Affinity chromatography on immobilised nucleotides. The synthesis, specificity and applications of immobilised inosine 5'-monophosphate.
The synthesis and characterisation of two IMP analogues, 8-(6-aminohexyl)-ionosine 5'-monophosphate, Ahx8IMP, and inosine 2',3'-O-[1-(6-aminohexyl)-levulinic acid amide]-acetyl 5'-monophosphate, (AhxLvn)2',3'IMP, is described. These analogues were attached to CNBr-activated agarose through the terminal amino group of the spacer molecule. The immobilised-IMP analogues displayed specificity for the inosine-nucleotide-dependent enzyme, IMP dehydrogenase (IMP:NAD+ oxidoreductase, EC 1.2.1.14) but not for the NAD+-dependent enzymes, L-alanine and L-acetate dehydrogenases. Escherichia coli IMP dehydrogenase could be eluted biospecifically from immobilised 8-substituted and ribose-substituted IMP adsorbents with IMP, XMP and GMP. Multiple peaks of enzyme activity in the elution profiles were interpreted in terms of aggregation of the enzyme. A protocol for the large-scale purification of E. coli IMP dehydrogenase is proposed. Homogeneous enzyme of specific activity 9.1 units/mg was obtained in 50% overall yield, representing 14 mg pure protein from a 20-1 culture of E. coli. The two IMP analogues were inactive as substrates in the IMP dehydrogenase reaction.
D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D002846:Chromatography, Affinity / Q000379:methods; D004926:Escherichia coli / Q000201:enzymology; D006157:Guanosine Monophosphate; D007168:IMP Dehydrogenase / Q000302:isolation & purification*; D007291:Inosine Monophosphate / Q000031:analogs & derivatives / Q000138:chemical synthesis*; D007292:Inosine Nucleotides / Q000138:chemical synthesis*; D007658:Ketone Oxidoreductases / Q000302:isolation & purification*; D011817:Rabbits; D012265:Ribonucleotides; D019820:Xanthine; D014970:Xanthines
D007292:Inosine Nucleotides; D012265:Ribonucleotides; D014970:Xanthines; D007291:Inosine Monophosphate; D019820:Xanthine; C011141:xanthosine monophosphate; C027942:8-((6-aminohexyl)amino)inosine 5'-monophosphate; C027944:inosine 2',3'-O-(1-(6-aminohexyl)levulinic acid amide)acetal 5'-monophosphate; D006157:Guanosine Monophosphate; D007168:IMP Dehydrogenase; D007658:Ketone Oxidoreductases