By incubating Tetrahymena mimbres cells with [3H]myristic acid, [3H]ethanolamine, [3H]inositol, and [3H]mannose, proteins having apparent molecular masses of 23 and 63 kDa were identified as the cells' principal glycosylphosphatidylinositol (GPI)-anchored proteins. These proteins accounted for as much as 2-5% of the whole cell proteins, with the higher levels being recovered from non-growing cells. The two proteins, gpi 23 and gpi 63, were purified to near homogeneity through Triton X-114/water partitioning followed by preparative SDS/PAGE. The lipid components of the GPI anchors were determined by chemical and enzymic hydrolysis. Both proteins were anchored by ceramides, with the principal long-chain base being C18 sphinganine containing an O-methyl group at the 3 position. O-Methylation was shown not to be an artifact of hydrolysis. When T. mimbres was cultured at 15 degrees C, the ceramide fatty acid component of the GPI anchors was principally palmitic acid (75% in gpi 23 and 76% in gpi 63). GPI anchors from 28 degrees C-grown cells contained mainly stearic acid (79% in gpi 23 and 70% in gpi 63). Temperature change had little effect on the long-chain-base composition. The direction of temperature-induced lipid change in the protein-bound anchors was the same as found in the inositolphosphorylceramide putative precursors of the protein anchors described in the accompanying paper [Hung, Ko and Thompson (1995) Biochem. J. 307, 107-113], but the detailed fatty acid compositions of the precursors and the protein-bound lipids were quite different. The precise metabolic regulation of anchor lipid chain length supports the concept that composition of the lipid anchor is important in the function and/or metabolism of the anchored protein.

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