Purification and properties of adenosine 5′-triphosphate-d-glucose 6-phosphotransferase from rat liver

Abstract

1. An 870-fold purification of glucokinase from rat liver is described which involves ammonium sulphate fractionation and the use of DEAE-Sephadex, DEAE-cellulose and polyacrylamide columns. 2. The preparation is free of any interfering enzymes and has a specific activity of 8mumoles/min./mg. of protein. 3. Glucokinase catalyses the phosphorylation of glucose, mannose and 2-deoxyglucose. 4. The enzyme is inhibited by high concentrations of glucose 6-phosphate only; ADP is an inhibitor whose effect depends on the Mg(2+) concentration. 5. The properties of glucokinase are compared briefly with those of other phosphotransferases.

Glycolytic and gluconeogenic enzyme activities in parenchymal and non-parenchymal cells from mouse liver

“…Hexokinase and glucokinase were assayed by the method of Parry & Walker (1966). The medium consisted of 50mM-tris chloride, I00mM-KCI, 7.5mM-MgSO4, 1.6mM-ATP, 0.5mM-NADP+, 2.5,ug of glucose 6-phosphate dehydrogenase, 10,ug of 6-phosphogluconate dehydrogenase and either 0.5mM-D-glucose (hexokinase) or 100mM-D-glucose (hexokinase plus glucokinase), in a final volume of 3.Oml, pH7.5.…”

Identification, characterization of levoglucosan kinase, and cloning and expression of levoglucosan kinase cDNA from Aspergillus niger CBX-209 in Escherichia coli

“…The high K m value (71.2 mM) of LG kinase for levoglucosan may account for the lower assimilation and conversion rates of levoglucosan in this fungus. For general hexokinases, their K m values ranged from only several millimolars in bacteria or fungi [34,45] up to 20 mM in mammals [35,46]. In the latter case, the glucokinase was suggested to regulate the blood sugar concentration with a high K m [47,48].…”

“…Sodium, potasium, and calcium ions had no effect on either activity or stability of this kinase (data not shown), while glucokinase from rat liver needed potasium ions for stability [35].…”

“…The amount of enzyme used was such that a measurable rate would have been obtained if any of these would react at a rate 0.5% of that of levoglucosan. Just like general glucokinases from bacteria and mammals [34,35], the phosphoryl donor most effective with levoglucosan as an acceptor was ATP. Nucleoside triphosphates at 2 mM in the presence of manganese ion gave relative rates of phosphorylation as: ATP, 100; GTP, 2; and ITP, 8.…”

Purification and Characterization of Glucokinase inEscherichia coliB

“…The addition of various metal ions such as Ba z +, Mnz+, Fe z +, Cu z +, Niz+, and Co z + caused decreases in the activity of the enzyme. (6) Interaction between substrates and products. The effects of G-6-P and ADP on· glucokinase reaction were investigated.…”