Purification of the 33,000-dalton ligand binding-protein constituent of the lymphoblast substance P receptor.
Substance P (SP) acts as an immunoregulator by binding to specific functional cell surface receptors on a subpopulation of human T-helper lymphocytes. Receptors with similar properties have also been characterized on the human IM-9 B-lymphoblast cell line. Four distinct proteins of molecular weight (MW) 33,000, 58,000, 78,000, and 116,000 can be specifically affinity labeled using [125I]-SP Bolton-Hunter reagent and disuccinymidyl suberate (DSS). Peptide-mapping studies of these individually purified affinity labeled proteins have shown that the 33,000 MW membrane protein is present in the higher molecular weight cross-linked proteins. In the present studies, the 33,000 MW affinity-labeled protein was purified using semipreparative sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by high-performance hydroxylapatite chromatography. Starting with 800 mg of affinity-labeled protein, the final yield was 39.0 micrograms of 33,000 MW affinity-labeled protein. Based on an estimate of 53 mg receptor per 800 mg membrane protein, this represents an overall yield of greater than 70%. Peptide mapping was done by digesting 20 micrograms of the receptor protein with bovine trypsin. The proteolytic fragments were separated by reverse-phase high-performance liquid chromatography, and the amino acid content of 13 distinct peptides was determined. With this procedure, sufficient receptor can now be purified so that partial amino acid sequences can be obtained for further structural studies.
McGillis
J P
JP
Howard Hughes Medical Institute, University of California Medical Center, San Francisco 94143-0724.
Organist
M L
ML
Scriven
K H
KH
Payan
D G
DG
D000596:Amino Acids / Q000032:analysis; D002460:Cell Line; D006801:Humans; D008024:Ligands; D008214:Lymphocytes / Q000032:analysis*; D008565:Membrane Proteins / Q000302:isolation & purification*; D008970:Molecular Weight; D018040:Receptors, Neurokinin-1; D017981:Receptors, Neurotransmitter / Q000302:isolation & purification*
D000596:Amino Acids; D008024:Ligands; D008565:Membrane Proteins; D018040:Receptors, Neurokinin-1; D017981:Receptors, Neurotransmitter