Characterization of covalently cross-linked somatostatin receptors in hamster beta cell insulinoma.
The selective binding of somatostatin-28 (SS-28) to beta cells of hamster insulinoma was characterized using HPLC-purified 125I-[Leu8,D-Trp22,Tyr25]SS-28 or 125I-SS-28. A single class of high-affinity sites (Kd = 53 +/- 5 pM) was observed with a binding capacity of 2.85 pmol/mg membrane protein. A large number of relatively low-affinity sites was found also. The order of potency of different peptides to inhibit 125I-SS-28 binding is SS-28 greater than SS-14 greater than SMS-201-995 and the respective half-maximal inhibitory doses are 0.16 nM, 10 nM and 1000 nM. CCK8 and other active pancreatic peptides (glucagon, insulin, gastric inhibitory peptide, vasoactive intestinal peptide, oxyntomodulin) do not inhibit the SS-28 receptor binding. 125I-SS-28-labeled beta membranes were successfully cross-linked using either the cleavable cross-linker dithiobis(succinimidylpropionate) (1 mM) alone or with a heterobifunctional agent, N-hydroxysuccinimidyl-4-azidobenzoate (HSAB). In both cases five molecular components were revealed, after polyacrylamide gel electrophoresis of the membrane proteins and autoradiography, with the following molecular mass: 196-kDa, 132 kDa, 69 kDa, 45 kDa and 28 kDa. The labeling of 196-kDa, 132-kDa and 45-kDa species was specific in that they could be inhibited by unlabeled SS-28. The major labeled species corresponds to the 132-kDa band and no change in the mobility of this HSAB covalently bound SS-28 receptor was found after addition of dithiothreitol, suggesting that this specific receptor does not contain interchain disulphide bonds. The molecular mass of SS-28 receptors differs markedly from that of guinea-pig pancreatic acinar membranes, where a single 93-kDa protein is identified as a 125I-SS-28 receptor site in comparative experiments. Both the binding kinetics and structural differences sustain the selective action of SS-28 in the endocrine pancreas.
D007516:Adenoma, Islet Cell / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D006224:Cricetinae; D003432:Cross-Linking Reagents; D007340:Insulinoma / Q000378:metabolism*; D007515:Islets of Langerhans / Q000378:metabolism; D008647:Mesocricetus; D010190:Pancreatic Neoplasms / Q000378:metabolism*; D017981:Receptors, Neurotransmitter / Q000378:metabolism*; D017481:Receptors, Somatostatin; D013004:Somatostatin / Q000378:metabolism
D003432:Cross-Linking Reagents; D017981:Receptors, Neurotransmitter; D017481:Receptors, Somatostatin; D013004:Somatostatin