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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
Abida N. Hussain,^1 Asifa Ahmed 2 and
Mohammad Ishaq^2
1 Department of Biochemistry, The Aga Khan University, Karachi, Pakistan 2 Department of Biochemistry, Karachi University, Karachi, Pakistan 3 Corresponding author
Accepted 7 January 1997
Abbreviations: LDH, lactate dehydrogenase; AGE, Agarose gel electrophoresis; KPE, potassium phosphate buffer containing EDTA
Abstract
Lactate dehydrogenase was purified 21-fold from liver of Varanus bengalensis using colchicine- sepharose column chromatography. The crude enzyme showed two isoenzymes (LDH-5 and LDH-4) by agarose gel electrophoresis (AGE). The purified enzyme showed a single band after SDS-PAGE corresponding to molecular mass of 35 kDa. The molecular mass of native enzyme was about 140 kDa. The optimum pH for the forward reaction was 7.5 while that for the reverse reaction was pH 9.5. The K (^) m values for pyruvate, NADH, lactate and NAD +^ were 0.17 ± 0.037, 0.02 ± 0.004, 12.4 ± 3.05 and 0.38 ± 0.032 mM, respectively. Pre-heating of enzyme showed that its t 50 was 40-50˚C. Oxalate and n-hexanediol were inhibitors for both forward and reverse reactions. Among divalent ions, Cu ++ was shown to be more effective inhibitor for the forward reaction.
Keywords: LDH isoenzyme, liver, properties, purification, Varanus bengalensis
Introduction
Lactate dehydrogenase ( L -lactate:NAD oxidoreductase, EC 1.1.1.27; LDH) plays an important role in the regu- lation of anaerobic glycolysis through reoxidation of NADH (Everse and Kaplan, 1973; Javed et al ., 1995). The structure of vertebrate LDH has been investigated by a number of investigators (Cahn et al ., 1962; Everse and Kaplan, 1973; Li et al ., 1989)) who have shown that it is a tetramer composed of two subunits, A (M or
muscle or type-5) and B (H or heart or type-1). The combinations of various forms of subunits produce five isoenzymes (Maekawa, 1988; Javed et al. 1995). These isoenzymes differ in various physicochemical, immunological and physiological properties (Everse and Kaplan, 1973; Maekawa, 1988; Javed and Waqar, 1993). LDH isoenzymes are not formed by random subunits combinations, and differences in the proportions of subunit for isoenzymes in different tissues (organs) suggest a physiological basis for their existence. LDH- and LDH-5 which contain mainly M subunits permit 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 information is available on LDH from reptiles. In our previous studies (Javed et al ., 1995), we have shown that LDH from a reptile, Uromastix hardwickii , has four isoenzymes in the liver which differ in physicochemical and electrophoretic properties from livers of other animals. Similarly, the characteristics of LDH isoenzyme purified from testes of U. hardwickii are also different from other animals (Javed et al ., 1994; Javed and Waqar, 1996). In this paper, we have described the properties of purified LDH from liver of another reptile, Varanus bengalensis.
Materials and Methods
Materials
Fresh livers were obtained from Varanus bengalensis and frozen at -20˚C until use. No distinctions were made between male and female animals. NADH, NAD +^ , agarose, lithium lactate, nitroblue tetrazolium (NBT), phenazine methosulfate (PMS), agarose type-1 low EEO, MW SDS-200 kit and sodium pyruvate were from 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 Kocha (Department of Hygienic Chemistry, Showa College of Pharmaceutical Sciences, Tokyo, Japan). All other chemicals were of analytical grade.
Purification of LDH
All purification procedures of LDH were performed at 0- 4 ˚C, otherwise indicated. Frozen liver was thawed and the LDH was purified to homogeneity by ammonium sulphate fractionation and colchicine-sepharose column
chromatography according to the methods previously described (Javed et al. 1995).
