title: Subunit ratios of separated hybrid hexamers of Neurospora NADP-specific glutamate dehydrogenase containing complementing mutationally modified monomers.
abstract: The am1 and am3 mutational variants of the Neurospora crassa NADP-specific glutamate dehydrogenase show complementation activity in hybrid hexamers. A freeze-thaw hybridization method was used to construct hybrids from purified enzymes and the products were separated into species of different monomer ratio by affinity chromatography. Hexamers with am1:am3 ratios of 1:5, 2:4, 3:3, 4:2 and 5:1 were all recovered as resolved or partially resolved peaks in quantities approximating to a binomial distribution. Reassociation of monomers during the hybridization process was random, except for some differential loss of am3 protein by precipitation and an apparent absence of reassociated am1 homohexamers. Complementation activity was shown by hybrids of all five monomer ratios, owing to activation of am3 monomers by conformational constraints arising from the intrinsically inactive am1 monomers. The activating effect of such constraints was greatest in hexamers containing only a single am1 monomer and least in the 5 am1:1am3 species. When fully activated by L-glutamate all am3 monomers were equivalent in intrinsic catalytic activity, irrespective of the number of am1 monomers per hexamer.
mesh_terms: D055598:Chemical Phenomena; D002621:Chemistry; D002846:Chromatography, Affinity; D005969:Glutamate Dehydrogenase / Q000378:metabolism*; D005971:Glutamates; D046911:Macromolecular Substances; D009154:Mutation; D009249:NADP; D009491:Neurospora / Q000201:enzymology*; D009492:Neurospora crassa / Q000201:enzymology*; D055503:Protein Multimerization; D013329:Structure-Activity Relationship
chemical_list: D005971:Glutamates; D046911:Macromolecular Substances; D009249:NADP; D005969:Glutamate Dehydrogenase