Antibodies to the nonnative forms of D-glyceraldehyde-3-phosphate dehydrogenase: Identification, purification, and influence on the renaturation of the enzyme

Monoclonal antibodies of two clones reacting with the nonnative forms of D- glyceraldehyde-3-phosphate dehydrogenase, EC 1.2.1.12 (GAPDH), were obtaine d. Antibodies of clone 6C5 belonged to IgG1 subtype; antibodies of clone 6G 7 belonged to IgM type. The interaction of antibodies of both clones with t he immobilized and soluble enzyme was studied. The specificity of antibodie s to the definite oligomeric: forms was demonstrated on immobilized monomer s, dimers, and tetramers of GAPDH. The affinity of antibodies to monomeric and dimeric forms of GAPDH, either active or not, was demonstrated. At the same time the antibodies did not react with the tetrameric enzyme. The bind ing of antibodies had no influence on the enzymatic activity. However, the addition of antibodies to the denatured enzyme blocked the spontaneous rena turation of GAPDH. The immobilized antibodies of both clones were successfu lly used for the purification of GAPDH solution from the denatured admixtur es. (C) 1999 Academic Press.