RESUMO
Covalent linking of two photoactivatable insulin derivatives, B2-(2-nitro,4-azidophenylacetyl)-des-PheB1-insulin and B29-(2-nitro,4-azidophenylacetyl)-insulin to viable rat adipocytes gives a system, which contains a fixed stoichiometry between hormone and receptor. The biological signal of prolonged lipogenesis has been used to study several aspects of insulin binding and action: the role of the site of the crosslink between insulin and receptor, recognition of bound photoinsulin by anti-insulin antibodies, the half-life of the biologically active complex, the pH-dependence of the biological signal, and the possible role of internalization. Furthermore, the effect of trypsin on the insulin receptor, as well as the insulin-receptor complex, has been investigated and a refined model of the receptor is presented.
Assuntos
Marcadores de Afinidade/metabolismo , Insulina/metabolismo , Receptor de Insulina/metabolismo , Tecido Adiposo/metabolismo , Animais , Anticorpos , Azidas/metabolismo , Sítios de Ligação , Eletroforese em Gel de Poliacrilamida , Meia-Vida , Concentração de Íons de Hidrogênio , Técnicas Imunológicas , Insulina/análogos & derivados , Insulina/imunologia , Metabolismo dos Lipídeos , Fragmentos de Peptídeos/metabolismo , Fotoquímica , Ratos , Tripsina/metabolismoRESUMO
Isolated rat adipocytes were photo-affinity-labelled with B2-(4-azido-2-nitrophenylacetyl)-des-PheB1-insulin or B29-(4-azido-2-nitrophenylacetyl)insulin. Four anti-insulin antibodies (3 monoclonal, 1 polyclonal) were tested for their ability to inhibit the persistent stimulation of lipogenesis caused by the covalently bound insulin [Brandenburg et al. (1980) Nature (London) 286, 821-822]. The polyclonal and 2 monoclonal antibodies, directed against the C-terminus of the B-chain, gave a significant depression, while one antibody, directed against the region A(8-10), was without effect. Under reversible conditions, without irradiation, all antibodies completely inhibited lipogenesis. For the polyclonal antibody this is shown in a dose-dependent way. It is concluded that the effective antibodies can recognize their epitope because it is accessible on the surface of the complex and does not represent part of the receptor-binding surface of insulin. This binding leads to interference with the generation and/or transmittance of the biological signal.