The Isolation and Partial Characterization of Two Novel Sphingolipids from Neurospora crassa: Di(Inositolphosphoryl)ceramide and [(gal)_3glu]ceramide

Abstract

Neurospora crassa strains labeled uniformly with ^(32)P_i and [^3H]inositol exhibit at least six phospholipid components containing ^3H when separated by paper chromatography. One of the major components is phosphatidylinositol. Other components, which account for 40 to 60% of the lipid-extractable ^3H in various strains, are stable to mild alkaline methanolysis and appear to be sphingolipids with equivalent amounts of inositol and phosphorus. The major phosphosphingolipid was purified by means of differential solubility and by column chromatography on porous silica beads. This substance contains equivalent amounts of hydroxysphinganine and hydroxytetracosanoic acid and 2 eq each of myoinositol, phosphorus, and sodium. Alkaline degradation yielded 2 eq of inositol monophosphate and periodate degradation gave a C-15 fragment. The elemental composition of this compound also fits the formulation, (inositol-P)_2-ceramide. A [^3H]inositol pulse-chase experiment carried out with an inositol-requiring mutant in exponential growth shows labeled inositol accumulating in the sphingolipid accompanied by decreased labeling in phosphatidylinositol and the acid-soluble fraction. These changes also occur when the chase is carried out during inositol starvation suggesting that degradation of phosphatidylinositol and formation of sphingolipid occurs in the absence of growth. A neutral glycosphingolipid was also obtained as a by-product of the phospholipid purification. This substance is provisionally formulated as the ceramide tetrahexoside: [(gal)_3glu]-N-hydroxytetracosonyl-hydroxysphinganine.