Specific binding of proinsulin C-peptide to intact and to detergent-solubilized human skin fibroblasts

Proinsulin C-peptide exerts physiological effects on kidney and nerve function, but the mechanisms involved remain incompletely understood. Using fluorescence correlation spectroscopy, we have studied binding of rhodamine-labelled human C-peptide to intact human skin fibroblasts and to detergent-solubilised extracts of fibroblasts, K-562, and IEC-6 cells. Specificity was shown by displacement of rhodamine-labelled human C-peptide with unlabelled human C-peptide. C-peptide was found to bind to the cell membranes of intact fibroblasts with an association constant of 3 x 10(9) M(-1), giving full saturation at about 0.9 nM, close to the physiological C-peptide plasma concentration. Treatment of all investigated cells with the zwitter-ionic detergent Chaps was found to release macromolecules that bind specifically to C-peptide. The binding in Chaps extracts of fibroblasts was sensitive to time but remained reproducible for up to 2 h at room temperature. Lysophosphatidylcholine, Triton X-100, beta-octylglucopyranoside, SDS, or cholate gave extracts with only low or nonspecific binding. It is concluded that C-peptide binding components can be solubilised from cells, and that Chaps appears to be a suitable detergent.