Determination of NAD pyrophosphorylase activity in biological samples

Edit Paulik, Hiremagalur N. Jayaram, George Weber

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11 Scopus citations

Abstract

A sensitive and simple method was developed for the accurate measurement of NAD pyrophosphorylase (NMN adenylyltransferase; EC 2.7.7.1) activity in biological samples. The reaction product of [4-3H]NAD was separated from the substrates [4-3H]NMN and ATP by HPLC. Under the standardized conditions of the assay, the enzyme activity in human chronic myelogenous leukemia K562 cells was found mainly in the nucleus (97%) with a sp act of 183.5 ± 3.5 nmol/h/mg protein. The Km's for substrates NMN and ATP were 0.11 ± 0.01 mm and 0.55 ± 0.04 mm, respectively. This technique is highly reproducible with a 5% variation (SD) in five separate determinations. The lowest number of cells used for this enzyme assay was 41,000 with a protein content of 4 μg. The range of NAD produced during the assay was 2 to 200 μm. NAD pyrophosphorylase activities in the mononuclear cells of leukemic patients, human ovarian carcinoma cells, and rat liver were assayed.

Original languageEnglish (US)
Pages (from-to)143-148
Number of pages6
JournalAnalytical biochemistry
Volume197
Issue number1
DOIs
StatePublished - Aug 15 1991

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ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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