Terbium, a fluorescent probe for insulin receptor binding. Evidence for a conformational change in the receptor protein due to insulin binding.
Terbium (Tb3+), an ion of the lanthanide series that has been used as a fluorescent probe for calcium (Ca2+) binding sites in proteins, binds to the proteins in both solubilized and purified human placental insulin receptor preparations. Tb3+ fluorescence was determined directly and the effect of insulin binding on Tb3+-enhanced fluorescence was studied without the need to separate bound and free ligands. Tb3+ behaved similarly to, but was more potent (50-fold) than, Ca2+ in increasing the insulin bound to its receptor. When insulin bound to its receptor, the Tb3+ fluorescence of the receptor preparation decreased. When various insulin analogues were tested, the decrease in Tb3+ fluorescence was proportional to the biologic activity of the insulin analogues. In addition, Tb3+ could be displaced from insulin-sensitive sites by Ca2+, indicating that there were Ca2+ (and Tb3+) binding sites on or near the insulin receptor. These sites, when filled, were responsible for the increased insulin receptor affinity. The decrease in Tb3+ fluorescence after insulin binding may be indicative of a conformational change in the insulin receptor precipitated by the binding of insulin. This conformational change may be related to the release of Ca2+ by insulin binding, is associated with a decrease in insulin receptor affinity, and suggests that an allosteric mechanism involving both Ca2+ and insulin binding sites may be responsible for the observed changes in insulin receptor affinity.
D001667:Binding, Competitive; D002850:Chromatography, Gel; D005456:Fluorescent Dyes; D006801:Humans; D066298:In Vitro Techniques; D007328:Insulin / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D008566:Membranes / Q000378:metabolism; D017830:Octoxynol; D011092:Polyethylene Glycols; D011972:Receptor, Insulin / Q000378:metabolism*; D013725:Terbium
D005456:Fluorescent Dyes; D007328:Insulin; D008565:Membrane Proteins; D013725:Terbium; D011092:Polyethylene Glycols; D017830:Octoxynol; D011972:Receptor, Insulin