Expression, purification, and characterization of a recombinant erythroid-specific hexokinase isozyme.
Hexokinase type I (HK I; ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1), the predominant glucose-phosphorylating enzyme in red blood cells, exists in human erythrocytes in multiple molecular forms that differ in isoelectric point and are separable by ion-exchange chromatography. The major forms, designated HK Ia, Ib and Ic, have similar kinetic properties but are characterized by different age-dependent decay and different intracellular distribution in reticulocytes. HK Ib, which elutes between HK I and HK II in the DEAE ion-exchange chromatography, appears to be unique to RBCs and different from any other hexokinase isozyme previously described. Indeed, Murakami and Piomelli recently reported the presence of a specific HK isozyme (named HKr) expressed in K562 cells and in human reticulocytes and, moreover, the resolution of the human HK I gene structure provided the direct evidence of an erythroid-specific exon 1. To further investigate the microheterogeneity of HK I in human RBCs we established a prokaryotic expression system for the HKr isozyme, using the pET plasmid, inducible with IPTG. The recombinant HKr, expressed in bacterial cells as a catalytically active enzyme, was purified to homogeneity by a combination of DEAE ionexchange chromatography followed by hydrophobic interaction chromatography and dye-ligand affinity chromatography. The kinetic and chromatographic properties of the homogeneous recombinant HKr suggest that this erythroid-specific HK isozyme in fact corresponds to the HK isoform previously described in human RBCs and referred to as HK Ib.
Bianchi
M
M
Institute of Biological Chemistry "G. Fornaini", University of Urbino, Italy.
Serafini
G
G
Bartolucci
E
E
Palma
F
F
Magnani
M
M
D004912:Erythrocytes / Q000201:enzymology*; D006593:Hexokinase / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D020014:K562 Cells; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D014162:Transfection
D007527:Isoenzymes; D011994:Recombinant Proteins; D006593:Hexokinase