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.
Henriksson
M
M
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
Pramanik
A
A
Shafqat
J
J
Zhong
Z
Z
Tally
M
M
Ekberg
K
K
Wahren
J
J
Rigler
R
R
Johansson
J
J
Jörnvall
H
H
D002096:C-Peptide / Q000378:metabolism*; D002457:Cell Extracts; D002462:Cell Membrane / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D002478:Cells, Cultured; D002793:Cholic Acids / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology*; D005347:Fibroblasts / Q000737:chemistry / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D006801:Humans; D020014:K562 Cells; D011485:Protein Binding / Q000187:drug effects; D011994:Recombinant Proteins / Q000378:metabolism; D015203:Reproducibility of Results; D012867:Skin / Q000737:chemistry / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D012995:Solubility / Q000187:drug effects; D013379:Substrate Specificity; D013696:Temperature; D013997:Time Factors
D002096:C-Peptide; D002457:Cell Extracts; D002793:Cholic Acids; D003902:Detergents; D011994:Recombinant Proteins; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate