Cross-linking with glutaraldehyde with subsequent NaDodSO4-polyacrylamide gel electrophoresis has been introduced as a convenient method for studying the association of oligomeric proteins [Hermann, R., Rudolph, R.,& Jaenicke, R. (1979) Nature (London) 277, 243-245]. In the present paper, an improved version of this approach was applied to the analysis of the complex association behavior of the tetrameric lactic dehydrogenase from pig muscle. Monomers, dimers (as intermediates of reconstitution), and tetramers could be quantitatively determined during reconstitution. The initial fast formation of dimers from monomers does not reach completion; a certain amount of monomers remains during the whole reconstitution process. Monomers and dimers disappear parallel to the formation of tetramers. The reassociation behavior of lactic dehydrogenase is described by a kinetic mechanism comprising a dissociation-association equilibrium of monomers and dimers [characterized by an equilibrium constant K = (3 +/- 1) X 10(8) L mol-1] followed by the rate-limiting association of dimers to tetramers [described by a second-order rate constant k = (3.15 +/- 0.15) X 19=0(4) L mol-1 s-1]. Tetramerization is found to strictly parallel reactivation.
The reconstitution of the tetrameric phosphoglycerate mutase from bakers' yeast after denaturation in guanidine hydrochloride has been studied. When assays are performed in the presence of trypsin, it is found that reactivation parallels the regain of tetrameric structure. However, in the absence of trypsin, the regain of activity is more rapid, suggesting that monomeric and dimeric intermediates possess partial activity (35% of the value of native enzyme) which is sensitive to trypsin. When reconstitution is studied in the presence of substrates, it is again found that monomeric and dimeric intermediates possess 35% activity. Under these latter conditions, the activity of the monomer but not of the dimer is sensitive to trypsin.
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