Evidence that somatostatin (SRIF14) is the primary coligand in pancreas required for specific binding of [3H]estradiol in pancreatic tissue: demonstration that [3H]estradiol and [125]SRIF14 form complexes of varying size with a specific binding protein

There is present in rat pancreas a protein that requires an accessory factor in order to bind [3H]estradiol. To identify this accessory factor 874g of dog pancreas were acid extracted, and following selective filtration and dialysis, the low molecular weight constituents (less than 10,000) were concentrated by lyophilization. Samples of this lyophilizate were fractionated by high performance liquid chromatography (HPLC) and eluate fractions analyzed for their capacity to enhance binding of [3H]estradiol to a protein fraction from rat pancreas that had been purified relatively free of endogenous accessory factor. Such enhancement of [3H]estradiol-binding activity eluted predominantly in one peak that coincided with the elution profile of pure somatostatin (SRIF14). Analysis of eluate fractions for somatostatin-like immunoreactive material (SLIM) indicated coincidence of SLIM with the factor that enhanced binding of [3H]estradiol. It appears likely that accessory factor in pancreas is primarily somatostatin (SRIF14). Following incubation of [125I]SRIF14 and [3H]estradiol with a partially purified binding-protein fraction from rat pancreas, a complex containing labeled [125I] and [3H] was separated by Sephadex G-200 column chromatography. In the presence of 25 microM SRIF14, which activates [3H]estradiol-binding maximally in the presence of 10 nM steroid, a protein peak containing both radiolabeled ligands eluted in the void volume indicating an apparent molecular size in excess of 200,000 Daltons. At a concentration of 1 microM SRIF14, a complex eluted at a position corresponding to an apparent Mr of 120,000. Evidently, the steroid and polypeptide mutually enhance binding to this pancreatic protein, and depending on their concentrations form structures of widely varying sizes.