3-MINUTE HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHIC ASSAY FOR NMN ADENYLYLTRANSFERASE USING A 20-MM-LONG REVERSED-PHASE COLUMN

NMN adenylyltransferase (NAD pyrophosphorylase; NMNAT) reversibly cata lyzes the synthesis of NAD from ATP and NMN. In this paper, we describ e a rapid and sensitive high-performance liquid chromatographic assay for NMNAT, which uses a 20-mm-long C-18 reversed-phase (RP) column. Th e activity was measured by separating in less than 3 min the substrate s (NMN and ATP) from the product (NAD) with 0.1 M potassium phosphate, pH 6.0, at a 2 ml/min flow-rate and 22 degrees C, NAD was directly qu antitated from its ultraviolet absorbance. Amounts of NAD as small as 25 pmol could be measured. The activity value closely agreed with that determined by the spectrophotometric assay. This method was successfu lly applied to the determination of NMNAT activity in human placental and bull testis extracts, as well as in rat pheochromocytoma (PC12) ce lls.