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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.
M K Gabriel, and
E T McGuinness
July 1982,
Journal of forensic sciences,
M K Gabriel, and
E T McGuinness
September 1980,
Analytical biochemistry,
M K Gabriel, and
E T McGuinness
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Analytical biochemistry,
M K Gabriel, and
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July 1980,
Journal of biochemical and biophysical methods,
M K Gabriel, and
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M K Gabriel, and
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December 1990,
Clinica chimica acta; international journal of clinical chemistry,
M K Gabriel, and
E T McGuinness
February 2002,
RNA (New York, N.Y.),
M K Gabriel, and
E T McGuinness
August 1991,
Lipids,
M K Gabriel, and
E T McGuinness
February 2000,
Analytical chemistry,
M K Gabriel, and
E T McGuinness
May 1997,
Analytical biochemistry,
