[Use of immobilization in the study of glyceraldehyde 3-phosphate dehydrogenase. Immobilized monomers].
Active immobilized monomers of glyceraldehyde 3-phosphate dehydrogenase were prepared by means of dissociation of the tetrameric enzyme molecule covalently bound to Sepharose via a single subunit. The conditions were elaborated to achieve the inactivation and solubilization of the non-covalently bound subunits leaving the monomer coupled to the matrix intact. This procedure differs from the previously developed method of matrix-bound oligomeric enzymes dissociation in a detail which was found to be essentially important. The widely used method includes complete denaturation of all subunits during treatment with urea followed by reactivation of the immobilized one, whereas only the non-covalently bound subunits suffer denaturation under the conditions developed in the present work. The immobilized monomers of glyceraldehyde 3-phosphate dehydrogenase exhibit Vmax and Km (for NAD and substrate) values similar to those found for the immobilized tetramer. Reassociation of the immobilized monomers with soluble enzyme subunits obtained in the presence of urea produces matrix-bound tetrameric species. Immobilized trimers ae formed upon incubation of matrix-bound monomers in a diluted apoenzyme solution. The immobilized monomeric, trimeric and tetrameric enzyme species were used to study the role of subunit interactions in cooperative phenomena exhibited by the dehydrogenase.
D004800:Enzymes, Immobilized / Q000378:metabolism*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D012685:Sepharose; D015003:Yeasts / Q000201:enzymology
D004800:Enzymes, Immobilized; D046911:Macromolecular Substances; D012685:Sepharose; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases
Ispol'zovanie immobilizatsii dlia izucheniia glitseral'degid-3-fostfat-degidrogenazy. Immobilizirovannye monomery.