Ion-pair reverse-phase high-performance liquid chromatography. Application to the study of chicken liver NAD+ kinase

✍ Scribed by Mina K. Gabriel; Eugene T. McGuinness

• Publisher: Elsevier Science
• Year: 1985
• Tongue: English
• Weight: 412 KB
• Volume: 149
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(85)90579-2

✦ Synopsis

An ion-pair, reverse-phase, high-performance liquid chromatography method of assay was developed and used in a series of rate studies carried out with the enzyme chicken liver NAD+ kinase (ATP:NAD+ 2'-phosphotransferase, EC 2.7.1.23). Complete separation of all products and reactants was achieved within 15 min. ATP, NAD+, ADP, and NADP+ were monitored at 260 nm as they eluted from a Zorbax (Dupont) ODS (4.6 X 250-mm) column using an acetonitrile and 0.01 mM NH4(H2PO4)/0.005 M tetrabutylammonium phosphate (pH 7.0) gradient. The enzyme shows a marked preference for ATP (and dATP) and Mg2+ (or Mn2+) relative to other trinucleotides and divalent metal ions. It exhibits residual adenylate kinase and ATPase activity, but no NADH kinase activity. When polyphosphate replaced ATP, NADP+ production dropped to 2.5%. The addition of Ca2+ and/or bovine brain calmodulin did not significantly enhance the rate of NADP+ production.