Rabbit red blood cell hexokinase. Evidence for two distinct forms, and their purification and characterization from reticulocytes

Abstract

Rabbit hexokinase (EC 2.7.1.1) has been shown to exist in the soluble fraction of reticulocytes as two distinct molecular forms, designated hexokinase Ia and hexokinase Ib, which are separable by ion exchange chromatography and polyacrylamide gel electrophoresis. Hexokinase Ia was found to be similar to the brain enzyme, while hexokinase Ib differs from every other previously reported hexokinase isozyme. Reticulocyte hexokinase Ia and Ib have been purified 55,000-and 50,000-fold, respectively, by a combination of ion exchange chromatography, affinity chromatography, and preparative polyacrylamide gel electrophoresis, as proteins homogeneous by sodium dodecyl sulfate-gel electrophoresis. The native proteins have the same molecular weight of 105,000 by gel filtration and sedimentation velocity on sucrose density gradients. Sodium dodecyl sulfate-polyacrylamide gels have a molecular weight of 104,000, indicating that the two forms are monomers. Hexokinase Ia had a pI of 6.2 to 6.3 pH units while hexokinase Ib had a pI of 5.7 to 5.8 pH units by isoelectric focusing. The two enzymes were specific for Mg.ATP and Mg.ITP as the nucleotide substrates. Several hexoses could be phosphorylated by hexokinase Ia and Ib with different affinities.