Limited Proteolysis of Porine‐Muscle Latic Dehydrogenase by Thermolysin during Reconstitution Yields Dimers

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Abstract
Reassociation of lactic dehydrogenase [LDH] from porcine skeletal muscle at 10.degree. 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 LDH: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: the protease is essentially inactive towards native LDH; it splits intermediates of reconstitution at a high rate; it can be inactivated instantaneously by the addition of 10 mM EDTA. As demonstrated by gel chromatography, thermolysin digestion stabilizes dimers of porcine muscle LDH as intermediates of reconstitution. These dimers consist of virtually intact polypeptide chains of MW .apprx. 35,000, apart from nicked subunits with MW of 18,000 and 12,000, 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 .fwdarw. tetramer transition is found to be rate-limiting for both reassociation and reactivation. This result corroborates a previously proposed model for the reconstitution of LDH based on either cross-linking experiments or the reassembly of dimeric intermediates of dissociation.

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