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Cellular and Molecular Biology Letters

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Volume 12, Issue 3


Ultracentrifugation studies of the location of the site involved in the interaction of pig heart lactate dehydrogenase with acidic phospholipids at low pH. A comparison with the muscle form of the enzyme

Grzegorz Terlecki / Elżbieta Czapińska / Katarzyna Hotowy
Published Online: 2007-03-05 | DOI: https://doi.org/10.2478/s11658-007-0010-5


Lactate dehydrogenase (LDH) from the pig heart interacts with liposomes made of acidic phospholipids most effectively at low pH, close to the isoelectric point of the protein (pH = 5.5). This binding is not observed at neutral pH or high ionic strength. LDH-liposome complex formation requires an absence of nicotinamide adenine dinucleotides and adenine nucleotides in the interaction environment. Their presence limits the interaction of LDH with liposomes in a concentration-dependent manner. This phenomenon is not observed for pig skeletal muscle LDH. The heart LDH-liposome complexes formed in the absence of nicotinamide adenine dinucleotides and adenine nucleotides are stable after the addition of these substances even in millimolar concentrations. The LDH substrates and studied nucleotides that inhibit the interaction of pig heart LDH with acidic liposomes can be ordered according to their effectiveness as follows: NADH > NAD > ATP = ADP > AMP > pyruvate. The phosphorylated form of NAD (NADP), nonadenine nucleotides (GTP, CTP, UTP) and lactate are ineffective. Chemically cross-linked pig heart LDH, with a tetrameric structure stable at low pH, behaves analogously to the unmodified enzyme, which excludes the participation of the interfacing parts of subunits in the interaction with acidic phospholipids. The presented results indicate that in lowered pH conditions, the NADH-cofactor binding site of pig heart LDH is strongly involved in the interaction of the enzyme with acidic phospholipids. The contribution of the ATP/ADP binding site to this process can also be considered. In the case of pig skeletal muscle LDH, neither the cofactor binding site nor the subunit interfacing areas seem to be involved in the interaction.

Keywords: Lipid-protein interaction; Lactate dehydrogenase isoenzymes; Acidic phospholipids; Cardiolipin; Phosphatidylserine

  • [1] Aubert, A., Costalat, R., Magistretti, P.J. and Pellerin, L. Brain lactate kinetics: Modeling evidence for neuronal lactate uptake upon activation. Proc. Natl. Acad. Sci. USA 102 (2005) 16448–16453. http://dx.doi.org/10.1073/pnas.0505427102CrossrefGoogle Scholar

  • [2] Crawford, R.M., Budas, G.R., Jovanovic, S., Ranki, H.J., Wilson, T.J., Davies, A.M. and Jovanovic, A. M-LDH serves as a sarcolemmal K(ATP) channel subunit essential for cell protection against ischemia. EMBO J. 21 (2002) 3936–3948. http://dx.doi.org/10.1093/emboj/cdf388CrossrefGoogle Scholar

  • [3] Pioli, P.A., Hamilton, B.J., Connolly, J.E., Brewer, G. and Rigby, WF. Lactate dehydrogenase is an AU-rich element-binding protein that directly interacts with AUF1. J. Biol. Chem. 277 (2002) 35738–35745. http://dx.doi.org/10.1074/jbc.M204002200Google Scholar

  • [4] Li, S.S. Lactate dehydrogenase isoenzymes A (muscle), B (heart) and C (testis) of mammals and the genes coding for these enzymes. Biochem. Soc. Trans. 2 (1989) 304–307. CrossrefGoogle Scholar

  • [5] Li, S.S. Human and mouse lactate dehydrogenase genes A (muscle), B (heart), and C (testis): protein structure, genomic organization, regulation of expression, and molecular evolution. Prog. Clin. Biol. Res. 344 (1990) 75–99. Google Scholar

  • [6] Read, J.A., Winter, V.J., Eszes, C.M., Sessions, R.B. and Brady, R.L. Structural basis for altered activity of M-and H-isozyme forms of human lactate dehydrogenase. Proteins 43 (2001) 175–185. http://dx.doi.org/10.1002/1097-0134(20010501)43:2<175::AID-PROT1029>3.0.CO;2-#CrossrefGoogle Scholar

