ABSTRACT
The antibodies specific to an inactive glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus prepared by the treatment of the tetrameric holoenzyme with glutaraldehyde were obtained. They were purified from the pool of polyclonal rabbit antibodies to GAPDH with the use of immobilized GAPDH cross-linked by glutaraldehyde as an affinity sorbent. Such antibodies were capable of interacting with the native enzyme, inducing its time-dependent inactivation; the effect was different with the apo- and holoenzyme forms. Differential scanning calorimetry of the purified [GAPDH].[antibody] complex revealed a large shift of the temperature corresponding to the maximal heat capacity of the holoenzyme towards the lower temperature. Again, the effect appeared to be different with the apoenzyme. Together, the results are consistent with the hypothesis that a specific antibody is able to exercise a certain strain on the target protein, altering its conformation toward the structure of the species which served to select the antibody. The possibility of preparing selective enzyme inhibitors based on the antibodies specific to inactive enzyme conformations is considered.
Subject(s)
Antibodies/immunology , Antibodies/pharmacology , Geobacillus stearothermophilus/enzymology , Glyceraldehyde-3-Phosphate Dehydrogenases/immunology , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Animals , Calorimetry, Differential Scanning , Glyceraldehyde-3-Phosphate Dehydrogenases/antagonists & inhibitors , Glyceraldehyde-3-Phosphate Dehydrogenases/chemistry , Protein Conformation/drug effects , Protein Folding , Protein Renaturation , Rabbits , TemperatureABSTRACT
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a protein with various activities far from its enzymatic function. Here, we showed that the oxidation of SH-groups of the active site of GAPDH enhanced its binding with total transfer RNA or with total DNA. Both NAD and NADH-the cofactors of GAPDH-inhibited the GAPDH-RNA (DNA) interaction, though NAD was much less effective than NADH in the case of oxidized GAPDH. Oxidation of GAPDH strongly decreased its affinity to NAD but not to NADH. Immobilized tetramers of GAPDH dissociated into dimers during the incubation with total RNA but not DNA. The staining of HeLa cells with monoclonal antibodies specific to dimers, monomers or the denatured form of GAPDH revealed the condensation of non-native forms of GAPDH in the nucleus. The role of oxidation of GAPDH in the regulation of the quaternary structure of the enzyme and in its interaction with nucleic acids is discussed.
