Here we report an adapted protocol using the Promega NAD/NADH-Glo™ Assay kit. The assay normally allows quantification of trace amounts of both oxidized and reduced forms of nicotinamide adenine dinucleotide (NAD) by enzymatic cycling, but we now show that the NAD analog 3- acetylpyridine adenine dinucleotide (AcPyrAD) also acts as a substrate. In fact, AcPyrAD generates amplification signals of larger amplitude than those obtained with NAD. We exploited this finding to devise and validate a novel method for assaying the base exchange activity of SARM1 in reactions containing NAD and an excess of the free base 3-acetylpyridine (AcPyr), where AcPyrAD is the product. We also propose an application of this method based on competition between AcPyr and other free bases to rank their preference for SARM1. This has significant advantages over traditional methods for assaying SARM1 base exchange as it is rapid, sensitive, cost-effective, and easily scalable. This could represent a useful tool given current interest in the role of SARM1 base exchange in programmed axon death and related human disorders. It may also be applicable to other multifunctional NAD glycohydrolases (EC 3.2.2.6) that possess similar base exchange activity.

Adaptation of a commercial NAD quantification kit to assay the base exchange activity of SARM1 / Cirilli, Ilenia; Amici, Adolfo; Gilley, Jonathan; Coleman, Michael P.; Orsomando, Giuseppe. - ELETTRONICO. - (2023). [10.1101/2023.12.28.573537]

Adaptation of a commercial NAD quantification kit to assay the base exchange activity of SARM1

Ilenia Cirilli
Primo
;
Adolfo Amici;Giuseppe Orsomando
Ultimo
2023-01-01

Abstract

Here we report an adapted protocol using the Promega NAD/NADH-Glo™ Assay kit. The assay normally allows quantification of trace amounts of both oxidized and reduced forms of nicotinamide adenine dinucleotide (NAD) by enzymatic cycling, but we now show that the NAD analog 3- acetylpyridine adenine dinucleotide (AcPyrAD) also acts as a substrate. In fact, AcPyrAD generates amplification signals of larger amplitude than those obtained with NAD. We exploited this finding to devise and validate a novel method for assaying the base exchange activity of SARM1 in reactions containing NAD and an excess of the free base 3-acetylpyridine (AcPyr), where AcPyrAD is the product. We also propose an application of this method based on competition between AcPyr and other free bases to rank their preference for SARM1. This has significant advantages over traditional methods for assaying SARM1 base exchange as it is rapid, sensitive, cost-effective, and easily scalable. This could represent a useful tool given current interest in the role of SARM1 base exchange in programmed axon death and related human disorders. It may also be applicable to other multifunctional NAD glycohydrolases (EC 3.2.2.6) that possess similar base exchange activity.
2023
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11566/325987
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