We found a match
Your institution may have rights to this item. Sign in to continue.
- Title
Insulin binding monitored by fluorescence correlation spectroscopy.
- Authors
Zhong, Z-H.; Pramanik, A.; Ekberg, K.; Jansson, O. T.; Jörnvall, H.; Wahren, J.; Rigler, R.
- Abstract
Aim/hypothesis. The characteristics of insulin binding to its receptors have been extensively studied by the radioligand binding assay. We used fluorescence correlation spectroscopy to determine the distribution of diffusion times and further novel data on the kinetics of insulin's binding to its receptor. Methods: Cultured human renal tubular cells (HRTC) were incubated with tetramethyl rhodamine labelled insulin (Rh-Ins) for 60 min. Fluorescence intensity fluctuations and autocorrelation functions for Rh-Ins, free in the incubation medium and bound to the cell membrane, were studied at single-molecule detection sensitivity in a 0.2 fL confocal volume. Results: Measurements at the cell membrane revealed Rh-Ins binding with at least two diffusion components (diffusion times τDI=0.8 ms, τD2=20 ms) and corresponding weight fractions of y1=0.43 and y2=0.42. Specificity of the binding was shown by the dislocation of bound Rh-Ins when excess unlabelled insulin was added. Scatchard analysis showed a non-linear plot, revealing two binding processe with different affinities (Kass-2 · 1010 M-1b and -1 · 109 M-1, respectively. Conclusion/interpretation. The fluorescence correlation spectroscopy results show two classes of binding sites consistent with negative cooperativity. This conclusion is in agreement with studies of insulin binding using radioligand binding assays. Because of its high sensivity (single molecule detection), FCS, provides additional data allowing a more precise evaluation of the kinetics of ligand-receptors interactions at low expressions levels in living cells.
- Subjects
INSULIN; PANCREATIC secretions; HYPOGLYCEMIC agents; HORMONES; RECEPTOR-ligand complexes; LIGAND binding (Biochemistry); CELL receptors
- Publication
Diabetologia, 2001, Vol 44, Issue 9, p1184
- ISSN
0012-186X
- Publication type
Article
- DOI
10.1007/s001250100608