NMN 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 dependence with respect to enzyme concentration over the range investigated. The method yields accurate and reliable estimates of the enzyme activity in the direction of NAD(+) synthesis. Furthermore, we developed an HPLC-based method suitable for the assay of activity both in the forward and reverse directions of the enzymatic reaction. The method appears particularly useful for measuring the NMNAT activity when the product is not NAD(+) (e.g., in studies using alternative substrates), and offers the possibility of monitoring simultaneously both the NMNAT-catalyzed reaction and interfering side reactions. This is achieved through the HPLC identification and quantitation of metabolites and derivatives produced in the reaction mixture during the assay. The two methods described here should cover most needs for the assay of NMNAT activity.

Assay methods for nicotinamide mononucleotide adenylyltransferase of wide applicability

EMANUELLI, Monica;RAFFAELLI, Nadia;RUGGIERI, Silverio;AMICI, Adolfo;MAGNI, GIULIO;ORSOMANDO, Giuseppe;
1995-01-01

Abstract

NMN 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 dependence with respect to enzyme concentration over the range investigated. The method yields accurate and reliable estimates of the enzyme activity in the direction of NAD(+) synthesis. Furthermore, we developed an HPLC-based method suitable for the assay of activity both in the forward and reverse directions of the enzymatic reaction. The method appears particularly useful for measuring the NMNAT activity when the product is not NAD(+) (e.g., in studies using alternative substrates), and offers the possibility of monitoring simultaneously both the NMNAT-catalyzed reaction and interfering side reactions. This is achieved through the HPLC identification and quantitation of metabolites and derivatives produced in the reaction mixture during the assay. The two methods described here should cover most needs for the assay of NMNAT activity.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11566/81366
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