Persistence of a curvilinear Scatchard plot for insulin binding despite correcting for degradation

We evaluated whether correcting for insulin degradation products (both cell-associated and those recovered in the media) in the analysis of insulin binding to rat hepatocytes linearized the Scatchard plot. In the presence of bacitracin, the average amount of intact insulin in the cell pellet increased from 68% to 90% and in the media from 21% to 69% when assayed by G-50 Sephadex separation. However, using only the amount of intact insulin in Scatchard analysis still resulted in a curvilinear form whether bacitracin was present or not. Persistence of curvilinear Scatchard plots is probably due to the fact that binding of insulin to hepatocytes in our system is over twice as high as in the cells used in the two studies which reported linearization of the Scatchard plot when these corrections were made. Because of the relatively low binding in those studies, corrections for insulin degradative products resulted in an amount of bound insulin that fell on the initial linear portion of the typical curvilinear Scatchard plot. Bacitracin significantly inhibited insulin degradation and correcting for the amount of insulin degradation in Scatchard analysis does not yield a linear plot.