Glucose 6-phosphate-solubilized hexokinase: Some electrophoretic properties

Abstract

Abstract 1. 1.Electrophoresis of hexokinase (ATP: D-hexose-6-phosphate transferase, EC 2.7.1.1), solubilized from brain mitochondria by glucose 6-phosphate, (Glc-6-P) using conventional 7% polyacrylamide gels, after dialysis to remove Glc-6-P, resulted in aggregation and non-entry of hexokinase into the gels. If dialysis was omitted, hexokinase did enter the gel in the presence of Glc-6-P, however, once it had been removed from the soluble enzyme, aggregation could not be reversed. 2. 2. Hexokinase appears to be the only protein which is specifically solubilized from mitochondria by Glc-6-P, therefore the aggregate is not a multienzyme complex. 3. 3. The unpurified, Glc-6-P-solubilized enzyme has a mol. wt of 130 000 as judged by sodium dodecysulfate polyacrylamide gel electrophoresis, after treatment with 0.2% mercaptoethanol-1% sodium dodecylsulfate at 45°C for 1 h. However, the mol. wt drops to 100 000 when treated in a boiling-water bath or if the mercaptoethanol concentration is raised from 0.2 to 2%. 4. 4. Purification of hexakinase by DEAE-cellulose chromatography resulted in a loss of the aggregation phenomenon observed with the unpurified enzyme, using conventional 7% polyacrylamide gels. 5. 5. The DEAE-cellulose-purified enzyme had a mol. wt of 100 000, when treated at 45°C with 0.2% mercaptoethanol-1% sodium dodecylsulfate, with a minor component of mol. wt 130 000. Subunits of mol. wt 45 000 were obtained when the purified enzyme was pretreated with 2% mercaptoethanol in a boiling water bath before the addition of 1% sodium dodecylsulfate. 6. 6. Hexokinase, purified by DEAE-cellulose chromatography did not rebind to the mitochondrial membrane under the influence of MgCl2, whereas the unpurified enzyme did rebind.