Purification and properties of the cytoplasmic hexokinase from rabbit brain.
About 90% of the total hexokinase activity in rabbit brain was found to be associated with mitochondria while the remaining part was found in the cytosolic fraction. The soluble enzyme was purified 4,700-fold to near homogeneity by a combination of ion-exchange chromatography, dye-ligand chromatography and affinity chromatography. The purified enzyme showed a specific activity of 110 units/mg of protein and was obtained in 70% yield. The molecular weight of the purified hexokinase was found to be approximately 98,000 both for the native and the denatured enzyme. The isoelectric point, pI, was 6.2 pH units by isoelectric focusing and the enzyme was found to be able to phosphorylate several hexoses. Mg . ATP2-, among the nucleotide substrates, was the most effective phosphate donor. The properties of the purified cytoplasmatic hexokinase were compared with those of the solubilized mitochondrial enzyme. No significant differences were found in molecular weight, isoelectric point, pH dependence of activity, electrophoretic mobility and affinity for glucose and Mg.ATP2-. However, the temperature dependence of activity, and the specificity for several hexose substrates were markedly different.
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D003593:Cytoplasm / Q000201:enzymology; D004355:Drug Stability; D006593:Hexokinase / Q000032:analysis* / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000032:analysis; D011489:Protein Denaturation; D011817:Rabbits; D013347:Subcellular Fractions / Q000201:enzymology
D009419:Nerve Tissue Proteins; D006593:Hexokinase