Studies on the use of sepharose-N-(6-aminohexanoyl)-2-amino-2-deoxy-D-glucopyranose for the large-scale purification of hepatic glucokinase.
The synthesis of N-(6-aminohexanoyl)-2-amino-2-deoxy-D-glucose is described and it was shown to be a competitive inhibitor (Ki, 0.75 mM) with respect to glucose of rat hepatic glucokinase (EC 2.7.1.2). After attachment to CNBr-activated Sepharose 4B, this derivative was able to remove glucokinase quantitatively from crude liver extracts and release it when the columns were developed with glucose, glucosamine, N-acetyl-glucosamine or KC1. Repeated exposure of the columns to liver extracts led to rapid loss in their effectiveness as affinity matrices because proteins other than glucokinase are bound to the columns. The nature of such protein binding and methods for the rejuvenation of "used" columns are discussed along with the effect of the mode of preparation of the Sepharose-ligand conjugate and the concentration of bound ligand on the purification of glucokinase. Glucose 6-phosphate dehydrogenase is cited as an example of both non-specific protein binding to the affinity column and of the importance of the control of ligand concentration in removing such non-specifically bound proteins. Some guidelines emerged that should be generally applicable to other systems, particularly those which involve affinity chromatography of enzymes that are present in tissue extracts in very low amounts and possess only a relatively low association constant for the immobilized ligand.
D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods; D005941:Glucokinase / Q000302:isolation & purification*; D005944:Glucosamine / Q000031:analogs & derivatives* / Q000302:isolation & purification; D005947:Glucose; D005954:Glucosephosphate Dehydrogenase; D008024:Ligands; D008099:Liver / Q000201:enzymology*; D011134:Polysaccharides; D011189:Potassium Chloride; D011485:Protein Binding; D051381:Rats; D012685:Sepharose
D008024:Ligands; D011134:Polysaccharides; D011189:Potassium Chloride; D012685:Sepharose; D005954:Glucosephosphate Dehydrogenase; D005941:Glucokinase; D005947:Glucose; D005944:Glucosamine