Purification of cofactor-dependent enzymes by affinity chromatography

✍ Scribed by Chi-Yu Lee; Carl-Johan Johansson

• Publisher: Elsevier Science
• Year: 1977
• Tongue: English
• Weight: 778 KB
• Volume: 77
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(77)90293-7

✦ Synopsis

Several 8-(6aminohexyl)-amino adenine nucleotide derivatives, including ATP, 2',5'-ADP, 3',5'-ADP and desulfo-CoA (CoA, reduced coenzyme A), were prepared and immobilized on Sepharose by cyanogen bromide activation. 8-(6-Aminohexyb-amino-ATP-Sepharose was found to exhibit good aftinity for both NAD+-dependent dehydrogenases and kinases. Sequential biospecific elutions with NADH and ATP resulted in a good separation of dehydrogenases from kinases. As many as eight different dehydrogenases and kinases could be substantially purified from both porcine muscle and mouse kidney extracts by this new procedure. 8-(6Aminohexyl)-amino-2',5'-ADP-and -3',5'-ADP-Sepharose were shown to exhibit good affinity for many NADP-dependent dehydrogenases from yeast extracts and CoAdependent enzymes, respectively. Purification of citrate synthases from pig heart and Eschericia coli extracts by means of these 8-substituted adenine nucleotide affinity columns was atso presented. We also observed that Sepharose-bound-8-(6-aminohexyl)-amino-ATP exhibits good affinity not only for many kinases but also for many NAD+-dependent dehydrogenases and citrate synthases as well. The multi-90

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