Solubilization of active somatostatin receptors from rabbit retina.
Somatostatin receptors from rabbit retinal membranes were solubilized in an active form using a mixture of the detergent n-octyl b-D-glucopyranoside (OG) and CHAPS. The binding of [125I]-Try11-somatostatin to the soluble extract was saturable and of high affinity, with an apparent affinity constant (Kd) of 0.60 +/- 0.20 nM and a maximum number of binding sites (Bmax) of 80 +/- 48 fmol/mg protein. The specific binding of [125I]Tyr11-somatostatin was inhibited in a dose-dependent manner only by the somatostatinergic analogs. The biochemical characteristics of both the membrane-bound and soluble receptors were studied by photoaffinity labeling techniques. Analysis by SDS-PAGE and subsequent autoradiography revealed the presence of a major protein of similar relative molecular mass (M(r) 54,000 and 57,000 for membrane and soluble sites, respectively). The photolabeling of this protein was specifically inhibited by somatostatin-28, somatostatin-14, SMS 201-995 (a synthetic octapeptide analog of somatostatin) but not by bombesin and somatostatin-28(1-14). The non-hydrolysable GTP analog guanosine-5'-O-(3-thio-triphosphate) (GTP gamma S) regulated the photolabeling of [125I]Tyr11-somatostatin to the membrane and soluble receptors. These studies describe for the first time the successful solubilization of the somatostatin receptor and the biochemical characterization of both membrane-bound and soluble receptors from rabbit retina.
Department of Basic Sciences, School of Health Sciences, University of Crete, Stavrakia, Heraklion, Greece.
G
G
Liapakis
E
E
Politou
K
K
Thermos
D000818:Animals; D001665:Binding Sites; D002850:Chromatography, Gel; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism*; D011817:Rabbits; D017481:Receptors, Somatostatin / Q000378:metabolism*; D012160:Retina / Q000378:metabolism*; D012995:Solubility; D014020:Tissue Extracts / Q000378:metabolism
D003902:Detergents; D008565:Membrane Proteins; D017481:Receptors, Somatostatin; D014020:Tissue Extracts; D016244:Guanosine 5'-O-(3-Thiotriphosphate)