Lactate dehydrogenase: Electrophoretic behaviour electron microscopy and structure

Abstract

The covalent binding of GSH and cysteine to mouse lactate dehydrogenase by disulphide bonds, gives rise to all but five bands of the complex electrophoretograms of the enzyme [Dudman, N.P.B. (1969) Biochem. Biophys. Res. Commun., 36, 608]. Other explanations have been proposed for this complexity, including that the enzyme exists as a number of conformational isomers [Houssais, J.-F. (1966) Biochim. Biophys. Acta 128, 239], and that the enzyme arises from the expression of three different genes [Costello, L. A. and Kaplan, N. O. (1963) Biochim. Biophys. Acta 73, 658]. The basis for these proposals is examined critically. From electrophoretograms of mouse lactate dehydrogenase both fully reduced, and covalently bound to GSH and cysteine, it is concluded that each muscle-type subunit has one especially active -SH group, but that the heart-type subunit has none. The state of ionization of the covalently bound GSH and cysteine residues, and of the free enzymatic thiol residues is also derived. Mouse lactate dehydrogenase is found to be adsorbed by some batches of polyacrylamide. Cathodic sub-bands of Isoenzyme 5 are bound most firmly, and adsorption appears strongest when the enzyme is fully reduced. Acrylamide gels containing pyruvate and NADH appear not to adsorb the enzyme. The possible effects of differing geometrical arrangements of subunits upon the electrophoretic pattern of lactate dehydrogenase are considered. Subunits of the enzyme from beef heart are found by electron microscopy to be arranged in flattened tetrahedra. This observation, taken with the electrophoretic pattern of the enzyme, indicates that beef lactate dehydrogenase comprises subunits of at least four distinguishable types.