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

• Published in: Analytical biochemistry Vol. 149; no. 2; pp. 339 - 343
• Main Authors: Gabriel, Mina K., McGuinness, Eugene T.
• Format: Journal Article
• Language: English
• Published: San Diego, CA Elsevier Inc 01.09.1985; Elsevier
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Applied sciences; Biological and medical sciences; Calcium - pharmacology; Calmodulin - pharmacology; Chickens; Chromatography, High Pressure Liquid - methods; Diphosphates - pharmacology; Enzymes and enzyme inhibitors; Exact sciences and technology; Fundamental and applied biological sciences. Psychology; ion-pair HPLC; Liver - enzymology; Metals - pharmacology; NAD + kinase; Other techniques and industries; Phosphoric Acids - pharmacology; Phosphotransferases (Alcohol Group Acceptor); Phosphotransferases - isolation & purification; Polymers - pharmacology; reverse-phase HPLC; Substrate Specificity; Transferases; ion-pair HPLC; reverse-phase HPLC; NAD + kinase; Vertebrata; NAD+ kinase; Enzyme; Analysis; Ion pair; HPLC chromatography; Aves
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/0003-2697(85)90579-2

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.