ASSAY-METHODS FOR NICOTINAMIDE MONONUCLEOTIDE ADENYLYLTRANSFERASE OF WIDE APPLICABILITY

MMN adenylyltransferase (NMNAT) reversibly catalyzes the synthesis of NAD(+) or NaAD(+) from ATP and NMN or NaMN. In this work, we describe a continuous coupled spectrophotometric assay that can be rapidly and routinely used in place of the previous cumbersome two-step assay. The reaction rates measured with the coupled assay display a Linear depen dence with respect to enzyme concentration over the range investigated . The method yields accurate and reliable estimates of the enzyme acti vity in the direction of NAD(+) synthesis. Furthermore, we developed a n HPLC-based method suitable for the assay of activity both in the for ward and reverse directions of the enzymatic reaction. The method appe ars particularly useful for measuring the NMNAT activity when the prod uct is not NAD(+) (e.g., in studies using alternative substrates), and offers the possibility of monitoring simultaneously both the NMNAT-ca talyzed reaction and interfering side reactions. This is achieved thro ugh the HPLC identification and quantitation of metabolites and deriva tives produced in the reaction mixture during the assay. The two metho ds described here should cover most needs for the assay of NMNAT activ ity. (C) 1995 Academic Press, Inc.