Affinity chromatography purification and partial characterization of phytohemagglutinin-receptor glycoproteins from porcine splenic lymphocytes.
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)
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
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