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Endocrinology, Vol 124, 831-837, Copyright © 1989 by Endocrine Society

ARTICLES

Covalent labeling of the somatostatin receptor in rat anterior pituitary membranes

JF Bruno and M Berelowitz
Department of Medicine, State University of New York, Stony Brook 11794.

The molecular characteristics of the somatostatin (SRIF) receptor were investigated by covalently cross-linking [125I-Tyr11]SRIF to rat anterior pituitary membranes using three heterobifunctional cross- linking agents, N-5-azido-2-nitrobenzoyloxysuccinimide, N- hydroxysuccinimidyl-4-azidobenzoate, and N-succinimidyl-6-(4'-azido-2'- nitrophenylamino) hexanoate, and the homobifunctional agent disuccinimidyl suberate. Sodium dodecyl sulfate-gel electrophoresis followed by autoradiography revealed two SRIF-binding proteins with apparent mol wt (Mr) of 69,000 and 66,000 that were selectively labeled by the four cross-linking agents. When cross-linking was performed with N-5-azido-2-nitrobenzoyloxysuccinimide, both proteins migrated as a broad band centered at 68,000; however, with N-hydroxysuccinimidyl-4- azidobenzoate, the band was resolved into 69,000 and 66,000 Mr components. N-Succinimidyl-6-(4'-azido-2'-nitrophenylamino) hexanoate covalently labeled the 69,000 Mr protein and a minor species with a Mr of 45,000-47,000. Cross-linking with disuccinimidyl suberate labeled only the 66,000 Mr band. Labeling of both bands was specific, since affinity labeling with each of the four agents was abolished when 1 microM cyclic SRIF was included in the binding reaction. Binding of [125I-Tyr11]SRIF to membranes and labeling of the 69,000 and 66,000 Mr SRIF-binding species were similarly inhibited in a dose-dependent manner by unlabeled SRIF. Radiolabeling of both proteins was specifically displaced by 1 microM SRIF-28 and [D-Trp8,D-Cys14]SRIF, but not by oxytocin. Moreover, the extent of radiolabel incorporation into both components was dependent on the concentration of [125I- Tyr11]SRIF in the binding reaction. These results demonstrate the presence of two SRIF-binding proteins in rat anterior pituitary membranes that show characteristics of the SRIF receptor.




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Copyright © 1989 by The Endocrine Society