Insulin binding sites on rat liver nuclear membranes: biochemical and immunofluorescent studies

Preliminary investigations (Horvat et al., '75) indicated the nucleus of rat liver as a site for specific binding of insulin. In this report these observations are confirmed. Nuclei from rat liver were isolated in a highly purified state as verified by interference contrast and electron microscopy and by chemical analysis. Extensive scanning of the preparations did not reveal the presence of structures resembling plasma membranes. The nuclear envelope was isolated by a modification of the method of Kay et al. ('72). Electron micrographs showed the presence of nuclear "ghosts" and few other recognizable nuclear elements, but no plasma membranes (60--80 A thick) were detected. The preparation was found to contain specific insulin binding activity. Specificity of the binding sites for insulin was demonstrated in competition studies with other polypeptide hormones and a synthetic insulin analog. Scatchard analysis of the binding data indicates the presence of a single class of high affinity receptors. In contrast to findings with plasma membranes the hormone-receptor complex is very stable and the kinetics of the dissociation of bound [125I]-insulin do not indicate negative cooperativity of the binding sites. Immunofluorescent labeling of intact, unfixed nuclei showed a specific fluorescent halo only around those nuclei that have been preincubated with insulin. All other controls were negative.