The nuclear envelope. The major site of insulin binding in rat liver nuclei.

Prior studies have demonstrated the presence of specific binding sites for insulin on nuclei isolated from rat liver. In the present study to determine the subnuclear location of these insulin binding sites, intact nuclei were first incubated with ‘ZzI-insulin and then subfractionated. Approximately 70% of specific insulin binding was associated with the nuclear membrane fractions. When highly purified nuclear membranes were prepared and incubated directly with InsIinsulin, specific binding sites were demonstrable. When compared on the basis of protein content, nuclear membranes specifically bound 10 to 15 times more labeled insulin than did intact nuclei. The binding of insulin to nuclear membranes was time-dependent and saturable; Scatchard plots revealed a high affinity binding site with a K, of 6.1 + 0.1 nM and a lower affinity binding site with a K, of 65.6 f. 11.8 nM. As has been reported for insulin binding sites on plasma membranes, proinsulin and desoctapeptide insulin competed for insulin binding sites on nuclear membranes in proportion to their reduced biological activities; unrelated hormones were without effect. In contrast to insulin binding sites on plasma membranes, insulin binding sites on nuclear membranes displayed neither negative cooperativity nor an enhancement of binding activity when incubated with 2 M NaCl. The pH optimum for binding of insulin to nuclear membranes was a plateau between 6.5 and 7.25; in comparison, the pH optimum for binding to plasma membranes was a sharp peak at 8.0. These studies indicate that (a) the nuclear envelope is the major site of insulin binding to the cell nucleus; (b) the binding sites for insulin on the nuclear envelope have high affinities and are hormone specific; and (c) these binding sites have characteristics differing from those on the plasma membrane.