Immunoaffinity purification of membrane protein constituents of the IM-9 lymphoblast receptor for substance P

Abstract

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.