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

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 × 250-mm) column using an acetonitrile and 0.01 m m NH 4(H 2PO 4)/0.005 m tetrabutylammonium phosphate (pH 7.0) gradient. The enzyme shows a marked preference for ATP (and dATP) and Mg 2+ (or Mn 2+) 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 Ca 2+ and/or bovine brain calmodulin did not significantly enhance the rate of NADP + production.