title: Affinity chromatography on immobilised nucleotides. Some applications to the purification of thermophilic dehydrogenases and kinases.
abstract: The effect of pH and temperature on the capacity and binding of Bacillus stearothermophilus, alcohol dehydrogenase and phosphofructokinase to N6-(6-aminohexyl)-5'-AMP-Sepharose has been examined. Specific elution from the substituted AMP-Sepharose was examined using a variety of cofactors, fragments of cofactors and substrates. A purification scheme for each enzyme on the substituted AMP-Sepharose using nucleotides and gradients of pH and salt is presented. Interestingly, elevated temperature increased the affinity of both enzymes for N6-(6-aminohexyl)-5'-AMP-Sepharose, however, the Michaelis constant for nucleotide determined at various temperatures remained constant. The effect of pH and salt concentration on the binding of B. stearothermophilus glyceraldehyde-3-phosphate dehydrogenase to 6-aminohexanoyl-NAD+-Sepharose was also examined; raising the pH above 7.5 lowers the capacity of the matrix and the effect of a range of ammonium sulphate concentrations on the adsorption of the enzyme was examined. A specific purification of glyceraldehyde-3-phosphate dehydrogenase from partially purified extracts of this organism was achieved.
mesh_terms: D000249:Adenosine Monophosphate; D000429:Alcohol Oxidoreductases / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D009243:NAD; D009249:NADP; D009994:Osmolar Concentration; D010732:Phosphofructokinase-1 / Q000302:isolation & purification*; D011485:Protein Binding; D012685:Sepharose; D013696:Temperature
chemical_list: D009243:NAD; D000249:Adenosine Monophosphate; D009249:NADP; D012685:Sepharose; D000429:Alcohol Oxidoreductases; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D010732:Phosphofructokinase-1