Biochemical characterization of phosphoglucose isomerase and genetic variants from mouse and Drosophila melanogaster.
A simple and unique procedure was developed to purify phosphoglucose isomerase variants from the whole mouse body extracts and Drosophila homogenate. It involved the use of an 8-(6-aminohexyl)-amino-ATP-Sepharose column followed by a preparative isoelectric focusing. In each case, the enzyme in the homogenate was adsorbed by ionic interaction on the ATP-Sepharose column. Substantial purification was achieved by the affinity elution with the substrate-glucose-6-phosphate. Mouse and Drosophila phosphoglucose isomerase as well as the corresponding variants were shown to be dimers of similar molecular weight and to exhibit similar kinetic properties. The isoelectric points for the variants from DBA/2J and C57BL/6J mice were determined to be 8.4 and 8.7 respectively, while they were 6.8 and 6.3 respectively for Drosophila and 4/4 variants. Differential thermal stability was observed for the two mouse variants but not for the Drosophila ones. Amino acid composition analysis was performed for both mouse and Drosophila enzymes. Rabbit antisera for mouse (DBA/2J) and Drosophila (2/2) enzymes were raised. Within each species, complete immunological identity was observed between the variants. The antisera were used to characterize the null mutants of phosphoglucose isomerase identified in the mouse and Drosophila populations. By rocket immunoelectrophoresis, the null allele of the naturally occurring heterozygous null variant of Drosophila was shown to express no cross-reacting materials (CRM).
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002852:Chromatography, Ion Exchange; D004331:Drosophila melanogaster / Q000201:enzymology; D014644:Genetic Variation; D005956:Glucose-6-Phosphate Isomerase / Q000235:genetics* / Q000302:isolation & purification; D006358:Hot Temperature; D005779:Immunodiffusion; D007122:Immunoelectrophoresis; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D007700:Kinetics; D051379:Mice; D008970:Molecular Weight; D009154:Mutation
D000596:Amino Acids; D005956:Glucose-6-Phosphate Isomerase