Effect of the insulin degradation inhibitors bacitracin and chloroquine on insulin binding data to H35 rat hepatoma cells.

The influence of correction for internalization and of inhibition degradation by the use of bacitracin or chloroquine on the insulin binding data of H35 rat hepatoma cells has been assessed. Internalized insulin represented about 50% of total cell bound insulin at tracer concentration and is not affected by bacitracin or chloroquine. The use of the insulin degradation inhibitors bacitracin and chloroquine resulted in Scatchard plots that were strikingly less curvilinear than control cells (as indicated by a decreased K1/K2 ratio (p less than 0.001) and Ke/Kf ratio (p less than 0.01)). In the two site model there was a significant increase in the number of high affinity binding sites concomitant with a significant decrease in the high affinity association constant; there was also a significant decrease in the number of the low affinity binding sites. The total number of binding sites did not change. For the negative cooperativity model the use of each drug resulted in a significant increase in the affinity constant of the filled site. These results indicate that insulin degradation induces negative cooperativity and that the results are inconsistent with the two site model.