title: Hybridization as a technique for studying interchain interactions in the catalytic trimers of aspartate transcarbamoylase.
abstract: Since subunit interactions in regulatory enzymes mediate the ligand-promoted conformational changes responsible for their allosteric properties, it is necessary to have techniques for determining the effects of ligands and mutational alterations on the strength of the interchain interactions. In aspartate transcarbamoylase from Escherichia coli, the multiple interchain interactions are so linked that it is difficult to study them separately. Therefore, we have focused on the nonallosteric catalytic trimers isolated from the holoenzyme and have used the rate of hybrid formation between native and succinylated protein as a measure of the dissociation of the trimers into single polypeptide chains. Although catalytic trimers exhibit no evident dissociation in sedimentation studies at 10(-8) M, incubation of mixtures of native and succinylated trimers for long periods of time (days) yielded hybrids which are readily detected by polyacrylamide gel electrophoresis. This sensitive technique was used to demonstrate that the substrate, carbamoylphosphate, and the bisubstrate analog, N-(phosphonacetyl)-L-aspartate, cause a marked strengthening of the interchain interactions, whereas the inhibitor, sodium pyrophosphate, at concentrations as low as 10 mM, promotes dissociation of the trimers. This weakening of the interchain interactions by pyrophosphate facilitated the isolation and purification of functionally competent hybrid trimers by a technique which was much more convenient and provided higher yields than previous, more drastic methods which employed urea or guanidine hydrochloride to cause dissociation of the trimers. The hybridization technique was useful in studying the effects of mutational alterations on the strength of the interchain interactions and the ability of active and inactive mutants to bind pyrophosphate.
mesh_terms: D001221:Aspartate Carbamoyltransferase / Q000032:analysis* / Q000235:genetics; D002384:Catalysis; D055598:Chemical Phenomena; D002621:Chemistry; D011756:Diphosphates / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology; D008024:Ligands; D046911:Macromolecular Substances; D009154:Mutation
chemical_list: D011756:Diphosphates; D008024:Ligands; D046911:Macromolecular Substances; D001221:Aspartate Carbamoyltransferase