Gel electrophoresis
To resolve the types of LDH isoenzymes in liver extract and chromatographic fractions, we used horizontal agarose gel electrophoresis. One percent ultrathin (0. mm) horizontal agarose gel (7.5ለ10cm) was prepared on gel bond and electrophoresis and LDH staining were carried out as described elsewhere (Javed et al ., 1995). To check the purity of the LDH, SDS-PAGE was done according to the method described by Gorg et al. (1985) using a gradient gel from 7.5% to 25%.
Molecular mass determination
The molecular mass of the LDH was estimated using a 1.5ለ73 cm column packed with approximately 125 ml of Sepharose CL-6B in phosphate buffer containing EGTA (KPE) buffer. All standard proteins and dextran blue were passed through column separately.
Enzyme and protein assays
Enzyme activity was measured in the forward reaction (NADH to NAD+^ ) at 25˚C in a reaction mixture containing 50 mM phosphate buffer (pH 7.5), 0.18 mM NADH, 0. mM sodium pyruvate and a suitable amount of enzyme 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
26 Exp. Mol. Med.
enzyme that produced one micromol of NAD +^ per min under the assay conditions. A molar absorption coefficient for NADH of 6.22ለ 103 M -1^ cm-1^ at 340 nm was used for calculations (Voorter et al ., 1993). For the reverse reaction (NAD +^ to NADH), the enzyme activity was determined by measuring an increase in absorbance at 340 nm. The final concentration of the reactants in a standard reaction mixture was 50 mM Tris-HCl buffer (pH 9.5), 50 mM lithium lactate and 0.1 mM NAD +^. It contained 20 μl of suitably diluted enzyme. The volume of reaction mixtures in both cases was 1 ml. K m and V max ( K cat ) values were calculated from initial velocity measurements, using Enzfitter (R. J. Leather-barrow), a non-linear regression program that integrates the data into the Michaelis-Menton equation, with simple weighting. Proteins were determined by the method of Lowry et al. (1951) with bovine serum albumin as standard. For inhibition studies, a known concentration of the test modulator (after adjusting pH 7.5 for forward reaction and pH 9.5 for reverse reaction) was added to the reaction mixture. The activity of the enzyme was then measured by the forward or reverse reactions. The results are expressed as remaining activity (in %) compared to control (100%). The effect of these chemicals were also observed on the oxidation/reduction of NADH/NAD +^ in the absence of enzyme. The nature of oxalate inhibition was determined using Lineweaver-Burk plots.
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. To wash out glyceraldehyde 3-phosphate dehydrogenase, KPE buffer without NaCl was passed through the column. LDH was then eluted with 1 mM NADH in KPE buffer. The flow rate was about 40 ml/h and 1 ml fractions were collected. The protein concentration (ᆨ) was measured as A 280. The enzyme activity (ᆩ) was determined by measuring the change in absorbance at 340 nm min-1 at 25˚C.
Figure 2. A. Agarose gel electrophoresis (AGE) of LDH from Varanus liver. Lanes 69, 73 and 77 are tube number after elution of LDH from colchicine-Sepharose column by NADH. Crude sample is liver extract. B. SDS-PAGE of purified LDH. a, reference proteins are; 1, carbonic anhydrase (29 kDa); 2, egg albumin (45 kDa); 3, BSA ( kDa); 4, phosphorylase B (97.4 kDa); 5, β-galactosidase (116 kDa); 6, myosin ( kDa); b, purified LDH.
28 Exp. Mol. Med.
at which 50% of the activity remains after an incubation of 5 min) for forward-reaction is about 50˚C and for reverse-reaction, it is 40˚C.
Effect of Inhibitors
The effect of various modulators on the LDH activity is shown in Table 4. Oxalate was found to be a strong inhibitor, both for the forward and reverse directions particularly at 1 mM concentrations. The apparent IC (^50) (concentration where 50% inhibition was observed) was found to be about 0.05 mM (Figure 5). Glutamate was a more effective inhibitor in the reverse reaction (100% inhibition) as compared to the forward reaction (22.3% inhibition) at 20 mM concentration. Cu ++^ and Co ++^ were
more effective inhibitors for forward reactions as compared to the reverse reactions. Mn 2+^ and Mg 2+^ had almost no effect on LDH activity on either sides of reaction. n -Hexanediol has also shown inhibition for forward and reverse reactions with IC 50 of about 1.5 M and 0.75 M, respectively (Figure 6).
