| Popis: |
Particulate glucokinases (ATP: d -glucose 6-phosphotransferase, EC 2.7.1.2) from rat liver were localized in sucrose density gradient subfractions of the microsomal fraction. Most of this activity was extracted in soluble form with 0.1% (w/v) Triton X-100. A purification is described which involved (NH4)2SO4 fractionation and the use of DEASE-Sephadex and DEAE-cellulose. By gel filtration on Bio-gel, two active fractions, G1 and G2, were obtained with a 760-fold purification for G1 and 250-fold one for G2. As assessed by polyacrylamide column, the molecular weights of rat hepatic glucokinases were 120 000 for G1 and 50 000 for G2, respectively. The preparations had specific activities of 1.15 μmoles/min per mg of protein for G1 and 0.37 for G2. The enzymes were found to have different temperature stabilities and pH sensitivites. They catalyzed the phosphorylation of glucose, glucosamine and, to a lesser extent, fructose, but significant differences were noted between the interactions of G1 and G2 with their substrates. Each purified glucokinase presented two apparent Km values for glucose, glucosamine and ATP. These results suggested an activation of the catalytic activity of G1 and G2 by high concentrations of the substrate. The other sugars tested, 2-deoxy- d -glucose, N- acetyl- d -glucosamine , d -mannose, d -galactose and d -xylose, were not phosphorylated. The properties of glucokinases were briefly compared with those of other phosphotransferases. |
