Determination of NAD pyrophosphorylase activity in biological samples

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 micrograms. The range of NAD produced during the assay was 2 to 200 microM. NAD pyrophosphorylase activities in the mononuclear cells of leukemic patients, human ovarian carcinoma cells, and rat liver were assayed.