title: Affinity chromatography purification and partial characterization of phytohemagglutinin-receptor glycoproteins from porcine splenic lymphocytes.
abstract: We describe the isolation of pig spleen lymphocyte glycoproteins that interact with phytohemagglutinin (PHA), the lectin from Phaseolus vulgaris. Purification was achieved by affinity chromatography of a Nonidet P-40 extract of the cells on a PHA--Affi-Gel 10 column. The retained glycoproteins were eluted with an acidic (pH 3.0) glycine buffer and represented 1.9-2.4% of the amount of protein applied to the column. They contained 20 +/- 1.3% hexose and 1.7 +/- 0.7% fatty acids, on a weight basis. Electrophoretic analyses (sodium dodecyl sulfate--polyacrylamide gel electrophoresis) showed the presence of major Coomassie blue positive bands with apparent molecular masses of 50-55, 75, 95, 130, and 155 kdaltons along with minor bands of 20-40, 42, 45, 60-65, 175, and 200-250 kdaltons. The purified PHA-receptor glycoproteins inhibited, in a dose-dependent manner, the incorporation of [3H]thymidine in pig lymphocytes cultured at a concentration of 10(6) cells/mL in the presence of PHA. A 50% inhibition was observed when 20 micrograms/mL of the glycoproteins was added to the lymphocyte cultures containing 0.5 microgram/mL of PHA. Scatchard analysis of the binding of 125I-labelled PHA, in the presence of increasing amounts of the purified glycoproteins, showed a suppression of the binding of the lectin to high affinity sites of the cells, as evidenced by a change from biphasic to a linear profile. Results of binding suggested a competitive inhibition by a population of purified glycoproteins with a similar affinity for the lectin. The purified glycoproteins decreased PHA-dependent interleukin 2 (IL-2) production by pig lymphocytes as assayed with a IL-2 dependent murine cell line.(ABSTRACT TRUNCATED AT 250 WORDS)
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D007106:Immune Sera / Q000494:pharmacology; D007376:Interleukin-2 / Q000096:biosynthesis; D008213:Lymphocyte Activation; D008214:Lymphocytes / Q000032:analysis*; D010835:Phytohemagglutinins / Q000378:metabolism* / Q000494:pharmacology; D019038:Platelet Glycoprotein GPIb-IX Complex; D010980:Platelet Membrane Glycoproteins; D011971:Receptors, Immunologic / Q000276:immunology / Q000302:isolation & purification* / Q000502:physiology; D011975:Receptors, Mitogen / Q000302:isolation & purification*; D013154:Spleen / Q000166:cytology*; D013552:Swine
chemical_list: D007106:Immune Sera; D007376:Interleukin-2; D010835:Phytohemagglutinins; D019038:Platelet Glycoprotein GPIb-IX Complex; D010980:Platelet Membrane Glycoproteins; D011971:Receptors, Immunologic; D011975:Receptors, Mitogen; C032193:glycoprotein receptor GPIb-IX