Purification and characterization of the bombesin/gastrin-releasing peptide receptor from Swiss 3T3 cells.

摘要: The bombesin/gastrin-releasing peptide (GRP) receptor was solubilized from Swiss mouse 3T3 cell membranes in an active form and purified about 90,000-fold to near homogeneity by a combination of wheat germ agglutinin-agarose ligand affinity chromatography. displayed single diffuse band with Mr 75,000-100,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After treatment the N-glycanase, removing N-linked oligosaccharide moieties, protein yielded = 38,000 band. These results agree value estimated for GRP that labeled cells cross-linking 125I-GRP1-27. GRP1-27 bound Kd 0.038 +/- 0.019 nM. By comparison, soluble unfractionated extracts intact 0.036 0.003 nM 0.13 0.04 nM, respectively. relative potencies series analogs indicated extraction procedure did not significantly alter receptor's binding specificity. However coupling its guanyl nucleotide regulatory maintained extract, G-protein co-purify receptor. Physiological concentrations NaCl greatly inhibited some receptor, while other affected. A domain molecule involving Lys-13 or Arg-17 identified which promoted under conditions low ionic strength. findings aided development effective resin purification