Abstract
The receptor for somatostatin present in rat pancreatic plasma membranes was characterized by affinity labeling with [125I-Tyr11]somatostatin utilizing three different heterobifunctional cross-linking agents: N-5-azido-2-nitrobenzoyloxy-succinimide, N-succinimidyl 6-(4-azido 2'-nitrophenylamine)hexanoate, and N-hydroxysuccinimidyl 4-azido-benzoate. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed a broad band of Mr = 92,000 when any of the three cross-linkers was used; N-succinimidyl 6-(4-azido 2'-nitrophenylamine), however, was most efficient. Labeling of the Mr = 92,000 protein band was not affected by reducing agents but was sensitive to somatostatin and guanine nucleotides, particularly GTP gamma S, at concentrations which reduced binding to the receptor. The affinity-labeled protein could be solubilized completely with Zwittergent 3-12, partially with Triton X-100 and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid, and poorly with Zwittergent 3-08 and digitonin. When exposed to agarose-coupled lectins, the detergent solubilized, labeled Mr = 92,000 protein was completely adsorbed to wheat germ agglutinin, partially to ricin communis II, and not at all to concanavalin A or lotus or lentil lectin. The Mr = 92,000 protein bound to wheat germ agglutinin-agarose was not eluted by N-acetylglucosamine but was by triacetylchitotriose, providing a considerable purification of the somatostatin receptor. These data allow us to conclude that the somatostatin receptor is a monomeric glycoprotein with an Mr = 90,000 binding subunit which probably contains a polymeric arrangement of N-acetylglucosamine residues.
Highlights
The receptor for somatostatin present in rat pan- action by which somatostatin alters cellular processes after creatic plasma membranes was characterized by af- binding toits receptors is stillpoorly understood
Labeling of the M, = 92,000 protein band was not affected by reducing agents but was sensitive to somatostatin and Recently we demonstrated, using different radiolabeled ligands, that pancreatic acinar cells possess a single class of high affinity cell surface somatostatin receptors [17,18,19]
SANAH, showed a similarbroad band centeredat M, = 92,000 when analyzed by electrophoresis and autoradiography (Fig. 1).Most efficient cross-linking was observed with ANB-NOS, which differs from SANAH by the shorterlength between the
Summary
After centrifugation at 1,000 X g for 10 min, the pellet was resuspended in the aforementioned buffer enriched with 1.35 M sucrose. The membrane protein content was measured following solubilization in 0.1 N NaOH by the method of Bradford [23] using bovine serum albumin as a standard. Binding of ['251-Tyr'']somatostatin to pan- obtained from Behring Diagnostics and Sigma, bifunctional crosscreatic membranes was carried out in mM Tris-HC1 buffer (pH 8) linking reagents from Pierce, acrylamide, bisacrylamide, SDS, and a containing 0.2 mM CaC12, 0.2mg/ml bacitracin, 0.5 mM benzamidine, standard mixture of proteins of known molecular weight from Bio-. Membranebound labeled somatostatin was separated by centrifugation a t 10,000 X g for 1 min in a microcentrifuge and washed twice with ice-cold
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
