Identification of a sphingolipid‐specific phospholipase D activity associated with the generation of phytoceramide‐1‐phosphate in cabbage leaves

The structure and biosynthetic route for an unidentified lipid (lipid X) detected by TLC of cabbage ( Brassica oleracea ) lipids was determined. Lipid X is a phospholipid that is resistant to mild alkali and detectable by MALDI ‐ TOF MS as an adduct with Phos‐tag, a phosphate‐capture zinc complex. Various α‐hydroxy fatty acids (16:0, 22:0, 24:0 and 24:1) were detected by GC ‐ MS of fatty acid methyl esters prepared from lipid X. The deacyl derivative of lipid X was determined to be 4‐hydroxysphingenine (dehydrophytosphingosine)‐1‐phosphate by MALDI ‐ TOF MS with Phos‐tag. From these results, lipid X was determined to be phytoceramide‐1‐phosphate ( PC 1P) with an α‐hydroxy fatty acid. When cabbage homogenates were incubated, PC 1P was formed, with a concomitant decrease in the amount of glycosylinositol phosphoceramide ( GIPC ). The formation of PC 1P from GIPC was confirmed by treatment of purified cabbage GIPC with a membrane fraction of cabbage homogenates. Using a partially purified enzyme fraction, we found that the enzyme hydrolyzes GIPC specifically, but not glycerophospholipids and sphingomyelin. Arabidopsis thaliana also had this enzyme activity. From these results, we conclude that a previously uncharacterized phospholipase D activity that specifically hydrolyzes GIPC produces PC 1P in brassicaceous plants.