Effect of pH on binding kinetics and biological effect of insulin in rat adipocytes.

The pH dependence of binding of [TyrA14-125I]monoiodoinsulin to isolated rat adipocytes was investigated at 37 degrees C. Although the amount of tracer bound at steady state at pH 7.8 was 6 times that at pH 6.8, the fractional degradation of receptor-bound iodoinsulin remained the same. The decreased binding at acidic pH could be accounted for by a decrease in the affinity. The pH-induced change in affinity was similar for iodoinsulin and insulin. The half-time of dissociation was not changed in the pH range 7.0 to 7.8. The presence of 25 nM insulin in the wash out medium caused a small increase in the dissociation of tracer independent of pH. Kinetic experiments revealed that the decreased affinity at acidic pH was mainly due to a decrease in the association rate constant. The decreased binding at acidic pH was reflected in a shift to the right of the insulin dose-response curve for initial rate of uptake of 3-O-[methyl-14C]glucose and the rate of conversion of glucose to lipids. It is concluded that the degradation of bound iodoinsulin is independent of pH (6.8 to 8.8), that the decreased binding at acidic pH is due to a decreased association rate constant, and that cells in acidic milieu are relatively insulin-resistant.