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.