Limited proteolysis of porcine-muscle lactic dehydrogenase by thermolysin during reconstitution yields dimers.
Reassociation of lactic dehydrogenase from porcine skeletal muscle at 10 degrees C was studied by limited proteolysis during reconstitution, applying thermolysin as proteolytic enzyme. Proteolysis was achieved by short incubation of the reassociating enzyme with the protease in a ratio lactic dehydrogenase:thermolysin = 10:1 (0.1 M sodium phosphate buffer pH 7.6 plus 1 mM dithioerythritol). Thermolysin was found to satisfy all the obligatory requirements for proteolytic studies of reconstituting enzymes: (a) the protease is essentially inactive towards native lactic dehydrogenase; (b) it splits intermediates of reconstitution at a high rate; (c) it can be inactivated instantaneously by the addition of 10 mM EDTA. As demonstrated by gel chromatography, thermolysin digestion stabilizes dimers of porcine muscle lactic dehydrogenase as intermediates of reconstitution. These 'dimers' consist of virtually intact polypeptide chains of molecular weight about 35000, apart from nicked subunits with molecular weights of 18000 and 12000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Analyzing the kinetics of reassociation by thermolysin digestion at various times during reconstitution, and subsequent gel filtration, the dimer replaced by tetramer transition is found to be rate-limiting for both reassociation and reactivation. This result corroborates a previously proposed model for the reconstitution of lactic dehydrogenase, based on either cross-linking experiments [R. Hermann et al. (1979) Nature (Lond.) 277, 243--245], or the reassembly of dimeric intermediates of dissociation [R. Jaenicke et al. (1981) Eur. J. Biochem. 114, 525--531].
D000818:Animals; D004789:Enzyme Activation; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism*; D046911:Macromolecular Substances; D009132:Muscles / Q000201:enzymology*; D013552:Swine; D013820:Thermolysin / Q000378:metabolism*
D046911:Macromolecular Substances; D007770:L-Lactate Dehydrogenase; D013820:Thermolysin