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Titolo:
THE INTERACTION OF EPIDERMAL GROWTH-FACTOR WITH ITS RECEPTOR IN A431 CELL-MEMBRANES - A SLOPPED-FLOW FLUORESCENCE ANISOTROPY STUDY
Autore:
ROUSSEAU DL; STAROS JV; BEECHEM JM;
Indirizzi:
VANDERBILT UNIV,DEPT MOLEC BIOL,221 KIRKLAND HALL NASHVILLE TN 37232 VANDERBILT UNIV,DEPT MOLEC BIOL NASHVILLE TN 37232 VANDERBILT UNIV,DEPT BIOCHEM NASHVILLE TN 37232 VANDERBILT UNIV,DEPT MOLEC PHYSIOL & BIOPHYS NASHVILLE TN 37232
Titolo Testata:
Biochemistry
fascicolo: 44, volume: 34, anno: 1995,
pagine: 14508 - 14518
SICI:
0006-2960(1995)34:44<14508:TIOEGW>2.0.ZU;2-W
Fonte:
ISI
Lingua:
ENG
Soggetto:
RESONANCE ENERGY-TRANSFER; PROTEIN TYROSINE KINASE; HIGH-AFFINITY RECEPTORS; EGF-RECEPTOR; FACTOR BINDING; SIGNAL TRANSDUCTION; CARCINOMA-CELLS; DIRECT VISUALIZATION; MONOCLONAL-ANTIBODY; HUMAN FIBROBLASTS;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
68
Recensione:
Indirizzi per estratti:
Citazione:
D.L. Rousseau et al., "THE INTERACTION OF EPIDERMAL GROWTH-FACTOR WITH ITS RECEPTOR IN A431 CELL-MEMBRANES - A SLOPPED-FLOW FLUORESCENCE ANISOTROPY STUDY", Biochemistry, 34(44), 1995, pp. 14508-14518

Abstract

We describe a quantitative examination of the interaction of epidermal growth factor (EGF) with the EGF receptor using A431 cell membrane vesicles as a receptor source. Using T-format steady-state fluorescenceanisotropy detection coupled with stopped-flow mixing, we measured the association and EGF-induced dissociation kinetics of fluorescein 5-isothiocyanate-labeled mEGF (FITC-EGF) with the EGF receptor over a wide range of FITC-EGF concentrations, membrane dilutions, and time scales (milliseconds to minutes). Fluorescence anisotropy-based equilibriumbinding titrations were also performed. All studies utilized the samereceptor preparation, ligand preparation, and detection system. The entire data surface (approximate to 78 000 data points) was simultaneously analyzed using global analysis techniques with a variety of kinetic models. Our analysis identified with a high level of confidence receptor populations with two association rate constants (k(on) = 1.2 x 10(6) M(-1) s(-1), 7.2 x 10(6) M(-1) s(-1)) and three dissociation rate constants (k(off) = 0.95 x 10(-2) s(-1), 0.13 x 10(-2) s(-1), 0.32 x 10(-3) s(-1)), which reflect the presence of at least two distinct receptor populations in A431 cell membranes. Analysis of the kinetic data was found to be much more sensitive to the presence of multiple receptor populations than was the analysis of the equilibrium binding data.

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Documento generato il 03/04/20 alle ore 11:08:53