General ligands in affinity chromatography. Cofactor-substrate elution of enzymes bound to the immobilized nucleotides adenosine 5'-monophosphate and nicotinamide-adenine dinucleotide.
1. Two different gels have been prepared suitable for the separation of a number of enzymes, in particular NAD(+)-dependent dehydrogenases, by affinity chromatography. For both the matrix used was Sepharose 4B. For preparation (a), NAD(+)-Sepharose, 6-aminohexanoic acid has been coupled to the gel by the cyanogen bromide method and then NAD(+) was attached by using dicyclohexylcarbodi-imide; for preparation (b), AMP-Sepharose, N(6)-(6-aminohexyl)-AMP has been coupled directly to cyanogen bromide-activated gel. 2. Affinity columns of both gels retain only the two enzymes when a mixture of bovine serum albumin, lactate dehydrogenase and glyceraldehyde 3-phosphate dehydrogenase is applied. Subsequent elution with the cofactor NAD(+) yields glyceraldehyde 3-phosphate dehydrogenase whereas lactate dehydrogenase is eluted by applying the same molarity of the reduced cofactor. 3. The binding of both glyceraldehyde 3-phosphate dehydrogenase and lactate dehydrogenase to the gel tested, AMP-Sepharose, is strong enough to resist elution by gradients of KCl of up to at least 0.5m. A 0.0-0.15m gradient of the competitive inhibitor salicylate, however, elutes both enzymes efficiently and separately. 4. The elution efficiency of lactate dehydrogenase from AMP-Sepharose has been examined by using a series of eluents under comparable conditions of concentration etc. The approximate relative efficiencies are: 0 (lactate); 0 (lactate+semicarbazide); 0 (0.5mm-NAD(+)); 80 (lactate+NAD(+)); 95 (lactate+semicarbazide+NAD(+)); 100 (0.5mm-NADH). 5. All contaminating lactate dehydrogenase activity can be removed from commercially available crude pyruvate kinase in a single-step procedure by using AMP-Sepharose.
D000249:Adenosine Monophosphate; D002845:Chromatography; D002846:Chromatography, Affinity; D004798:Enzymes / Q000302:isolation & purification*; D005782:Gels; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000302:isolation & purification; D007770:L-Lactate Dehydrogenase / Q000302:isolation & purification; D008722:Methods; D009243:NAD; D011134:Polysaccharides; D011189:Potassium Chloride; D011770:Pyruvate Kinase / Q000302:isolation & purification; D012710:Serum Albumin, Bovine
D004798:Enzymes; D005782:Gels; D011134:Polysaccharides; D009243:NAD; D012710:Serum Albumin, Bovine; D000249:Adenosine Monophosphate; D011189:Potassium Chloride; D007770:L-Lactate Dehydrogenase; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D011770:Pyruvate Kinase