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.