  • [7] Wang, X.C., Jiang, L. and Zhou, H.M. Minimal functional unit of lactate dehydrogenase. J. Protein Chem. 3 (1997) 227–231. http://dx.doi.org/10.1023/A:1026382926299CrossrefGoogle Scholar

  • [8] King, L. and Weber, G. Conformational drift of dissociated lactate dehydrogenases. Biochemistry 25 (1986) 3632–3637. http://dx.doi.org/10.1021/bi00360a023CrossrefGoogle Scholar

  • [9] Dabrowska, A. and Gutowicz, J. Interaction of bovine heart lactate dehydrogenase with erythrocyte lipids. Biochim. Biophys. Acta 855 (1986) 99–104. http://dx.doi.org/10.1016/0005-2736(86)90193-8CrossrefGoogle Scholar

  • [10] Dabrowska, A., Terlecki, G. and Gutowicz, J. Interaction of bovine skeletal muscle lactate dehydrogenase with liposomes. Comparison with the data for the heart enzyme. Biochim. Biophys. Acta 980 (1989) 357–360. http://dx.doi.org/10.1016/0005-2736(89)90324-6CrossrefGoogle Scholar

  • [11] Terlecki, G., Czapińska, E., Rogozik, K., Lisowski, M. and Gutowicz, J. Investigation of the interaction of pig muscle lactate dehydrogenase with acidic phospholipids at low pH. Biochim. Biophys. Acta 1758 (2006) 133–144. http://dx.doi.org/10.1016/j.bbamem.2006.02.013CrossrefGoogle Scholar

  • [12] Terlecki, G., Czapinska, E. and Gutowicz J. The role of lipid phase structure in the interaction of lactate dehydrogenase with phosphatidylserine. Activity studies. Cell. Mol. Biol. Lett. 7 (2002) 895–903. Google Scholar

  • [13] Terlecki, G. and Gutowicz, J. Further evidence for the importance of lipid bilayers in the interaction between lactate dehydrogenase and phosphatidylserine. Cell. Mol. Biol. Lett. 7 (2002) 905–910. Google Scholar

  • [14] Marsh, D. Handbook of Lipids Bilayers, CRC Press, Boca Raton, FL, 1990. Google Scholar

  • [15] Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72 (1976) 248–254. http://dx.doi.org/10.1016/0003-2697(76)90527-3CrossrefGoogle Scholar

  • [16] Gottschalk, N. and Jaenicke, R. Chemically crosslinked lactate dehydrogenase: stability and reconstitution after glutaraldehyde fixation. Biotechnology and Applied Biochemistry 9 (1987) 387–400. Google Scholar

  • [17] 18. Rouser, G., Siakatos, A.N. and Fleischer, S. Quantitative analysis of phospholipids by thin-layer chromatography and phosphorus analysis of spots. Lipids 1 (1966) 85–86. http://dx.doi.org/10.1007/BF02533000CrossrefGoogle Scholar

  • [18] Cabiaux, V., Vandenbranden, M., Falmagne, P. and Ruysschaert, J.M. Aggregation and fusion of lipid vesicles induced by diphtheria toxin at low pH: possible involvement of the P site and the NAD+ binding site. Biosci. Rep. 3 (1985) 243–250. http://dx.doi.org/10.1007/BF01119594CrossrefGoogle Scholar

  • [19] Cabiaux, V., Vandenbranden, M., Falmagne, P. and Ruysschaert, J.M. Diphtheria toxin induces fusion of small unilamellar vesicles at low pH. Biochim. Biophys. Acta 775 (1984) 31–36. http://dx.doi.org/10.1016/0005-2736(84)90231-1CrossrefGoogle Scholar

  • [20] Wittenberger, C.L. Kinetic studies on the inhibition of a (D(-)-specific lactate dehydrogenase by adenosine triphosphate. J. Biol. Chem. 243 (1968) 3067–3075. Google Scholar

