title: Combined use of strain construction and affinity chromatography in the rapid, high-yield purification of 6-phosphogluconate dehydrogenase from Escherichia coli.
abstract: A rapid, high-yield method for purification of 6-phosphogluconate dehydrogenase from Escherichia coli K-12 is described. Sonic extracts prepared from heat-induced cultures of strain RW184, doubly lysogenic for the specialized transducing bacteriophage lambdacI857St68h80dgndhis and bearing a deletion of the gene for glucose 6-phosphate dehydrogenase, contained levels of 6-phosphogluconate dehydrogenase 15- to 20-fold higher than cultures of wild-type cells. Affinity chromatography on blue dextran-Sepharose with batchwise elution with 1 mM nicotinamide adenine dinucleotide phosphate affected a further 10-fold purification. Enzyme prepared in this manner was homogeneous according to electrophoresis on sodium dodecyl sulfate-polyacrylamide gels and immunoelectrophoresis using antiserum directed against it. Fructose 1,6-diphosphate is an inhibitor of enzyme activity.
mesh_terms: D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000201:enzymology*; D005635:Fructosediphosphates / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008722:Methods; D009249:NADP / Q000494:pharmacology; D010734:Phosphogluconate Dehydrogenase / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D005635:Fructosediphosphates; D009249:NADP; D010734:Phosphogluconate Dehydrogenase