Adaptation of a Commercial NAD<sup>+</sup> Quantification Kit to Assay the Base-Exchange Activity and Substrate Preferences of SARM1
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 di...
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| Format: | Article |
| Language: | English |
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MDPI AG
2024-02-01
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| Series: | Molecules |
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| Online Access: | https://www.mdpi.com/1420-3049/29/4/847 |
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| author | Ilenia Cirilli Adolfo Amici Jonathan Gilley Michael P. Coleman Giuseppe Orsomando |
| author_facet | Ilenia Cirilli Adolfo Amici Jonathan Gilley Michael P. Coleman Giuseppe Orsomando |
| author_sort | Ilenia Cirilli |
| collection | DOAJ |
| description | 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 for this enzyme-cycling assay. In fact, AcPyrAD generates amplification signals of a 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 the product is AcPyrAD. We then used this assay to study competition between AcPyr and other free bases to rank the preference of SARM1 for different base-exchange substrates, identifying isoquinoline as a highly effect substrate that completely outcompetes even AcPyr. This has significant advantages over traditional HPLC 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. |
| first_indexed | 2024-03-07T22:19:32Z |
| format | Article |
| id | doaj.art-15de90121f724784acf0e38f8755f98a |
| institution | Directory Open Access Journal |
| issn | 1420-3049 |
| language | English |
| last_indexed | 2024-03-07T22:19:32Z |
| publishDate | 2024-02-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Molecules |
| spelling | doaj.art-15de90121f724784acf0e38f8755f98a2024-02-23T15:29:03ZengMDPI AGMolecules1420-30492024-02-0129484710.3390/molecules29040847Adaptation of a Commercial NAD<sup>+</sup> Quantification Kit to Assay the Base-Exchange Activity and Substrate Preferences of SARM1Ilenia Cirilli0Adolfo Amici1Jonathan Gilley2Michael P. Coleman3Giuseppe Orsomando4Department of Clinical Sciences (DISCO), Section of Biochemistry, Polytechnic University of Marche, Via Ranieri 67, 60131 Ancona, ItalyDepartment of Clinical Sciences (DISCO), Section of Biochemistry, Polytechnic University of Marche, Via Ranieri 67, 60131 Ancona, ItalyJohn van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge, Forvie Site, Robinson Way, Cambridge CB2 0PY, UKJohn van Geest Centre for Brain Repair, Department of Clinical Neurosciences, University of Cambridge, Forvie Site, Robinson Way, Cambridge CB2 0PY, UKDepartment of Clinical Sciences (DISCO), Section of Biochemistry, Polytechnic University of Marche, Via Ranieri 67, 60131 Ancona, ItalyHere, 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 for this enzyme-cycling assay. In fact, AcPyrAD generates amplification signals of a 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 the product is AcPyrAD. We then used this assay to study competition between AcPyr and other free bases to rank the preference of SARM1 for different base-exchange substrates, identifying isoquinoline as a highly effect substrate that completely outcompetes even AcPyr. This has significant advantages over traditional HPLC 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.https://www.mdpi.com/1420-3049/29/4/847enzyme assaySARM1NAD(P)asebase exchangepyridine basesacetylpyridine |
| spellingShingle | Ilenia Cirilli Adolfo Amici Jonathan Gilley Michael P. Coleman Giuseppe Orsomando Adaptation of a Commercial NAD<sup>+</sup> Quantification Kit to Assay the Base-Exchange Activity and Substrate Preferences of SARM1 Molecules enzyme assay SARM1 NAD(P)ase base exchange pyridine bases acetylpyridine |
| title | Adaptation of a Commercial NAD<sup>+</sup> Quantification Kit to Assay the Base-Exchange Activity and Substrate Preferences of SARM1 |
| title_full | Adaptation of a Commercial NAD<sup>+</sup> Quantification Kit to Assay the Base-Exchange Activity and Substrate Preferences of SARM1 |
| title_fullStr | Adaptation of a Commercial NAD<sup>+</sup> Quantification Kit to Assay the Base-Exchange Activity and Substrate Preferences of SARM1 |
| title_full_unstemmed | Adaptation of a Commercial NAD<sup>+</sup> Quantification Kit to Assay the Base-Exchange Activity and Substrate Preferences of SARM1 |
| title_short | Adaptation of a Commercial NAD<sup>+</sup> Quantification Kit to Assay the Base-Exchange Activity and Substrate Preferences of SARM1 |
| title_sort | adaptation of a commercial nad sup sup quantification kit to assay the base exchange activity and substrate preferences of sarm1 |
| topic | enzyme assay SARM1 NAD(P)ase base exchange pyridine bases acetylpyridine |
| url | https://www.mdpi.com/1420-3049/29/4/847 |
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