N2630

The NADase reaction was performed in black 96-well half area plates (Corning, 3694). In each microwell, ThsA protein which was purified as previously described (Ofir et al., 2021), was added to cell lysate or to a positive control of 100 mM sodium phosphate buffer pH 8 supplemented with 1′′–3′ gcADPR standards. ThsA was added to a final concentration of 100 nM protein in a 50 μl final volume reaction. Five microlitres of 5 mM nicotinamide 1,N⁶ -ethenoadenine dinucleotide (εNAD, Sigma,N2630) solution was added to each well immediately before the beginning of measurements and mixed by pipetting to reach a concentration of 500 μM in the 50 μl final reaction volume. Plates were incubated inside a Tecan Infinite M200 plate reader at 25°C, and measurements were taken every 1 min at 300 nm excitation wavelength and 410 nm emission wavelength. Reaction rate was calculated from the linear part of the initial reaction.

In the fluorescence assay, 1, N⁶-etheno-adenine dinucleotide (εNAD⁺, Sigma Aldrich, N2630), the fluorescent analogue of NAD⁺, was used as the substrate. Enzymatic activity was characterized by monitoring the increase in fluorescence resulting from cleavage of the quenching nicotinamide group in 1, N⁶-ethenoadenine diphosphate ribose. εNAD⁺ stocks were made using a buffer (100 mM Tris 7.5). The reaction buffer was supplemented with 10 mM MES pH 6.5, 75 mM KCl, 2 mM MgCl₂. The concentrations of proteins and the substrate were optimized to be 400 nM and 50 μM, respectively.
The reaction procedure was carried out in triplicate at 37 °C in a 96-well half area plate (Corning, 3690). Fluorescence intensity was measured by Agilent BioTek Synergy H1, with an excitation wavelength of 300 nm and an emission wavelength of 410 nm, with readings taken every minute over a 0.5 h-1 h duration. The change in fluorescence over time was determined by calculating the slopes of the linear component of the curves. Data analysis was performed using GraphPad Prism 9.

eNAD (nicotinamide 1,N6-ethenoadenine dinucleotide, SIGMA – N2630) was solubilized in water and mixed with native NAD+ in a ratio of 1:10 (mol:mol) to a final stock concentration of 10 mM. Serial dilutions were made with 25 mM HEPES pH 7.5, 150 mM NaCl buffer, and the final mix was transferred to a 384-well black plate (Corning). Reaction started by the addition of hSARM1 to a final concentration 400 nM. Then, eNAD degradation rate was monitored by fluorescence reading (330-nm excitation and 405-nm emission wavelengths) of the plate using a SynergyHI (BioTek) in 25°C for 3 hr. For each NAD+ concentration, a control reaction without SARM1 was measured and subtracted from the +hSARM1 reading and the slope of the linear area was calculated. For the final plot, average of slopes from three separate assays for each concentration was calculated.