EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 29, No 1, 25-30, March 1997 Purification and characterization of lactate dehydrogenase from Varanus liver Masood H. Javed,1,3 Syed M. I. Azimuddin,2 muscle or type-5) and B (H or heart or type-1). The Abida N. Hussain,1 Asifa Ahmed2 and combinations of various forms of subunits produce five Mohammad Ishaq2 isoenzymes (Maekawa, 1988; Javed et al. 1995). These isoenzymes differ in various physicochemical, 1 Department of Biochemistry, The Aga Khan University, immunological and physiological properties (Everse and Karachi, Pakistan Kaplan, 1973; Maekawa, 1988; Javed and Waqar, 2 Department of Biochemistry, Karachi University, Karachi, Pakistan 1993). 3 Corresponding author LDH isoenzymes are not formed by random subunits combinations, and differences in the proportions of Accepted 7 January 1997 subunit for isoenzymes in different tissues (organs) suggest a physiological basis for their existence. LDH-4 Abbreviations: LDH, lactate dehydrogenase; AGE, Agarose gel electrophoresis; KPE, and LDH-5 which contain mainly M subunits permit potassium phosphate buffer containing EDTA rapid accumulation of lactate, and are found in skeletal muscle where anaerobic glycolysis predominates, whereas LDH-1 and LDH-2 containing mainly H subunits are found in heart, where pyruvate is oxidized via the Krebs cycle (Basaglia, 1989). Very little Abstract information is available on LDH from reptiles. In our previous studies (Javed et al., 1995), we have shown Lactate dehydrogenase was purified 21-fold from that LDH from a reptile, Uromastix hardwickii, has four liver of Varanus bengalensis using colchicine- isoenzymes in the liver which differ in physicochemical sepharose column chromatography. The crude and electrophoretic properties from livers of other enzyme showed two isoenzymes (LDH-5 and LDH-4) animals. Similarly, the characteristics of LDH isoenzyme by agarose gel electrophoresis (AGE). The p u r i f i e d purified from testes of U. hardwickii are also different enzyme showed a single band after SDS-PAGE from other animals (Javed et al., 1994; Javed and corresponding to molecular mass of 35 kDa. The Waqar, 1996). In this paper, we have described the molecular mass of native enzyme was about 140 properties of purified LDH from liver of another reptile, kDa. The optimum pH for the forward reaction was Varanus bengalensis. 7.5 while that for the reverse reaction was pH 9.5. The Km values for pyruvate, NADH, lactate and NAD+ were 0.17 ± 0.037, 0.02 ± 0.004, 12.4 ± 3.05 and Materials and Methods 0.38 ± 0.032 mM, respectively. Pre-heating of enzyme showed that its t5 0 was 40-50˚C. Oxalate Materials and n-hexanediol were inhibitors for both forward Fresh livers were obtained from Varanus bengalensis + + and reverse reactions. Among divalent ions, Cu and frozen at -20˚C until use. No distinctions were was shown to be more effective inhibitor for the made between male and female animals. NADH, NAD+, forward reaction. agarose, lithium lactate, nitroblue tetrazolium (NBT), phenazine methosulfate (PMS), agarose type-1 low Ke y w o r d s : LDH isoenzyme, liver, properties, purification, EEO, MW SDS-200 kit and sodium pyruvate were from Varanus bengalensis Sigma Chemical Co., U.S.A. 1,6-Hexanediol (extra pure) was from Aldrich Chemical Co. Inc., U.S.A. Colchicine-CH-Sepharose was a gift from Dr. Tomoji Introduction Kocha (Department of Hygienic Chemistry, Showa Lactate dehydrogenase (L-lactate:NAD oxidoreductase, College of Pharmaceutical Sciences, Tokyo, Japan). All EC 1.1.1.27; LDH) plays an important role in the regu- other chemicals were of analytical grade. lation of anaerobic glycolysis through reoxidation of NADH (Everse and Kaplan, 1973; Javed et al., 1995). Purification of LDH The structure of vertebrate LDH has been investigated All purification procedures of LDH were performed at 0- by a number of investigators (Cahn et al., 1962; Everse 4˚C, otherwise indicated. Frozen liver was thawed and and Kaplan, 1973; Li et al., 1989)) who have shown that the LDH was purified to homogeneity by ammonium it is a tetramer composed of two subunits, A (M or sulphate fractionation and colchicine-sepharose column 26 Exp. Mol. Med. chromatography according to the methods previously enzyme that produced one micromol of NAD+ per min described (Javed et al. 1995). under the assay conditions. A molar absorption coefficient for NADH of 6.22ለ103 M-1cm-1 at 340 nm was used for Gel electrophoresis calculations (Voorter et al., 1993). For the reverse To resolve the types of LDH isoenzymes in liver extract reaction (NAD+ to NADH), the enzyme activity was and chromatographic fractions, we used horizontal determined by measuring an increase in absorbance at agarose gel electrophoresis. One percent ultrathin (0.4 340 