Abstract
Changes induced on addition of the coenzyme, NADH or NAD+, to porcine lactic dehydrogenase isoenzymes H4 and M4 have been studied by hydrodynamic and spectroscopic methods. As shown by ultracentrifugal analysis, the native subunit structure remains unchanged on holoenzyme formation; a ∼5% increase of the sedimentation coefficient, parallelled by a slight decrease of the partial specific volume (<1%) indicate a significant change in the native tertiary and/or quaternary structure of the enzymes, corroborating earlier calorimetric data (Hinz and Jaenicke, 1975). The binding constant for the enzyme from skeletal muslce (M4) and NADH are found to be in agreement with K D-values obtained from equilibrium dialysis, as well as spectroscopic and thermal titration experiments (8 ΜM). Far UV circular dichroism measurements do not show significant changes on ligand binding, indicating unchanged helicity or compensatory conformational effects. In the near UV, ligand binding is reflected by an extrinsic Cotton effect around 340 nm; in the range of aromatic absorption no changes are detectable.
The experimental results suggest that there are gross structural changes on coenzyme binding to lactic dehydrogenase which do not affect the intrinsic spectral properties normally applied to analyze transconformation reactions in protein molecules.
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Holbrook, J. J., Liljas, A., Steindel, S. J., Rossmann, M. G.: Lactate dehydrogenase. In: The enzymes, 3rd. ed., Vol. XI Boyer, P. (ed.), pp. 191–292. New York: Academic Press 1975
Czerlinski, G. H., Schreck, G.: Fluorescence detection of the chemical relaxation of the reaction of lactate dehydrogenase with reduced nicotinamide adenine dinucleotide. J. Biol. Chem. 239, 913–921 (1964)
Heck, H. d'A.: Porcine heart lactate dehydrogenase: Optical rotatory dispersion, thermodynamics, and kinetics of binding reactions. J. Biol. Chem. 244, 4375–4381 (1969)
Hinz, H.-J., Jaenicke, R.: Thermodynamics of complex formation between nicotinamide adenine dinucleotide and pig skeletal muscle lactate dehydrogenase. Biochemistry 14, 24–27 (1975)
Schmid, F. X., Hinz, H.-J., Jaenicke, R.: Thermodynamic studies of binary and ternary complexes of pig heart lactate dehydrogenase. Biochemistry 15, 3052–3059 (1976)
Hinz, H.-J., Schmidt, R., Scheurmann, W., Jaenicke, R.: Thermodynamic studies of binary and ternary complexes of pig skeletal muscle lactate dehydrogenase. Eur. J. Biochem. 80, 543–550 (1977)
Privalov, P. L., Khechinashvili, N. N., Atanasov, B. P.: Thermodynamic analysis of thermal transitions in globular proteins. Biopolymers 10, 1865–1890 (1971)
Sturtevant, J. M.: Heat capacity and entropy changes in processes involving proteins. Proc. Natl. Acad. Sci. USA 74, 2236–2240 (1977)
Jeckel, D., Pfleiderer, G.: Änderung der optischen Rotationsdispersion von Lactat-Dehydrogenase aus Schweineherzmuskel nach Zugabe von Harnstoff, Coenzym und SH-blockierenden Reagenzien. Hoppe Seyler's Z. Physiol. Chem. 350, 903–914 (1969)
Bolotina, I. A., Markovich, D. S., Volkenstein, M. V., Závodzky, P.: Investigation of the conformation of lactate dehydrogenase and of its catalytic activity. Biochim. Biophys. Acta 132, 271–281 (1976)
Jaenicke, R., Knof, S.: Molecular weight and quaternary structure of lactate dehydrogenase, 3. Comparative Determination by sedimentation analysis, light scattering, and osmosis. Eur. J. Biochem. 4, 157–163 (1968)
Dawson, R. M. C., Elliot, D. C., Elliot, W. H., Jones, K. M. (eds.): Data for biochemical research, 2nd ed. Oxford: Clarendon Press 1969
Kratky, O., Leopold, H., Stabinger, H.: Dichtemessungen an Flüssigkeiten und Gasen auf 10−6 g/cm3 bei 0,6 cm3 PrÄparatvolumen. Z. Angew. Phys. 27, 273–277 (1969)
Jaenicke, R.: Quaternary structure and conformation of lactic dehydrogenase and glyceraldehyde- 3-phosphate dehydrogenase. In: Pyridine nucleotide dependent dehydrogenases. Sund, H. (ed.), pp. 71–90. Berlin, Heidelberg, New York: Springer 1970
Bartholmes, P., Durchschlag, H., Jaenicke, R.: Molecular properties of lactic dehydrogenase under the conditions of the enzymatic test. Eur. J. Biochem. 39, 101–108 (1973)
Pfleiderer, G., Jeckel, D., Wieland, T.: Zum Wirkungsmechanismus der MilchsÄure-Dehydrogenase: Die schützende Wirkung von Coenzym-Addukten gegenüber denaturierenden Agentien. Biochem. Z. 329, 104–111 (1975)
Jaenicke, R., Gratzer, W. B.: Cooperative and non-cooperative conformational effects of the coenzyme on yeast glyceraldehyde-3-phosphate dehydrogenase. Eur. J. Biochem. 10, 158–164 (1969)
Stinson, R. A., Holbrook, J. J.: Equilibrium binding of nicotinamide nucleotide to lactate dehydrogenase. Biochem. J. 131, 719–728 (1973)
Závodszky, P.: Quantitative characterization of the structural fluctuation of LDH. Abstracts 9th FEBS-Meeting, Budapest, p. 44 (1974)
Rudolph, R., Jaenicke, R.: Kinetics of reassociation and reactivation of pig-muscle lactic dehydrogenase after acid dissociation. Eur. J. Biochem. 63, 409–417 (1976)
Chen, Y.-H., Yang, J. F., Martinez, H. M.: Determination of the secondary structures of proteins by circular dichroism and optical rotatory dispersion. Biochemistry 11, 4120–4131 (1972)
Rossmann, M. G., Liljas, A., BrÄndén, C.-I., Banaszak, L. T.: Evolutionary and structural relationships among dehydrogenases. In: The enzymes, 3rd. ed., Vol. XI. Boyer, P. (ed.), pp. 62–102. New York: Academic Press 1975
Schmid, F. X.: Thermodynamisehe Untersuchungen an Lactatdehydrogenase und Alkoholdehydrogenase. Thesis, Regensburg (1977)
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Jaenicke, R., Gregori, E. & Laepple, M. Conformational effects of coenzyme binding to porcine lactic dehydrogenase. Biophys. Struct. Mechanism 6, 57–65 (1979). https://doi.org/10.1007/BF00537595
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DOI: https://doi.org/10.1007/BF00537595