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
相关论文
共 19 条
[1]   Evidence for specific interactions between kavain and human cortical neurons monitored by fluorescence correlation spectroscopy [J].
Boonen, G ;
Pramanik, A ;
Rigler, R ;
Häberlein, H .
PLANTA MEDICA, 2000, 66 (01) :7-10
[2]   SORTING SINGLE MOLECULES - APPLICATION TO DIAGNOSTICS AND EVOLUTIONARY BIOTECHNOLOGY [J].
EIGEN, M ;
RIGLER, R .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1994, 91 (13) :5740-5747
[3]   SPECIFIC BINDING OF THE C-PEPTIDE OF PROINSULIN TO CULTURED B-CELLS FROM A TRANSPLANTABLE RAT ISLET CELL TUMOR [J].
FLATT, PR ;
SWANSTONFLATT, SK ;
HAMPTON, SM ;
BAILEY, CJ ;
MARKS, V .
BIOSCIENCE REPORTS, 1986, 6 (02) :193-199
[4]   Unordered structure of proinsulin C-peptide in aqueous solution and in the presence of lipid vesicles [J].
Henriksson, M ;
Shafqat, J ;
Liepinsh, E ;
Tally, M ;
Wahren, J ;
Jörnvall, H ;
Johansson, J .
CELLULAR AND MOLECULAR LIFE SCIENCES, 2000, 57 (02) :337-342
[5]   Prevention of vascular and neural dysfunction in diabetic rats by C-peptide [J].
Ido, Y ;
Vindigni, A ;
Chang, K ;
Stramm, L ;
Chance, R ;
Heath, WF ;
DiMarchi, RD ;
DiCera, E ;
Williamson, JR .
SCIENCE, 1997, 277 (5325) :563-566
[6]   Differential effects of proinsulin C-peptide fragments on Na+, K+-ATPase activity of renal tubule segments [J].
Ohtomo, Y ;
Bergman, T ;
Johansson, BL ;
Jörnvall, H ;
Wahren, J .
DIABETOLOGIA, 1998, 41 (03) :287-291
[7]   C-peptide stimulates rat renal tubular Na+, K+-ATPase activity in synergism with neuropeptide Y [J].
Ohtomo, Y ;
Aperia, A ;
Sahlgren, B ;
Johansson, BL ;
Wahren, J .
DIABETOLOGIA, 1996, 39 (02) :199-205
[8]  
Pramanik A., 1999, Biomedical Chromatography, V13, P119, DOI 10.1002/(SICI)1099-0801(199904)13:2<119::AID-BMC835>3.0.CO
[9]  
2-P
[10]   Fluorescence correlation spectrometry of the interaction kinetics of tetramethylrhodamin alpha-bungarotoxin with Torpedo californica acetylcholine receptor [J].
Rauer, B ;
Neumann, E ;
Widengren, J ;
Rigler, R .
BIOPHYSICAL CHEMISTRY, 1996, 58 (1-2) :3-12