Effect of temperature on phospholipase C induced alterations in insulin binding.

Regulation of insulin-receptor affinity is at present poorly understood. In this study membrane structure was modified by exposing placental membranes to phospholipase C and the subsequent effect on insulin binding and dissociation was examined. As previously described, when insulin-receptor binding was performed at 4 degrees C, phospholipase C treated membranes showed an increase in the apparent number of insulin receptors. However, when binding was performed at 24 degrees C, the predominant effect of prior phospholipase C treatment was to increase the affinity of binding. At this temperature, phospholipase C pretreatment also increased membrane fluidity. Cytochalasin B, an agent which disrupts receptor aggregates, lowered average affinity at 24 degrees C. Phospholipase C treatment slowed the dissociation rate in 'infinite dilution' at 24 degrees C but the accelerating effect of 100 ng/ml insulin (negative cooperativity) was greatly enhanced. These studies suggest that in addition to its effect in unmasking receptor sites, increased membrane fluidity induced by phospholipase C may cause higher affinity binding by allowing greater receptor or receptor subunit aggregation. Receptor or subunit aggregation within a fluid membrane structure may determine in part the affinity of insulin-receptor binding and the kinetic phenomenon known as negative cooperativity.