  • [21] Torres-da Matta, J., Batista e Silva, C. and Hasson-Voloch, A. Effect of ATP on purified L(+) lactate dehydrogenase from electric organ of Electrophorus electricus (L.). Int. J. Biochem. 18 (1986) 191–194. http://dx.doi.org/10.1016/0020-711X(86)90156-4CrossrefGoogle Scholar

  • [22] Busto, F., de Arriaga, D. and Soler, J. ATP, ADP and AMP on the regulation of lactate dehydrogenase activity of Phycomyces blakesleeanus. Int. J. Biochem. 15 (1983) 73–78. http://dx.doi.org/10.1016/0020-711X(83)90013-7CrossrefGoogle Scholar

  • [23] Nemat-Gorgani, M. and Wilson, J.E. Acidic phospholipids may inhibit rat brain hexokinase by interaction at the nucleotide binding site. Arch. Biochem. Biophys. 236 (1985) 220–227. http://dx.doi.org/10.1016/0003-9861(85)90621-6CrossrefGoogle Scholar

  • [24] Craig, D.B. and Wallace, C.J. ATP binding to cytochrome c diminishes electron flow in the mitochondrial respiratory pathway. Protein Sci. 2 (1993) 966–976. http://dx.doi.org/10.1002/pro.5560020610CrossrefGoogle Scholar

  • [25] .Kimelberg, H.K and Lee, C.P. Binding and electron transfer to cytochrome c in artificial phospholipid membranes. Biochem. Biophys. Res. Commun. 34 (1969) 784–790. http://dx.doi.org/10.1016/0006-291X(69)90248-4CrossrefGoogle Scholar

  • [26] Vanderkooi, J., Erecinska, M. and Chance, B. Cytochrome c interaction with membranes. II. Comparative study of the interaction of c cytochromes with the mitochondrial membrane. Arch. Biochem. Biophys. 152 (1973) 531–540. http://dx.doi.org/10.1016/0003-9861(73)90672-3CrossrefGoogle Scholar

  • [27] Mustonen, P., Virtanen, J.A., Somerharju, P.J. and Kinnunen, P.K. Binding of cytochrome c to liposomes as revealed by the quenching of fluorescence from pyrene-labeled phospholipids. Biochemistry 26 (1987) 2991–2997. http://dx.doi.org/10.1021/bi00385a006CrossrefGoogle Scholar

  • [28] Demel, R.A., Jordi, W., Lambrechts, H., van Damme, H., Hovius, R. and de Kruijff, B. Differential interactions of apo-and holocytochrome c with acidic membrane lipids in model systems and the implications for their import into mitochondria. J. Biol. Chem. 264 (1989) 3988–3997. Google Scholar

  • [29] Nicholls, P. Cytochrome c binding to enzymes and membranes. Biochim. Biophys. Acta 346 (1974) 261–310. Google Scholar

  • [30] Brown, L.R. and Wuthrich, K. NMR and ESR studies of the interactions of cytochrome c with mixed cardiolipin-phosphatidylcholine vesicles. Biochim. Biophys. Acta 468 (1977) 389–410. http://dx.doi.org/10.1016/0005-2736(77)90290-5CrossrefGoogle Scholar

  • [31] Rytomaa, M., Mustonen, P. and Kinnunen, P.K. Reversible, nonionic, and pH-dependent association of cytochrome c with cardiolipin-phosphatidylcholine liposomes. J. Biol. Chem. 267 (1992) 22243–22248. Google Scholar

  • [32] Rytomaa, M. and Kinnunen, P.K. Evidence for two distinct acidic phospholipid-binding sites in cytochrome c. J. Biol. Chem. 269 (1994) 1770–1774. Google Scholar

  • [33] Lovell, S.J. and Winzor, D.J. Effects of phosphate on the dissociation and enzymatic stability of rabbit muscle lactate dehydrogenase. Biochemistry 13 (1974) 3527–3531. http://dx.doi.org/10.1021/bi00714a018CrossrefGoogle Scholar

