Multiple forms of Drosophila hexokinase. Purification, biochemical and immunological characterization.
Major forms of hexokinases (Hex A and Hex C) in Drosophila melanogaster were purified to homogeneity by utilizing affinity chromatography and preparative isoelectric focusing column as the key steps. Three different affinity ligands immobilized on Sepharose (3-amino pyridine adenine dinucleotide, glucosamine, and 8-(6-aminohexyl)-amino-ATP) were employed during different stages of enzyme purification. Antisera against purified Hex A and Hex C were raised in rabbits. Hex A, the major form in adults, and Hex B, the predominant form in larvae, showed complete immunological identity by double immunodiffusion and enzyme immunoinactivation studies. No cross-reactivity was observed between the antiserum to Hex A and Hex C or between the antiserum to Hex C and Hex A. By gel filtration chromatography and sodium dodecyl sulfate-acrylamide gel electrophoresis, all of the Drosophila hexokinases were shown to be monomers of molecular weights ranging from 40,000 to 50,000. Multiple forms of Drosophila hexokinase were studied extensively with respect to their biochemical properties including Michaelis constants, substrate and coenzyme specificities, pH-dependent activity, and thermal stability. Consistent with previous genetic evidence, the results of our studies also suggest that Hex A and Hex B (with subforms B1 and B2) are products of a single structural gene but are modified post-translationally, whereas the allelic forms of Hex C (C1 and C2) are derived from a different structural gene from that of Hex A and Hex B.
D000818:Animals; D004331:Drosophila melanogaster / Q000201:enzymology*; D006593:Hexokinase / Q000302:isolation & purification / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007106:Immune Sera; D007118:Immunoassay; D005779:Immunodiffusion; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D013379:Substrate Specificity
D007106:Immune Sera; D007527:Isoenzymes; D006593:Hexokinase