Discussion Lactate dehydrogenase from various mammalian species have been shown to be made up of five isoenzymes. We have observed that LDH from skeletal muscles of a reptile, Uromastix hardwickii consisted of six isoenzymes and they can not be separated by usual methods of electrophoresis (Javed et al ., 1992). The purified LDH isoenzymes from liver and testes of Uromastix were quite different from other species (Javed et al ., 1994; Javed et al ., 1995). Considering the results of Uromastix LDH, we have decided to study the properties of LDH purified from another reptile, Varanus. It has been well established that the liver of rat, and man contains two LDH isoenzymes (LDH-5 and LDH-4) with 98% as LDH- 5, i.e., M-type. The rabbit liver showed three isoenzymes (LDH-5, LDH-4 and LDH-3) with LDH-5 as predominant isoenzymes (Maly and Toranelli, 1993). The cattle liver showed three isoenzymes (LDH-4, LDH-3 and LDH-2) while pig liver showed four isoenzymes (LDH-4, LDH-3,
Figure 4. Effect of pre-heating upon activity of LDH. The purified LDH was incubated for 5 min at various temperatures. An aliquot was then taken and kept in ice. After completing the experiment, the activity of LDH was measured both for forward and reverse reactions as explained in the ‘Materials and Methods’.
Remaining activitya(%) Inhibitors Concentration (mM) Forward Reverse reaction reaction Control 0.0 100.0 100. Oxalate 0.5 19.0 98. 1.0 7.5 35. Glutamate 10.0 89.0 5. 20.0 77.7 0. Cu 2+^ 0.5 59.0 80. 1.0 23.8 70. Co 2+^ 0.5 95.0 100. 1.0 0.0 100. Mn 2+^ 0.5 100.0 100. 1.0 84.0 100. Mg2+^ 0.5 99.9 100. 1.0 88.8 90. n -Hexanediol 400.0 81.5 75. 800.0 68.5 55.
Table 4. Effect of inhibitors on LDH activity
a (^) A known amount of modulator was added in the reaction mixture and then the activity of LDH was determined as explained in the ‘Materials and Methods’. Values are mean for three determinations for all except for n -hexanediol which were repeated for four times.
Figure 5. Effect of oxalate on inhibition of LDH. An indicated amount of oxalate was added in the reaction mixture, and then the activity of LDH was determined as described for the forward reaction in the ‘Materials and Methods’.
Varanus liver LDH 29
LDH-2 and LDH-1) with LDH-3 and LDH-2 as predomi- nant isoenzymes (Maly and Toranelli, 1993). Varanus liver showed two isoenzymes (LDH-5 and LDH-4) same as in human liver. The K m values for pyruvate, NADH and NAD +^ were quite different in compared to Uromastix liver LDH which are about 15 μM, 40 μM and 10 μM, respectively (Javed et al. 1995). The reason is probably the type of LDH isoenzyme. We purified LDH-1 from uromastix liver previously (Javed et al ., 1995). It has been known that the kinetic properties of LDH-1 and LDH-5 are quite different (Everse and Kaplan, 1973; Maekawa, 1988). However, the K m for lactate in Varanus LDH was almost similar to LDH from Uromastix (Javed et al ., 1995). One of the main functions of liver is to convert lactate into pyruvate for energy or gluconeoge- nesis (Stryer, 1995). The pH and temperature effects on Varanus LDH were almost similar to LDH-5 from other sources (Hayashi et al. 1985; Javed, et al. 1995). The molecular weight of native LDH (140 kDa) from Varanus liver was same as previously reported. (Everse and Kaplan, 1973; Maekawa, 1988) and that of subunits was found to be about 35 kDa. Thus the Varanus liver LDH is also made up of four subunits. The inhibition of this LDH by oxalate is in accordance with previous reports (Everse and Kaplan, 1973). In most of the cases, oxalate has been shown to be either com- petitive or non-competitive types of inhibitor (Saxena et al., 1986; Javed and Waqar, 1993). Glutamate has been shown to protect the inactivation of LDH from the skeletal muscles of Carp (Nakajima et al., 1993). In Varanus liver LDH it was strong inhibitor for the reverse-direction only. We had also observed less inhibition by glutamate
