| Abstract: | Soluble hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1) was purified from human heart. 1 kg of tissue provided 25 mg hexokinase with a specific activity of 58 units/mg, representing a 1700-fold purification and 47% yield. The purification involved six steps, including affinity chromatography with glucosamine attached to Sepharose. The material was homogeneous according to electrophoresis, gel-filtration and sedimentation in the ultracentrifuge, but gave two main components on electrophoresis in denaturing conditions. From determination of the sedimentation and diffusion coefficients, the relative molecular mass was calculated to be 105 000. The enzyme is monomeric, but glucose 6-phosphate promotes an association to dimers. This effect is reversible and is independent of the concentrations of glucose or inorganic phosphate. The results support the postulate that soluble and mitochondrion-bound hexokinases are identical. |
