Hormone binding site of the insulin receptor: analysis using photoaffinity-mediated avidin complexing.
Biol Chem Hoppe Seyler
; 370(3): 251-8, 1989 Mar.
Article
em En
| MEDLINE
| ID: mdl-2540769
ABSTRACT
A trifunctional reagent was designed which allows derivatization of ligands, particularly peptides and proteins, for subsequent photoaffinity labelling of receptors and specific isolation of the covalent complex or its fragments. B29-(2-nitro-4-azidophenyl)-biocytinyl-insulin (NB-insulin) was synthesized, radioiodinated, and the B26-mono-iodo derivative isolated by HPLC. It was used to photoaffinity label human placental membranes and the purified insulin receptor. Extensive digestion of the covalent insulin-receptor complex with trypsin (EC 3.4.21.4) led to the generation of a fragment of Mr 14,000. Specific complexing with avidin, derivatized avidin or streptavidin could be demonstrated for the photoaffinity labelled alpha-subunit and the 14,000 core fragment. The latter was isolated (approx. 100 pmol from 3-4 placentae) by streptavidin affinity chromatography and HPLC. According to microsequencing based on the known primary structure of the insulin receptor, the N-terminus of the core peptide appears to be Leu20-His21-Glu22-Leu23. We thus conclude a part of the insulin-binding region of the receptor is located close to the N-terminus of its alpha-subunit in a remarkably stable domain of the sequence 20--(approx.) 120.
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Base de dados:
MEDLINE
Assunto principal:
Receptor de Insulina
/
Avidina
/
Receptores de Superfície Celular
Limite:
Animals
Idioma:
En
Revista:
Biol Chem Hoppe Seyler
Assunto da revista:
BIOQUIMICA
Ano de publicação:
1989
Tipo de documento:
Article