Mutant analysis of glyceraldehyde 3-phosphate dehydrogenase in Escherichia coli.
NAD(+)-specific glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) from Escherichia coli was purified to homogeneity by a relatively simple procedure involving affinity chromatography on agarose-hexane-NAD(+) and repeated crystallization. Rabbit antiserum directed against this protein produced one precipitin line in double-diffusion studies against the pure enzyme, and two lines against crude extracts of wild-type E. coli strains. Both precipitin lines represent the interaction of antibody with determinants specific for glyceraldehyde 3-phosphate dehydrogenase. Nine independent mutants of E. coli lacking glyceraldehyde 3-phosphate dehydrogenase activity all possessed some antigenic cross-reacting material to the wild-type enzyme. The mutants could be divided into three groups on the basis of the types and amounts of precipitin lines observed in double-diffusion experiments; one group formed little cross-reacting material. The cross-reacting material in crude cell-free extracts of several of the mutant strains were also tested for alterations in their affinity for NAD(+) and their phosphorylative activity. The cumulative data indicate that the protein in several of the mutant strains is severely altered, and thus that glyceraldehyde 3-phosphate dehydrogenase is unlikely to have an essential, non-catalytic function such as buffering nicotinamide nucleotide or glycolytic-intermediate concentrations. Others of the mutants tested have cross-reacting material which behaved like the wild-type enzyme for the several parameters studied; the proteins from these strains, once purified, might serve as useful analogues of the wild-type enzyme.
D000255:Adenosine Triphosphate / Q000378:metabolism; D002474:Cell-Free System; D003429:Cross Reactions; D000939:Epitopes; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D005779:Immunodiffusion; D009154:Mutation; D009243:NAD / Q000378:metabolism; D011234:Precipitins
D000939:Epitopes; D011234:Precipitins; D009243:NAD; D000255:Adenosine Triphosphate; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases