Membrane protein synthesis in mitogen-stimulated human T lymphocytes.
Proteins were biosynthetically labeled with [35S]-methionine in resting and mitogen-stimulated human peripheral blood lymphocytes. Lymphocytes were disrupted by sonication, the membrane fraction was isolated by differential centrifugation, and radiolabeled membrane proteins were identified after 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and autoradiography. The T cell mitogen concanavalin A (Con A) produced selective increases in the incorporation of [35S]-methionine into a membrane protein (or family of 4 structurally related membrane proteins) with an apparent m.w. of 28,000 and approximate isoelectric points (pI) of 5.5, 5.7, 6.0, and 6.2 (p28). Quantitative microdensitometry was used to evaluate p28 synthesis in a large number of different lymphocyte donors. Consistent and highly significant differences between resting and stimulated cells were demonstrable whether the 4 p28 spots were considered individually (p less than 0.0025) or in sum (p less than 0.0005). The p28 response to Con A was shown to be contained within the T lymphocyte fraction of mononuclear cells, was dose dependent over the mitogenic concentration range for Con A, and was blocked by alpha-methyl-D-mannoside, a sugar inhibitor of Con A binding. Differences between stimulated and resting cells were readily apparent within 2 hr of activation and became progressively larger with time. Protein p28 was prominently represented in plasma membranes isolated on discontinuous sucrose density gradients and was identified as a cell surface glycoprotein based on studies in which intact stimulated cells were exposed to degradative enzymes. This protein was lost from cells treated with pronase, whereas neuraminidase digestion changed the pI of p28 from a range of 5.5 to 6.2 to a range of 6.1 to 6.4 without changing the apparent m.w.
D002469:Cell Separation; D003208:Concanavalin A / Q000494:pharmacology; D004306:Dose-Response Relationship, Immunologic; D006023:Glycoproteins; D006801:Humans; D008213:Lymphocyte Activation; D008214:Lymphocytes / Q000378:metabolism; D008565:Membrane Proteins / Q000096:biosynthesis*; D008934:Mitogens / Q000494:pharmacology*; D010649:Phenylalanine / Q000378:metabolism; D012397:Rosette Formation; D013601:T-Lymphocytes / Q000276:immunology*; D013997:Time Factors
D006023:Glycoproteins; D008565:Membrane Proteins; D008934:Mitogens; D003208:Concanavalin A; D010649:Phenylalanine