Identification of glycoinositol phospholipids in rat liver by reductive radiomethylation of amines but not in H4IIE hepatoma cells or isolated hepatocytes by biosynthetic labeling with glucosamine

The identification of free glycoinositol phospholipids (GPIs) following biosynthetic labeling with [³H]glucosamine in cultured cells has been reported by several laboratories. We applied this procedure to two of the cell types used in these studies, H4IIE hepatoma cells and isolated hepatocytes, but were unable to detect a [³H]glucosamine-containing lipid that met any of the criteria for GPIs, including sensitivity to phosphatidylinositol-specific phospholipase C (PIPLC) or GPI-specific phospholipase D. Part of the difficulty in radiolabeling a GPI by this procedure was the rapid metabolic conversion of [³H]glucosamine to galactosamine and neutral or anionic derivatives. A PIPLC-sensitive radiolabeled lipid was detected only after 16 h of labeling. The water-soluble fragments released from this lipid by PIPLC corresponded largely to myo-inositol 1,2-cyclic phosphate and myo-inositol 1-phosphate, products expected from PIPLC cleavage of phosphatidylinositol or lyso-phosphatidylinositol. In an alternative approach that we introduce here, free GPIs in lipid extracts from rat liver plasma membranes were labeled by reductive radiomethylation. This procedure, which radiomethylates primary and secondary amines, has been shown to label a glucosamine residue adjacent to inositol in all GPIs characterized to date. The labeled extracts were fractionated by two-dimensional thin-layer chromatography, and a cluster of polar labeled lipids were assigned as GPIs based upon the following observations. 1) They were cleaved by PIPLC, 2) after hydrolysis in 6 N HCl, both radiomethylated glucosamine and a glucosamine-inositol conjugate were identified by cation exchange chromatography, and 3) hydrolysis in 4 M trifluoroacetic acid generated a fragment consistent with glucosamine-inositol-phosphate. These results illustrate new criteria for the identification of GPIs. The labeled GPIs also contained radiomethylated ethanolamine, another component found in GPI anchors of proteins and in mature lipid precursors of GPI anchors, suggesting that the liver plasma membrane GPIs retained considerable structural homology to GPI anchors.