Hexokinase isoenzymes from the Novikoff hepatoma. Purification, kinetic and structural characterization, with emphasis on hexokinase C

Biochem J. 1987 Mar 15;242(3):895-903. doi: 10.1042/bj2420895.


The purification to homogeneity of hexokinases B and C from the cytosol of rat Novikoff hepatoma was achieved by a protocol using an initial chromatography on Blue 2-agarose to separate the isoenzymes from each other. After that step each hexokinase was subjected to chromatography on DEAE-cellulose, hydroxyapatite and Sephacryl S-300, followed by re-chromatography on hydroxyapatite. The final preparations of hexokinases B and C had specific activities of 86 and 23.5 units/mg of protein respectively, and gave single bands on electrophoresis under non-denaturing conditions or in SDS/polyacrylamide gels. Mr values of about 100,000 were found for both isoenzymes either by Sephacryl S-300 chromatography or by SDS/polyacrylamide-gel electrophoresis. Values of apparent Km for glucose and ATP of pure hexokinase B were similar to those reported for the enzyme from other sources. The apparent Km value for glucose of hexokinase C was 0.025 mM. Marked inhibition of hexokinase C by glucose concentrations above 0.2 mM was found. The effect was partially relieved by ATP concentrations above 1 mM and was independent of pH. Glucose 6-phosphate was inhibitory, but the Ki value (0.18 mM) is higher than those reported for other animal hexokinases. The amino acid composition of hexokinase C was found to be similar to those reported for hexokinases B and D. Also, an immune serum directed against hexokinase A was able, at low dilutions, to bind hexokinases B and C. An immune serum directed against hexokinase C was able, at low dilutions, to bind hexokinase B and also, but weakly, hexokinase A.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acids / analysis
  • Animals
  • Chromatography, Liquid
  • Cross Reactions
  • Glucose / metabolism
  • Hexokinase / immunology
  • Hexokinase / isolation & purification
  • Hexokinase / metabolism*
  • In Vitro Techniques
  • Isoenzymes / isolation & purification
  • Isoenzymes / metabolism*
  • Kinetics
  • Liver Neoplasms, Experimental / enzymology*
  • Phosphorylation
  • Rats


  • Amino Acids
  • Isoenzymes
  • Hexokinase
  • Glucose