in case of LDH from Uromastix liver for the forward- reaction (Javed et al ., 1995). Cu 2+^ and Co 2+^ inhibition was more evident for forward reaction as compared to reverse-reaction while Mn2+^ and Mg2+^ were least effective which were shown to inhibitors of LDH from some other sources (Yoshido, 1965; Saxena, 1986; Javed et al ., 1995). However, CO 2 +^ , Mg 2 +^ and Mn 2 +^ have been shown to be activators of LDH from Norcadia asteroides (Ike et al ., 1992). The reason may be that LDH from N. asteroides was shown to be a membrane-bound enzyme, while we have purified the LDH from the cytosol of liver. n -Hexanediol has been shown to be a non-competitive inhibitor for human LDH-5 with pyruvate as substrate (Tanishima et al ., 1985). From electrophoretic and other biochemical proper- ties, it seems that the LDH of Varanus liver resembles mostly human liver LDH and quite different from the liver LDH of Uromastix , another reptile (Javed et al ., 1995).
Acknowledgements The authors gratefully acknowledge Mrs. Seema Tariq Khan and Mr. Jack Fernandes for secretarial and transcription assistance.
References Basaglia, F. (1989) Some aspects of isoenzymes of LDH, MDH and glucose-phosphate isomerase in fish. Comp. Biochem. Physiol. 92B: 213- Cahn, R. D., Kaplan, N. O., Levine, L. and Zwilling, E. (1962) Nature and development of LDH. Science 136: 962- Everse, J. and Kaplan, N. O. (1973) Lactate dehydrogenase: structure and function. Adv. Enzymol. 37: 61- Gorg, A., Postel, W., Weser, J., Schiwara, H. W. and Boesken, W. H. (1985) Horizontal SDS electrophoresis in ultrathin pore-gradient gels for the analysis of urinary proteins. Sci. Tools 32: 5- Hayashi, S., Ooshiro, Z., Itakura, T. and Masuda, Y. (1985) Biochemical properties of LDH purified from the eel liver. Bull. Japan. Soc. Sci. Fisheries 51: 79- Ike, J., Sangam, P. and Gunawekaran, M. (1992) Purification and properties of LDH from Norcardia asteroides. Microbios. 69: 119- Javed, M. H., Hussain, A. N. and Waqar, M. A. (1992) Heat-stabilizing factor of LDH in the skeletal muscle of uromastix. Biochem. Soc. Trans. 20: 208S Javed, M. H. and Waqar, M. A. (1993) Properties of LDH isoenzyme-1 from goat brain. Korean J. Biochem. 25: 49- Javed, M. H., Qureshi, M. A. and Waqar, M. A. (1994) The isoenzyme forms of LDH form the testes of Uromastix hardwickii. Biochem. Mol. Biol. Int. 34: 963- Javed, M. H., Yousuf, F. A., Hussain, A. N., Ishaq, M. and Waqar, M. A. (1995) Purification and properties of LDH from liver of Uromastix hardwickii. Comp. Biochem. Physiol. 111B: 27- Javed, M. H. and Waqar, M. A. (1996) Inhibition studies on LDH isoenzyme purified from Uromastix testes. J. Enz. Inhibition 10: 187- Li, S., O’Brien, D.A., Hou, E. W., Versala, J., Rockett, D. L. and Eddy, E. M. (1989)
Figure 6. Effect of n-hexanediol on the inhibition of LDH. A known amount of n- hexanediol was added in the reaction mixture, and the LDH activity was determined for forward and reverse reactions as explained in the ‘Materials and Methods’.
