Complex sphingolipid synthesis in plants: characterization of inositolphosphorylceramide synthase activity in bean microsomes.
Complex glycophosphosphingolipids present in plants are composed of ceramide, inositolphosphate, and diverse polar oligosaccharide substituents. The activity of inositolphosphorylceramide (IPC) synthase (phosphatidylinositol:ceramide inositolphosphate transferase), the enzyme proposed to catalyze the initial committed step in the formation of these complex sphingolipids, was characterized in wax bean hypocotyl microsomes. Enzyme activity was assayed by monitoring the incorporation of fluorescent NBD-C(6) ceramide or [3H]inositolphosphate from radiolabeled phosphatidylinositol (PI) into product identified by TLC. IPC synthase was found to utilize nonhydroxy fatty acid-containing ceramide, hydroxy fatty acid-containing ceramide, and NBD-C(6) ceramide as substrate. Maximum product formation was observed at PI concentrations in excess of 600 microM (with half-maximum activity at approximately 200 microM). Both endogenous PI and ceramide appeared to serve as substrates. Aureobasidin A and rustmicin, two potent inhibitors of fungal IPC synthase, inhibited enzyme activity in bean microsomes with values for IC(50) of 0.4-0.8 and 16-20 nM, respectively. IPC synthase activity appeared most closely associated with the Golgi based on results using selected marker enzymes. Enzyme activity was detected in a variety of plant tissues. This report, the first to characterize IPC synthase in plant tissues, demonstrates the similarities between the plant enzyme and its yeast counterpart, and provides insight into plant glycophosphosphingolipid biology.
Bromley
Pamela E
PE
Department of Biology, Williams College, Williamstown, MA 01267, USA.
Li
Yuneng O
YO
Murphy
Shawn M
SM
Sumner
Catherine M
CM
Lynch
Daniel V
DV
D002518:Ceramides / Q000378:metabolism*; D018548:Cotyledon / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation; D006028:Glycosphingolipids / Q000737:chemistry* / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism; D008861:Microsomes / Q000737:chemistry / Q000378:metabolism*; D027805:Phaseolus / Q000737:chemistry / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000378:metabolism*; D013107:Sphingolipids / Q000096:biosynthesis*
D002518:Ceramides; D006028:Glycosphingolipids; D010716:Phosphatidylinositols; D013107:Sphingolipids; C477937:inositolphosphorylceramide