Blue-dextran--Sepharose affinity chromatography: recognition of a polynucleotide binding site of a protein.
Native Escherichia coli polynucleotide phosphorylase can be retained on blue-dextran--Sepharose. The bound enzyme cannot be displaced by its mononucleotide substrates such as ADP, UDP, CDP, GDP and IDP, but it is easily eluted by its polymeric substrates. Under identical conditions, lactate dehydrogenase, bound on blue-dextran--Sepharose, is not eluted by poly(I) but can be specifically displaced by NADH. On the other hand, the trypsinized polynucleotide phosphorylase, known to be an active enzyme which has lost its polynucleotide site, does not bind to the affinity column. The native polynucleotide phosphorylase can also be tightly bound to poly(U)--agarose and displaced from it only by high salt concentration. The trypsinized enzyme is not bound at all on poly(I)--AGAROSe. Moreover, the native enzyme linked on blue-dextran--Sepharose, remains active indicating a free access of nucleoside diphosphates to the active center. These results taken together show that the dye ligand is not inserted onto the mononucleotide binding site and suggest rather that it binds to the polynucleotide binding region. The implications of this study and the application of blue-dextran--Sepharose affinity chromatography to other proteins having affinity for nucleic acids are discussed.
D002846:Chromatography, Affinity / Q000379:methods; D004396:Coloring Agents; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D011117:Polyribonucleotide Nucleotidyltransferase / Q000302:isolation & purification* / Q000378:metabolism; D011131:Polyribonucleotides; D012265:Ribonucleotides; D012685:Sepharose
D004396:Coloring Agents; D011131:Polyribonucleotides; D012265:Ribonucleotides; D012685:Sepharose; D011117:Polyribonucleotide Nucleotidyltransferase