Extensive labeling with [3H]ethanolamine of a hydrophilic protein of animal cells.
Murine T-lymphomas and Thy-1- mutants were labeled overnight with [3H]ethanolamine to detect proteins which possess a glycophospholipid anchor. When labeled cells were treated with 10% trichloroacetic acid and then analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography, both Thy-1 and a second intensely labeled protein (46 kDa) were observed. The presence of the radiolabeled 46-kDa protein in wild type and class E Thy-1 negative cells (cells in which Thy-1 is synthesized but cannot be labeled with [3H]ethanolamine) suggested incorporation into a distinct moiety. Labeling of the 46-kDa protein with [3H]ethanolamine is rapidly inhibited by cycloheximide. Further characterization of the 46-kDa protein by subcellular fractionation and Triton X-114 partitioning indicated that the protein is located in the cytosol. The protein is basic and does not bind to either concanavalin A or wheat germ agglutinin. Labeling of a 46-kDa protein has also been demonstrated in Chinese hamster ovary, COS, rat myeloma, cloned human T-lymphocytes, and HeLa cells. Pronase digestion of the [3H]ethanolamine-labeled 46-kDa protein of wild type lymphoma cells generated a nonbasic and polar labeled fragment which is labile to strong acid and base ([3H]ethanolamine is liberated), insensitive to periodate oxidation and alkaline phosphatase, and does not bind to concanavalin A or wheat germ agglutinin. Judging from methylation studies, the labeled ethanolamine residue does not contain a free amino group. Based on these results, we report a novel post-translational modification of selected protein(s) by the covalent addition of [3H]ethanolamine.
Tisdale
E J
EJ
Institute of Pathology, Case Western Reserve University, Cleveland, Ohio 44106.
Tartakoff
A M
AM
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D019856:Ethanolamine; D004983:Ethanolamines / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D008970:Molecular Weight; D011402:Pronase / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000378:metabolism*
D000596:Amino Acids; D004983:Ethanolamines; D011506:Proteins; D019856:Ethanolamine; D011402:Pronase