseman et al., 1991) and from chicken liver by ammonium sulfate precipitation, affinity chromatography on 5'AMP-Sepharose and Blue-Sepharose CL-6B columns (Gelpi et al., 1988). The purification procedure of sheep liver MDH is summarized in Table 1. The chromatographic pattern of sheep liver MDH on the DEAE-cellulose resin manifested the existence of a major MDH enzyme activity peak that designated SLMDH eluted with 0.0 M NaCl and one minor peak exhibited the MDH activity was resolved and eluted with 0.05 M NaCl (Figure 1a). The major peak fractions exhibiting MDH activity were collected, concentrated and placed onto a Sephacryl S-300 column ( Figure  1b) which showed the appearance of one MDH enzyme activity peak. Next size-exclusion chromatography, the SLMDH specific activity was raised to 6.7 units/mg protein representing 11.6 folds purification and 27.7% output (Table 1). A wide range of purification fold and recovery percent for MDH were reported. MDH from rat liver had 566-folds with 32% yield (Wiseman et al., 1991) and from chicken liver 225-fold with 47% yield (Gelpi et al., 1988).

Molecular weight determination and electrophoretic analyses of SLMDH
The native molecular weight of the prepared SLMDH is calculated from its elution volume on the gel filtration chromatography column to be 68 ± 1.6 kDa ( Figure 1b). The purity of SLMDH isoenzyme eluted from the Sephacryl S-300 columns was investigated by analysis on 7% native-PAGE. SLMDH isoenzyme displayed one protein band which corresponded with the MDH activity band affirming the tentative purity of the preparation (Figure 2a and 2b). The SLMDH subunit mass was resolved by SDS-PAGE which revealed that the SLMDH consists of two homodimer subunits with molecular weights of 34 ± 1.2 kDa each (Figure 2c). In consistent with SLMDH, MDH from Phytomonas sp. is a dimeric two subunits molecule of 37 kDa each (Uttaro and Opperdoes, 1997), Pseudomonas stutzeri MDH molecular mass was 66.5 kDa consisting of two subunits 34 kDa each (Labrou and Clonis, 1997), MDH from the phototrophic bacterium, Rhodopseudomonas capsulata has molecular weight of 80 kDa and consists of two subunits of 35 kDa (Ohshima and Sakuraba, 1986) and MDH from leaves of the plant Aptenia cordifolia showed a subunit of 39.4 kDa and a native mass of 83 kDa (Trípodi and Podestá, 2003). In contrast, MDH native protein of the bacterium Corynebacterium glutamicum was 130 kDa which was homotetramer with 33-kDa subunit each (Genda et al., 2003). The isoelectric point (pI) value of SLMDH was visualized on isoelectrofocusing PAGE as single species at pH 6.2 ( Figure  2d). Two MDH isoenzymes from Phytomonas sp. had pI values of 6.0 and 7.2 (Uttaro and Opperdoes, 1997), MDH from rat liver with PI value of 5.2 (Wiseman et al., 1991) and MDH from leaves of the plant Aptenia cordifolia showed pI value of 5.8 (Trípodi and Podestá, 2003).

SLMDH optimum pH and Km value determination
The pH optimum was studied by using glycine-NaOH buffer pH range of 8.6-10.6. The SLMDH enzyme optimum activity was detected at pH 9.6 ( Figure 3a). Analogously, the pH optimum was set at pH 9.5 for MDH from the Phytomonas sp. (Uttaro and Opperdoes, 1997) and the phototrophic bacterium, Rhodopseudomonas capsulata (Ohshima and Sakuraba, 1986) while the bacterium Corynebacterium glutamicum MDH optimum activity was at pH 10.5 (Genda et al., 2003). The Lineweaver-Burk plot for the reverse of the reaction velocity (1/v) and substrate concentration (1/[S]) indicated the value of Km to be 1.4 mM for NAD ( Figure 3b) and 11 mM for sodium malate (Figure 3c). The Km value of the Phytomonas sp MDH two isoforms for malate and NAD were 3 and 0.246 mM for the first isoform and 0.45 and 0.091 mM for the second isoform (Uttaro and Opperdoes, 1997).
Km value of MDH from Pseudomonas stutzeri was 0.34 and 0.67 mM for NAD and malate respectively (Labrou and Clonis, 1997).

Effect of divalent cations and various inhibitors on SLMDH
The effect of metal compounds on the purified SLMDH activity was examined. FeCl 2 , NiCl 2 and ZnCl 2 inhibited the purified sheep liver malate dehydrogenase (SLMDH) activity vigorously, while CoCl 2 and CuCl 2 inhibited SLMDH activity moderately ( Figure 4a) et al., 1977). Also, the inhibition of purified SLMDH activity by several inhibitors was studied (Figure 4b). Pre-incubation of the inhibitors for 5 minutes with SLMDH at 37˚C was carried out and the inhibition percent was deduced as a proportion of a noninhibited control. The purified SLMDH is inhibited with the serine protease inhibitor PMSF indicating that the active site of this isoenzyme contains a serine residue. Iodoacetamide inhibited the purified SLMDH isoenzyme activity which indicates that residues of methionine, cysteine and histidine have significant effects on the enzyme structure and action. EDTA inhibited the purified SLMDH isoenzyme indicating that SLMDH is a metalloprotein.
β-mercaptoethanol and dithiothreitol didn't inhibit the SLMDH isoenzyme action indicating that thiol groups in the enzyme structure doesn't perform a job for its action. The purified SLMDH is inhibited with the N-Ethylmaleimide indicating that the enzyme contains thiol group(s) in the enzymatic reaction. A considerable resistance of the enzyme SLMDH was detected toward most of the other tested inhibitors.

Comparison of the constructed AST kit with the commercially available kit
The purified SLMDH is used in the construction of AST diagnostic kit. The constructed kit (prepared AST diagnostic kit with the purified SLMDS enzyme) has been compared with commercially available kit using five healthy individuals samples (1-5 samples) and twenty five patient samples (6-30 samples) ( Table 2). The AST diagnostic kit is commonly used to monitor the liver function and following of various liver diseases. The prepared kit was found to be comparable to the commercial kit. The variance between the constructed and commercial kit was found more or less within the experimental error.

CONCLUSION
In conclusions, this study is the first report on sheep liver malate dehydrogenase (SLMDH). An easy, suitable and reproducible procedure is described for purification of full differentiated MDH from sheep liver as a local, available and rich source. The purified SLMDH enzyme is applied in the preparation of AST diagnostic kit which was found to be sensitive in comparison to a commercially kit.