Journal: The Journal of Biological Chemistry
Article Title: Multiple activities of sphingomyelin synthase 2 generate saturated fatty acid– and/or monounsaturated fatty acid–containing diacylglycerol
doi: 10.1016/j.jbc.2024.107960
Figure Lengend Snippet: Effects of D609, a PC-PLC/SMS inhibitor, on PC-PLC, PE-PLC, SMS, and CPES activities of SMS2. A , DG-generating activities of SMS2, which was purified in the presence of detergents, were analyzed in the presence of various concentrations of D609 (0, 0.25, 0.5, and 1 mM). Purified WT SMS2-TS (approximately 500 ng) was incubated in the presence of 16:0/18:1-PC alone, 16:0/18:1-PC and d18:1/18:0-Cer, 16:0/18:1-PE alone, or 16:0/18:1-PE and d18:1/18:0-Cer–containing detergent mixed micelles for 2 h at 37 °C. Produced 16:0/18:1-DG was quantified using LC–MS/MS. Values are presented as percentages of the activity of SMS2 in the absence of D609 (set at 100%). Values are presented as the mean ± SD (n = 6, technical replicates). ∗∗∗, p < 0.005 ( versus control (without D609)). One-way ANOVA with Dunnett’s post hoc test was used. B , SM-producing activities of SMS2, which was purified in the presence of detergents, in the presence of 16:0/18:1-PC and d18:1/18:0-Cer–containing detergent mixed micelles were analyzed in the presence of various concentrations of D609 (0, 0.25, 0.5, and 1 mM). Values are presented as percentages of the SMS activity of SMS2 in the absence of D609 (set to 100%). The values are presented as the mean ± SD (n = 4, technical replicates). ∗, p < 0.05; ∗∗, p < 0.01 ( versus control (without D609)). One-way ANOVA with Dunnett’s post hoc test was used. C , CPE-producing activities of SMS2, which was purified in the presence of detergents, in the presence of 16:0/18:1-PE and d18:1/18:0-Cer–containing detergent mixed micelles were analyzed in the presence of various concentrations of D609 (0, 0.25, 0.5, and 1 mM). Values are presented as percentages of the CPES activity of SMS2 in the absence of D609 (set at 100%). Values are presented as the mean ± SD (n = 6, technical replicates). ∗∗, p < 0.01; ∗∗∗, p < 0.005 ( versus control (without D609)). One-way ANOVA with Dunnett’s post hoc test was used. Cer, ceramide; CPE, ceramide phosphoethanolamine; CPES, CPE synthase; DG, diacylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PLC, phospholipase C; SM, sphingomyelin; SMS, sphingomyelin synthase; TS, Twin-Strep.
Article Snippet: Tricyclodecan-9-yl-xanthogenate (D609) was obtained from Chemscene.
Techniques: Purification, Incubation, Produced, Liquid Chromatography with Mass Spectroscopy, Activity Assay, Control