Solubilization and partial purification of somatostatin-28 preferring receptors from hamster pancreatic beta cells.
Somatostatin-28 (SRIF-28) preferring receptors were solubilized from hamster beta cell insulinoma using the zwitterionic detergent 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate. The binding of the iodinated [Leu8-D-TRP22-Tyr25]SRIF-28 analog (referred to as 125I[LWY] SRIF-28) to the solubilized fraction was time-dependent, saturable, and reversible. Scatchard analysis of equilibrium binding data indicated that the solubilized extract contained two classes of SRIF-28-binding sites: a high affinity site (Kd = 0.3 nM and Bmax = 1 pmol/mg protein) and a low affinity site (Kd = 13 nM and Bmax = 4.7 pmol/mg protein). The binding of 125I[LWY]SRIF-28 to solubilized SRIF-28 receptors was sensitive to the GTP analog guanosine-5'-O-thiotriphosphate, suggesting that receptors are functionally linked to a G-protein. By anion-exchange chromatography of the solubilized extract followed by chromatography on wheat germ agglutinin, a 46-fold purification of SRIF-28 receptors was obtained. At this stage of purification, only high affinity sites were found (Kd = 1 nM) and the GTP effect was not maintained. A specific protein of 37 kDa was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after photoaffinity labeling. We suggest that this protein is the putative SRIF-28 receptor or a subunit thereof.
Maletti
M
M
Institut National de la Santé et de la Recherche Médicale U. 55, Hôpital Saint-Antoine, Paris, France.
Andersson
M
M
Marie
J C
JC
Rosselin
G
G
Mutt
V
V
D000818:Animals; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D007515:Islets of Langerhans / Q000378:metabolism*; D007700:Kinetics; D008647:Mesocricetus; D008970:Molecular Weight; D010190:Pancreatic Neoplasms / Q000378:metabolism*; D011498:Protein Precursors / Q000378:metabolism; D017981:Receptors, Neurotransmitter / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D017481:Receptors, Somatostatin; D012995:Solubility; D013004:Somatostatin / Q000378:metabolism*; D053561:Somatostatin-28; D013379:Substrate Specificity
D011498:Protein Precursors; D017981:Receptors, Neurotransmitter; D017481:Receptors, Somatostatin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D013004:Somatostatin; D053561:Somatostatin-28