nm. The final concentration of the reactants in a mm) horizontal agarose gel (7.5ለ10cm) was prepared standard reaction mixture was 50 mM Tris-HCl buffer on gel bond and electrophoresis and LDH staining were (pH 9.5), 50 mM lithium lactate and 0.1 mM NAD+. It carried out as described elsewhere (Javed et al., 1995). contained 20 μl of suitably diluted enzyme. The volume To check the purity of the LDH, SDS-PAGE was done of reaction mixtures in both cases was 1 ml. Km an d according to the method described by Gorg et al. (1985) Vm a x (Kc a t) values were calculated from initial velocity using a gradient gel from 7.5% to 25%. measurements, using Enzfitter (R. J. Leather-barrow), a non-linear regression program that integrates the data Molecular mass determination into the Michaelis-Menton equation, with simple weighting. The molecular mass of the LDH was estimated using a Proteins were determined by the method of Lowry et al. (1951) with bovine serum albumin as standard. 1.5ለ73 cm column packed with approximately 125 ml of Sepharose CL-6B in phosphate buffer containing For inhibition studies, a known concentration of the test EGTA (KPE) buffer. All standard proteins and dextran modulator (after adjusting pH 7.5 for forward reaction and blue were passed through column separately. pH 9.5 for reverse reaction) was added to the reaction mixture. The activity of the enzyme was then measured Enzyme and protein assays by the forward or reverse reactions. The results are expressed as remaining activity (in %) compared to Enzyme activity was measured in the forward reaction + control (100%). The effect of these chemicals were also (NADH to NAD ) at 25˚C in a reaction mixture containing observed on the oxidation/reduction of NADH/NAD+ i n 50 mM phosphate buffer (pH 7.5), 0.18 mM NADH, 0.6 the absence of enzyme. The nature of oxalate inhibition mM sodium pyruvate and a suitable amount of enzyme was determined using Lineweaver-Burk plots. to obtain a measurable decrease in absorbance at 340 nm using Beckman DU70 UV-Vis spectrophotometer. One unit of enzyme was defined as the amount of Figure1. Colchicine-Sepharose column chromatography of LDH from Varanus liver. Sample was loaded onto a column (1.5ለ2 cm) of immobilized colchicine-Sepharose equilibrated with 2.5 M NaCl-KPE buffer. The column was washed with the same buffer. Figure 2. A. Agarose gel electrophoresis (AGE) of LDH from Varanus liver. Lanes 69, To wash out glyceraldehyde 3-phosphate dehydrogenase, KPE buffer without NaCl was 73 and 77 are tube number after elution of LDH from colchicine-Sepharose column by passed through the column. LDH was then eluted with 1 mM NADH in KPE buffer. The NADH. Crude sample is liver extract. B. SDS-PAGE of purified LDH. a, reference flow rate was about 40 ml/h and 1 ml fractions were collected. The protein concentration proteins are; 1, carbonic anhydrase (29 kDa); 2, egg albumin (45 kDa); 3, BSA (66 (ᆨ) was measured as A280. The enzyme activity (ᆩ) was determined by measuring kDa); 4, phosphorylase B (97.4 kDa); 5, β-galactosidase (116 kDa); 6, myosin (205 the change in absorbance at 340 nm min-1 at 25˚C. kDa); b, purified LDH. Varanus liver LDH 27 Results The results from a representative purification according to the procedure described in the “Materials and Colchicine-Sepharose chromatography Methods” section are shown in Table 2. The specific activity of purified LDH was 145.45 U/mg protein. The When dialyzed liver extract was applied on a colchicine- purified enzyme was stable at 4˚C for at least 2 years sepharose column, the entire LDH activity was retained while it was unstable at room temperature. by the column. Addition of 1 mM NADH in KPE buffer eluted LDH in a sharp peak (Figure 1). The agarose gel Kinetic studies electrophoresis (AGE) of LDH is shown in Figure 2. Two bands of LDH (LDH4 and 5) were identified in 40-80% The pH profiles of LDH for the forward and reverse ammonium sulphate fraction, while five LDH isoenzymes directions are shown in Figure 3. In the forward direction were resolved in peak fraction (Number 73) after the optimum pH was 7.0. For the reverse reaction, the c o l c h i c i n e -Sepharose column chromatography. The enzyme showed an optimum pH at 9.5. The activity was major isoenzyme in all cases was LDH-5 (about 70%). still very high (about 55%) even at pH 11.0. The purity of LDH was checked by SDS-PAGE which Table 3 summarizes the results of experiments in showed a single band corresponding to the molecular which kinetic parameters of LDH for pyruvate, lactate, + mass of about 35 kDa (Figure 2B, lane b). The molecular NADH and NAD . Before analyzing the effects of the mass of native LDH was calculated to be about 140 kDa concentrations of various substrates, LDH was dialyzed (Table 1) by Sepharose column chromatography.