Solubilization and immunopurification of a somatostatin receptor from the human gastric tumoral cell line HGT-1.
The human gastric tumoral cell line HGT-1 was previously shown to contain a membrane somatostatin receptor negatively coupled to adenylate cyclase through a pertussis toxin-sensitive inhibitory GTP-binding regulatory protein (Gi) (Reyl-Desmars, F., Laboisse, C., and Lewin, M. J. M. (1986) Regul. Pept. 16, 207-215). In this study, we have solubilized this receptor in a free unoccupied form using Triton X-100 as detergent and [125I-Tyr11]somatostatin-14 to monitor specific binding. Furthermore, we have prepared a monoclonal antibody against a chromatographically enriched soluble receptor fraction and used this antibody (30F3) to immunopurify the receptor in conjunction with Sepharose-somatostatin-14 immunopurification and steric exclusion high pressure liquid chromatography (HPLC). The purified fraction showed 18,600-fold enrichment in terms of specific binding (i.e. from 0.6 +/- 0.05 to 11,300 +/- 830 pmol/mg of protein) and a single dissociation constant (kappa D) of 76 +/- 8 nM. On HPLC, it migrated as a single and symmetric 90-kDa peak. Moreover, after 125I-protein labeling, it gave a single 90-kDa band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography. On the other hand, the 30F3 monoclonal antibody immunoblotted with a single 90-kDa band contained in the HGT-1 cell membrane. We therefore suggest that this antibody is specific to the HGT-1 membrane somatostatin receptor, that this receptor has a molecular mass of 90 kDa, and that we have obtained a homogeneous preparation of nondenatured receptor suitable for further cloning studies.
Reyl-Desmars
F
F
Unité de Recherches de Gastroenterologie, Institut National de la Santé et de la Recherche Medicalé Hôpital, Bichat, Paris, France.
Le Roux
S
S
Linard
C
C
Benkouka
F
F
Lewin
M J
MJ
D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D002462:Cell Membrane / Q000032:analysis / Q000378:metabolism; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D007118:Immunoassay; D015151:Immunoblotting; D008970:Molecular Weight; D017830:Octoxynol; D011092:Polyethylene Glycols; D017981:Receptors, Neurotransmitter / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D017481:Receptors, Somatostatin; D012995:Solubility; D013004:Somatostatin / Q000378:metabolism / Q000031:analogs & derivatives; D013274:Stomach Neoplasms / Q000032:analysis*; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D017981:Receptors, Neurotransmitter; D017481:Receptors, Somatostatin; D011092:Polyethylene Glycols; D013004:Somatostatin; C032367:somatostatin, Tyr(11)-; D017830:Octoxynol