Immunoaffinity purification of membrane protein constituents of the IM-9 lymphoblast receptor for substance P.
Substance P (SP) is an undecapeptide neuromediator that stimulates human T-lymphocyte function by binding to stereospecific membrane receptors. Human IM-9 cultured B-lymphoblasts express approximately 20,000 receptors per cell for [125I]SP with a Kd of 0.3 nM. [125I]SP was specifically crosslinked by disuccinimidyl suberate to IM-9 cell membrane proteins of 78, 58, and 33 kDa. An indirect immunoaffinity purification procedure has now been developed based on immunoabsorption of detergent-solubilized [125I]SP-labeled IM-9 cell membrane proteins to anti-SP antibody that was bound to an epoxide ultraffinity high-performance liquid chromatography column, followed by elution in acidic 8 M urea. The 58- and 33-kDa SP-receptor complexes were purified to apparent homogeneity by immunoaffinity chromatography and identified by autoradiography and silver staining of sodium dodecyl sulfate-polyacryl-amide gels.
McGillis
J P
JP
Howard Hughes Medical Institute, San Francisco, California.
Organist
M L
ML
Payan
D G
DG
D000345:Affinity Labels; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006801:Humans; D008214:Lymphocytes / Q000032:analysis*; D008565:Membrane Proteins / Q000302:isolation & purification; D008970:Molecular Weight; D018040:Receptors, Neurokinin-1; D017981:Receptors, Neurotransmitter / Q000302:isolation & purification*; D012995:Solubility
D000345:Affinity Labels; D008565:Membrane Proteins; D018040:Receptors, Neurokinin-1; D017981:Receptors, Neurotransmitter