Interaction of ACTH synthetic fragments with rat adrenal cortex membranes

Abstract

Synthetic peptide, corresponding to the amino acid sequence 11-24 of human adrenocorticotropic hormone (ACTH), was labeled with tritium (specific activity of 22 Ci/mmol). [(3)H]ACTH (11-24) was found to bind to rat adrenal cortex membranes with high affinity and specificity (K(d) = 1.8 +/- 0.1 nM). Twenty nine fragments of ACTH (11-24) have been synthesized and their ability to inhibit the specific binding of [(3)H]ACTH (11-24) to adrenocortical membranes has been investigated. Unlabeled fragment ACTH 15-18 (KKRR) was found to replace in a concentration-dependent manner [(3)H]ACTH (11-24) in the receptor-ligand complex (K(i) = 2.3 +/- 0.2 nM). ACTH (15-18) was labeled with tritium (specific activity of 20 Ci/mmol). [(3)H]ACTH (15-18) was found to bind to rat adrenal cortex membranes with high affinity (K(d) = 2.1 +/- 0.1 nM). The specific binding of [(3)H]ACTH (15-18) was inhibited by unlabeled ACTH (11-24) (K(i) = 2.2 +/- 0.1 nM). ACTH (15-18) at the concentration range of 1-1000 nM did not affect the adenylate cyclase activity in adrenocortical membranes.

From Bioinactive ACTH to ACTH Antagonist: The Clinical Perspective

“…First, the expression at the cell membrane of human MC2R in cell lines was not possible until the discovery of the melanocortin receptor-associated protein (MRAP) ( 46 ), which is required to address MC2R at the cell membrane. Second, studies on putative ACTH antagonists failed to perform a systematic analysis of ACTH antagonists’ activation and binding against all cognate MCRs to assess specificity ( 83 , 84 ).…”

New Development in the Solid-State Isotope Exchange with Spillover Hydrogen in Organic Compounds

“…They are employed for the screening and analysis of the molecular mechanisms underlying the action of the prospective medical drugs. 17,18 The HSCIE reaction makes it possible to produce evenly tritium-labeled peptides for use both in receptor binding analysis and for the determination of their peptidase biodegradation paths in vivo and in vitro. 19−21 Using this reaction between an organic compound applied onto an inorganic carrier and SH activated on the catalyst, it is possible to substitute hydrogen for tritium almost quantitatively in organic compounds and at the same time completely retain their physiological activity.…”

“…We analyzed the role of the structure of peptide chains and of their structural interactions on the hydrogen spillover reaction in peptides and proteins. , Using the HSCIE reaction, highly tritium- and deuterium-labeled ligands have been obtained for the subtypes of glutamate, serotonin, dopamine, nicotine, and melatonin brain receptors. They are employed for the screening and analysis of the molecular mechanisms underlying the action of the prospective medical drugs. , …”