Hexokinase type I multiplicity in human erythrocytes.
Hexokinase I in human erythrocytes exists in multiple molecular forms that differ in isoelectric points. By means of Western blotting and immunodetection of total glucose-phosphorylating activity by using an antibody raised in rabbit against homogeneous human placenta hexokinase I, a single protein band was detected. Identical results were also obtained by immunoaffinity chromatography of the partially purified enzyme. Separation of the three major hexokinase I subtypes (Ia, Ib and Ic) by h.p.l.c. ion-exchange chromatography and immunodetection following electrophoretic blotting confirmed that each hexokinase subtype showed the same apparent Mr of 112,000, which is the value obtained for the high-Mr hexokinase I from human placenta. Purification of erythrocyte hexokinase by a combination of several procedures including dye-ligand and affinity chromatography that were previously successfully applied to the purification of other mammalian hexokinases type I produced a 35,000-fold-purified enzyme that showed several contaminants after SDS/polyacrylamide-gel electrophoresis. Only one of these peptides was found to be recognized by anti-(hexokinase I) IgG, suggesting that proteolytic degradation does not occur and that hexokinases Ia, Ib and Ic have the same apparent Mr.
Magnani
M
M
Istituto di Chimica Biologica, Università degli Studi, Urbino, Italy.
Serafini
G
G
Stocchi
V
V
D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000201:enzymology*; D006593:Hexokinase / Q000097:blood* / Q000276:immunology / Q000302:isolation & purification; D006801:Humans; D007122:Immunoelectrophoresis; D007527:Isoenzymes / Q000097:blood / Q000276:immunology / Q000302:isolation & purification; D008970:Molecular Weight
D007527:Isoenzymes; D006593:Hexokinase