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 funct ion, but the mechanisms involved remain incompletely understood. Using fluo rescence correlation spectroscopy, we have studied binding of rhodamine-lab elled human C-peptide to intact human skin fibroblasts and to detergent-sol ubilised extracts of fibroblasts, K-562, and IEC-B cells. Specificity was s hown by displacement of rhodamine-labelled human C-peptide with unlabelled human C-peptide, C-peptide was found to bind to the cell membranes of intac t fibroblasts with an association constant of 3 x 10(9) M-1, giving full sa turation at about 0.9 nM, close to the physiological C-peptide plasma conce ntration, Treatment of all investigated cells with the zwitter-ionic deterg ent Chaps was found to release macromolecules that bind specifically to C-p eptide, The binding in Chaps extracts of fibroblasts was sensitive to time but remained reproducible for up to 2 h at room temperature. Lysophosphatid ylcholine, Triton X-100, beta -octylglucopyranoside, SDS, or cholate gave e xtracts with only low or nonspecific binding. It is concluded that C-peptid e binding components can be solubilised from cells, and that Chaps appears to be a suitable detergent. (C) 2001 Academic Press.