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

被引:33
作者
Henriksson, M [1 ]
Pramanik, A
Shafqat, J
Zhong, ZH
Tally, M
Ekberg, K
Wahren, J
Rigler, R
Johansson, J
Jornvall, H
机构
[1] Karolinska Inst, Dept Med Biochem, Stockholm, Sweden
[2] Karolinska Inst, Dept Surg Sci, Stockholm, Sweden
[3] Karolinska Inst, Dept Mol Med, Stockholm, Sweden
关键词
diabetes mellitus; proinsulin C-peptide; peptide-lipid interactions; FCS;
D O I
10.1006/bbrc.2000.4135
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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-B 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. (C) 2001 Academic Press.
引用
收藏
页码:423 / 427
页数:5
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