Journal: iScience

Article Title: SARM1 is a multi-functional NAD(P)ase with prominent base exchange activity, all regulated bymultiple physiologically relevant NAD metabolites

doi: 10.1016/j.isci.2022.103812

Figure Lengend Snippet: Preliminary characterization of selected multifunctional NAD(P)ases (A) C18-HPLC UV profiles from time-course analyses of the three indicated multifunctional NAD(P)ase enzymes studied, all assayed with 250 μM NAD at 25°C under similar rates of substrate consumption. Both ADPR and cADPR products accumulating into diverse proportions are highlighted while unmarked in between, the substrate NAD peak declines in parallel and proportionally by time. (B) pH studies carried out by HPLC assays in universal buffer (Tris/Bis-Tris/Na-acetate) for 1 h at 25°C using 0.25 mM NAD and 7 μg/mL hSARM1 or 0.5 mM NAD and 0.07 μg/mL CD38 or 6 mM NAD and 0.08 μg/mL Aplysia cyclase. Data are Mean ± SEM from n = 3 and are normalized to relative maxima of each curve (see asterisks). (C) Optimum temperatures. HPLC assays were set for 1 h using 0.25 mM NAD and 11 μg/mL hSARM1 or 0.5 mM NAD and 0.1 μg/mL CD38 or 2.5 mM NAD and 0.14 μg/mL Aplysia cyclase. Data are Mean ± SEM from n = 3 and are normalized as in (B). (D and E) Preferred substrates (D) and effects of metal ions (E). Various dinucleotides (250 μM each) or metal ions (1 mM each) were assayed at 25°C by HPLC for 2–6 h using 5.5 μg/mL hSARM1 or 0.1 μg/mL CD38 or 0.028 μg/mL Aplysia cyclase. Data are Mean ± SEM from n ≥ 2. Dinucleotide analogs indicated are: NGD, nicotinamide guanine dinucleotide; NHD, nicotinamide hypoxanthine dinucleotide; AcPyAD, 3-acetylpyridine adenine dinuclotide; NaAD, nicotinic acid adenine dinuclotide; NaADP, NaAD phosphate; αNAD, alpha-NAD; ϵNAD, nicotinamide 1,N 6 -etheno adenine dinucleotide; VAD, vacor adenine dinucleotide; NADH or NADPH, reduced NAD or NADP. See also .

Article Snippet: NaAD (nicotinic acid adenine dinucleotide) , Merck , Cat#N4256.

Techniques:

Journal: iScience

Article Title: SARM1 is a multi-functional NAD(P)ase with prominent base exchange activity, all regulated bymultiple physiologically relevant NAD metabolites

doi: 10.1016/j.isci.2022.103812

Figure Lengend Snippet: Base exchange reactions typically catalyzed by SARM1 and other multifunctional NAD(P)ases at neutral pH (A and B) Rates were measured in 50 mM HEPES/NaOH pH 7.5 at 25°C by HPLC using 0.05 μg/mL of CD38 or 0.1 μg/mL of Aplysia cyclase or 0.5 μg/mL of SARM1 SAM-TIR (upper panel A) or 7 μg/mL of SARM1 full length (bottom panel B). Both NAD and NADP substrates were fixed at 250 μM. The free bases 3-acetylpyridine (AcPyr) and nicotinic acid (Na) were added at 2 mM final. Vacor was added at 0.5 mM given its low solubility at physiological pH. The assay in B (left panel) is also shown in Supplementary (see Figure S4 ) and was replicated in the presence of two known allosteric regulators of SARM1, NMN 0.2 mM and VMN 0.05 mM (B, middle and right panels), leading both to a maximum triggering effect on SARM1 activity at this concentration as reported ( Loreto et al., 2021 ). Multiple time stops from individual assays as above were analyzed for linearity, then extents of hydrolysis (white bars), cyclization (red bars), or base exchange (black bars) were calculated from each corresponding product as shown in Figure 1 B and . In detail, base exchanges led to form AcPyrAD from AcPyr, VAD from vacor, NaAD from Na in the presence of NAD or AcPyrADP from AcPyr, VADP from vacor, NaADP from Na in the presence of NADP (see Figure S4 ). Rates (Mean ± SEM, n = 2) are shown in histograms for comparison, referred to either NAD or NADP alone controls (arbitrarily fixed to 1). The whole dataset is also shown in . Asterisks (∗) indicate conditions where base exchange was below detection. See also , and

Article Snippet: NaAD (nicotinic acid adenine dinucleotide) , Merck , Cat#N4256.

Techniques: Solubility, Activity Assay, Concentration Assay

Journal: iScience

Article Title: SARM1 is a multi-functional NAD(P)ase with prominent base exchange activity, all regulated bymultiple physiologically relevant NAD metabolites

doi: 10.1016/j.isci.2022.103812

Figure Lengend Snippet:

Article Snippet: NaAD (nicotinic acid adenine dinucleotide) , Merck , Cat#N4256.

Techniques: Recombinant, Chromatography, FLAG-tag, Software