A mass spectrometry-based method for the assay of ceramide synthase substrate specificity.
The acyl composition of sphingolipids is determined by the specificity of the enzyme ceramide synthase (EC 2.3.1.24). Ceramide contains a long-chain base (LCB) linked to a variety of fatty acids to produce a lipid class with potentially hundreds of structural variants. An optimized procedure for the assay of ceramide synthase in yeast microsomes is reported that uses mass spectrometry to detect any possible LCB and fatty acid combination synthesized from unlabeled substrates provided in the reaction. The assay requires the delivery of substrates with bovine serum albumin for maximum activity within defined limits of substrate concentration and specific methods to stop the reaction and extract the lipid that avoid the non-enzymatic synthesis of ceramide. The activity of ceramide synthase in yeast microsomes is demonstrated with the four natural LCBs found in yeast along with six saturated and two unsaturated fatty acyl-coenzyme As from 16 to 26 carbons in length. The procedure allows for the determination of substrate specificity and kinetic parameters toward natural substrates for ceramide synthase from potentially any organism.
Department of Biochemistry and Center for Plant Science Innovation, University of Nebraska-Lincoln, Lincoln, NE 68588, USA.
Kyle D
KD
Luttgeharm
Department of Biochemistry and Center for Plant Science Innovation, University of Nebraska-Lincoln, Lincoln, NE 68588, USA.
Edgar B
EB
Cahoon
Department of Biochemistry and Center for Plant Science Innovation, University of Nebraska-Lincoln, Lincoln, NE 68588, USA. Electronic address: jemarkham@unl.edu.
Jennifer E
JE
Markham
D002518:Ceramides / Q000032:analysis / Q000378:metabolism; D057075:Enzyme Assays / Q000379:methods*; D005227:Fatty Acids / Q000032:analysis / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D010088:Oxidoreductases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000378:metabolism; D013107:Sphingolipids / Q000032:analysis / Q000378:metabolism*; D013379:Substrate Specificity
D002518:Ceramides; D005227:Fatty Acids; D013107:Sphingolipids; D010088:Oxidoreductases; C108138:dihydroceramide desaturase
Ceramide synthase; Enzyme assay; Mass spectrometry; Sphingolipids; Substrate specificity