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Inhibition of yeast microsome SPT activity by myriocin. Two hundred micrograms of yeast microsomes (∼0.0125 μM SPT) were incubated with 0.95 mM L-Ser (3,3-D2) in the presence of 0–128 nM myriocin for 5 min before stopping the reaction with NH4OH followed by lipid extraction and HPLC-ESI-MS/MS to quantify the 3KDS (+2D) products. Enzyme activity, which was measured by SPT velocity, was plotted against myriocin concentration using the <t>Morrison</t> equation function from GraphPad Prism 5. The estimated Ki for myriocin is 10.3 ± 3.2 nM.
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Inhibition of yeast microsome SPT activity by myriocin. Two hundred micrograms of yeast microsomes (∼0.0125 μM SPT) were incubated with 0.95 mM L-Ser (3,3-D2) in the presence of 0–128 nM myriocin for 5 min before stopping the reaction with NH4OH followed by lipid extraction and HPLC-ESI-MS/MS to quantify the 3KDS (+2D) products. Enzyme activity, which was measured by SPT velocity, was plotted against myriocin concentration using the Morrison equation function from GraphPad Prism 5. The estimated Ki for myriocin is 10.3 ± 3.2 nM.

Journal: Journal of Lipid Research

Article Title: Quantification of 3-ketodihydrosphingosine using HPLC-ESI-MS/MS to study SPT activity in yeast Saccharomyces cerevisiae [S]

doi: 10.1194/jlr.D078535

Figure Lengend Snippet: Inhibition of yeast microsome SPT activity by myriocin. Two hundred micrograms of yeast microsomes (∼0.0125 μM SPT) were incubated with 0.95 mM L-Ser (3,3-D2) in the presence of 0–128 nM myriocin for 5 min before stopping the reaction with NH4OH followed by lipid extraction and HPLC-ESI-MS/MS to quantify the 3KDS (+2D) products. Enzyme activity, which was measured by SPT velocity, was plotted against myriocin concentration using the Morrison equation function from GraphPad Prism 5. The estimated Ki for myriocin is 10.3 ± 3.2 nM.

Article Snippet: Enzyme activity, which was measured by SPT velocity, was plotted against myriocin concentration using the Morrison equation function from GraphPad Prism 5.

Techniques: Inhibition, Activity Assay, Incubation, Extraction, Tandem Mass Spectroscopy, Concentration Assay