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Identification of a sphingolipid‐specific phospholipase D activity associated with the generation of phytoceramide‐1‐phosphate in cabbage leaves
Author(s) -
Tanaka Tamotsu,
Kida Takashi,
Imai Hiroyuki,
Morishige Junichi,
Yamashita Ryouhei,
Matsuoka Hisatsugu,
Uozumi Sachika,
Satouchi Kiyoshi,
Nagano Minoru,
Tokumura Akira
Publication year - 2013
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.12374
Subject(s) - sphingomyelin , biochemistry , chemistry , phospholipid , glycerophospholipids , enzyme , brassica , phospholipase , sphingolipid , phospholipase d , phosphate , fatty acid , biology , botany , membrane
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.