Adaptation of a Commercial NAD+ 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 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.

Nicotine Adenine Dinucleotides: The Redox Currency of the Cell.

There has been tremendous and rapidly growing interest in understanding intermediary metabolism as a key aspect of both normal cellular function and as a participant in the molecular pathogenesis of many different complex diseases. This area of research naturally intersects at virtually every level with the substantial and expanding body of knowledge regarding mechanisms of cellular redox balance. In this Forum, the contributing authors address specifically the union of intermediary metabolism and redox biology through detailed consideration of the biochemistry and biology of nicotine adenine dinucleotides, the cell's "redox currency." From technical considerations of how to measure nicotine adenine dinucleotides all the way to detailed treatments of their potential roles in specific disease states, this Forum provides a thorough introduction to a topic that is positioned to be at the heart of the next wave of research in metabolism and redox biology. Antioxid. Redox Signal. 28, 165-166.