  • [34] Okeley, N.M. and Gelb, M.H. A designed probe for acidic phospholipids reveals the unique enriched anionic character of the cytosolic face of the mammalian plasma membrane. J. Biol. Chem. 279 (2004) 21833–21840. http://dx.doi.org/10.1074/jbc.M313469200CrossrefGoogle Scholar

  • [35] Korzeniewski, B. and Zoladz, J.A. Influence of rapid changes in cytosolic pH on oxidative phosphorylation in skeletal muscle: theoretical studies. Biochem. J. 365 (2002) 249–258. http://dx.doi.org/10.1042/BJ20020031CrossrefGoogle Scholar

  • [36] Korzeniewski, B. AMP deamination delays muscle acidification during heavy exercise and hypoxia. J. Biol. Chem. 281 (2006) 3057–3066. http://dx.doi.org/10.1074/jbc.M510418200CrossrefGoogle Scholar

  • [37] Kraayenhof, R., Sterk, G.J. and Sang, H.W. Probing biomembrane interfacial potential and pH profiles with a new type of float-like fluorophores positioned at varying distance from the membrane surface. Biochemistry 32 (1993) 10057–10066. http://dx.doi.org/10.1021/bi00089a022CrossrefGoogle Scholar

  • [38] Zhao, H., Tuominen, E.K. and Kinnunen, P.K. Formation of amyloid fibers triggered by phosphatidylserine-containing membranes. Biochemistry 43 (2004) 10302–10307. http://dx.doi.org/10.1021/bi049002cCrossrefGoogle Scholar

  • [39] Baba, N. and Sharma, H.M. Histochemistry of lactic dehydrogenase in heart and pectoralis muscles of rat. J. Cell. Biol. 51 (1971) 621–635. http://dx.doi.org/10.1083/jcb.51.3.621CrossrefGoogle Scholar

  • [40] Kline, E.S., Brandt, R.B., Laux, J.E., Spainhour, S.E., Higgins, E.S., Rogers, K.S., Tinsley, S.B. and Waters, M.G. Localization of L-lactate dehydrogenase in mitochondria. Arch. Biochem. Biophys. 246 (1986) 673–680. http://dx.doi.org/10.1016/0003-9861(86)90323-1CrossrefGoogle Scholar

  • [41] Brandt, R.B., Laux, J.E., Spainhour, S.E. and Kline, E.S. Lactate dehydrogenase in rat mitochondria. Arch. Biochem. Biophys. 259 (1987) 412–422 http://dx.doi.org/10.1016/0003-9861(87)90507-8CrossrefGoogle Scholar

  • [42] Gladden, LB. Lactate metabolism: a new paradigm for the third millennium. J. Physiol. 558 (2004) 5–30. http://dx.doi.org/10.1113/jphysiol.2003.058701CrossrefGoogle Scholar

  • [43] Brooks, G.A., Dubouchaud, H., Brown, M., Sicurello, J.P. and Butz, C.E. Role of mitochondrial lactate dehydrogenase and lactate oxidation in the intracellular lactate shuttle. Proc. Natl. Acad. Sci. USA 96 (1999) 1129–1134. http://dx.doi.org/10.1073/pnas.96.3.1129CrossrefGoogle Scholar

  • [44] Hashimoto, T., Hussien, R. and Brooks, G.A. Colocalization of MCT1, CD147, and LDH in mitochondrial inner membrane of L6 muscle cells: evidence of a mitochondrial lactate oxidation complex. Am. J. Physiol. Endocrinol. Metab. 290 (2006) 1237–1244. http://dx.doi.org/10.1152/ajpendo.00594.2005CrossrefGoogle Scholar

About the article

Published Online: 2007-03-05

Published in Print: 2007-09-01

Citation Information: Cellular and Molecular Biology Letters, Volume 12, Issue 3, Pages 378–395, ISSN (Online) 1689-1392, DOI: https://doi.org/10.2478/s11658-007-0010-5.

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© 2007 University of Wrocław, Poland. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License. BY-NC-ND 3.0

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