1981 Volume 20 Pages 35-40
We reported previously that the maximal binding sites of insulin receptor on human erythrocyte are significantly increased by diabetes. In the present study, we investigated the possibility that insulin binding may be regulated through the alteration of membrane phosphorylation. The mechanism regulating interaction of insulin with receptors was evaluated by studying the binding of insulin to intact and ATP-phosphorylated membranes and the characteristics of the binding sites for cAMP and cGMP on membrane. Specific insulin binding to membranes reached a maximum after a 2-hr incubation at 0°. Binding sites with relatively high affmity (K1: 1.2×109M-1) could be distinguished from those of lower affinity (K2: 4.2×106M-1). The phosphorylation of membranes with 1 mM ATP plus 20μM cAMP caused a significant increase in the binding of insulin to lower capacity sites with high affinity (117%), while the affinity constants were unaffected by phosphorylation of membranes. GTP plus cAMP produced an increased effect on specific binding of insulin. The amount of phosphorylation with ATP was estimated as 1.2μmol/g protein/hr and that with GTP as 0.6μmol/g protein/hr. Binding sites for both cAMP and cGMP were composed of more than two types of receptors with different affinity constants. Secondly, we compared kinetic constants of cAMP binding between normal and diabetic subjects. The high affinity constant and the maximal capacity of high affinity binding sites were 4.85±0.66 (×107M-1) and 11.83±1.39 (pmol/mg protein)(n=33) in normal group and 3.50±037(×107M-1) and 17.84±1.64 (pmol/mg protein)(n=52) in diabetic group. These results have led us to a conclusion that human erythrocytes contain specific receptors for insulin, cAMP and cGMP, and suggested that the increase of insulin binding in diabetes may be associated with more highly phosphorylated membrane.
