Solubilization and characterization of guinea‐pig pancreatic somatostatin receptors

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Abstract
The solubilization of somatostatin receptors from guinea‐pig pancreas by different non‐denaturing detergents was investigated after stabilization of the receptors by prior binding of 125I‐[Tyr11]somatostatin or its analogue 125I‐[Leu8, DTrp22, Tyr25]somatostatin 28, to pancreatic plasma membranes. The somatostatin‐receptor complexes were solubilized in a high yield by Zwittergent 3–14 (3‐[tetradecyldimethylammonio]‐1‐propanesulfonate), a zwitterionic detergent. Other detergents, digitonin, Triton X‐100, Chaps (3‐[cholamidopropyldimethylammonio]‐1‐propanesulfonate) and octyl β‐D‐glycopyranoside, achieved only partial solubilization. The recovery of receptor complexes was increased by glycerol. In order to characterize solubilized somatostatin‐receptor comples, membranes receptors were covalently labelled using N‐5‐azido‐2‐nitrobenzoyloxysuccinimide as cross‐linking reagent before solubilization. Gel filtration chromatography analysis resulted in the identification of a major protein component of apparent Mr= 93000 which interacted with the two radioligands. In addition, a similar component of Mr= 88000 was characterized after analysis by SDS‐PAGE of membrane receptors covalently cross‐linked with 125I‐[Leu8, DTrp22, Tyr25]somatostatin 28 by different heterobifunctional reagents: N‐5‐azido‐2‐nitrobenzoyloxysuccinimide, N‐hydroxysuccinimidyl 4‐azidobenzoate, N‐succinimidyl 6‐(4′‐azido‐2′‐nitrophenylamino)hexanoate. Optimal cross‐linking results were obtained with N‐5‐azido‐2‐nitrobenzoloxysuccinimide. The solubilized somatostatin‐receptor complex was adsorbed to wheat‐germ agglutinin‐agarose column and eluted by specific sugars. We concluded that the guinea‐pig pancreatic somatostatin receptor in the membrane and in the non‐denaturing detergent solution behaves as a protein monomer of apparent Mr∼ 85000–90000. The somatostatin receptor is a glycoprotein which contains complex‐type carbohydrate chains.