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 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.
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000494:pharmacology; D002645:Chickens; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D011756:Diphosphates / Q000494:pharmacology; D008099:Liver / Q000201:enzymology*; D008670:Metals / Q000494:pharmacology; D010756:Phosphoric Acids / Q000494:pharmacology; D010770:Phosphotransferases / Q000302:isolation & purification*; D017853:Phosphotransferases (Alcohol Group Acceptor); D011108:Polymers / Q000494:pharmacology; D013379:Substrate Specificity
D002147:Calmodulin; D011756:Diphosphates; D008670:Metals; D010756:Phosphoric Acids; D011108:Polymers; C047269:polyphosphoric acid; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C021844:NAD kinase; D002118:Calcium