Hexokinase isoenzymes from the Novikoff hepatoma. Purification, kinetic and structural characterization, with emphasis on hexokinase C.
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.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002853:Chromatography, Liquid; D003429:Cross Reactions; D005947:Glucose / Q000378:metabolism; D006593:Hexokinase / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D066298:In Vitro Techniques; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008114:Liver Neoplasms, Experimental / Q000201:enzymology*; D010766:Phosphorylation; D051381:Rats
D000596:Amino Acids; D007527:Isoenzymes; D006593:Hexokinase; D005947:Glucose