Study of the interaction between NADP-dependent dehydrogenase and immobilized adenosine 2'-monophosphate by means of affinity electrophoresis.
A water-soluble 8-substituted adenosine 2'-monophosphate-polyacrylamide (8-sub-2'-AMP-PA) was prepared as a new affinity ligand for the determination of the dissociation constants of the interactions between immobilized 8-sub-2'-AMP and NADP-dependent dehydrogenases (NADP-dependent DH), NAD-dependent dehydrogenases (NAD-dependent DH) and phosphorylase by means of affinity electrophoresis. From the dissociation constants, it was found that NADP-dependent DH had a much stronger affinity to immobilized 8-sub-2'-AMP than did NAD-dependent DH and phosphorylase. On the other hand, NADP-dependent DH had a much weaker affinity to immobilized 8-sub-5'-AMP than did NAD-dependent DH. The effects of NADP+ and NAD+ on the interaction between immobilized 8-sub-2'-AMP and NADP-dependent DH were also investigated by means of affinity electrophoresis. NADP+ inhibited the interaction specifically, but NAD+ did not inhibit the interaction. These results indicate that 8-sub-2'-AMP binds to the coenzyme binding site of NADP-dependent DH, while other compounds, such as 8-sub-5'-AMP and NAD+, do not bind to NADP-depenndent DH. Such a difference suggests that the phosphate group at position 2' in 2'-AMP and NADP+ is important for the binding at the coenzyme binding site of NADP-dependent DH.
D000249:Adenosine Monophosphate / Q000378:metabolism*; D001921:Brain / Q000201:enzymology; D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005954:Glucosephosphate Dehydrogenase / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008024:Ligands; D008099:Liver / Q000201:enzymology; D009245:NADH Dehydrogenase / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D009252:NADPH Dehydrogenase / Q000378:metabolism*; D013379:Substrate Specificity
D008024:Ligands; D000249:Adenosine Monophosphate; D007770:L-Lactate Dehydrogenase; D005954:Glucosephosphate Dehydrogenase; D009247:NADH, NADPH Oxidoreductases; D009252:NADPH Dehydrogenase; D009245:NADH Dehydrogenase