Interaction of insulin receptors with lipid bilayers and specific and nonspecific binding of insulin to supported membranes.
In the first part, we study the interaction of the insulin receptor with model membranes of dimyristoylphosphatidylcholine (DMPC) by various techniques, including calorimetry, densitometry, static light scattering, and electron microscopy. By analyzing the pronounced depression of the lipid chain melting transition in terms of the Van Laar-Hildebrand theory of regular dilute solutions, an (exothermic) interaction energy of Wp = 2000 kJ.mol-1 is found for the receptor and of WL = 0.6 kJ.mol-1 for the lipid. This is interpreted in terms of an adsorption of the 2 hydrophilic head groups of the receptor to the membrane surface so that 1 protein interacts with about 2000 lipids. This number is verified by freeze-fracture electron microscopy. Binding of insulin induces a remarkable decoupling of the receptor head group from the membrane, pointing to a pronounced conformational change. In the second part, we introduce a simple fluorescence technique by which adsorption isotherms of water-soluble and fluorescent-labeled substrates, such as insulin, to membranes may be determined. It is based on the selective evanescent field excitation of ligands adsorbed to supported planar bilayers on argon-sputtered glass plates. These are deposited by the monolayer transfer technique or by vesicle condensation. The reconstituted receptor exhibits a weak (binding constant Kw = 3 X 10(9) L.M-1) and a strong (binding constant Ks greater than 10(10) L.M-1) binding site. Insulin exhibits a weak but remarkable nonspecific binding to bilayers of pure DMPC and DMPC containing 20% positively charged lipid and a strong binding to DMPC containing negatively charged lipids such as phosphatidylserine or ganglioside (GT1b).(ABSTRACT TRUNCATED AT 250 WORDS)
Sui
S F
SF
Physik Department (Biophysics Group), Technische Universität München, Garching, FRG.
Urumow
T
T
Sackmann
E
E
D002151:Calorimetry; D003208:Concanavalin A; D003720:Densitometry; D004134:Dimyristoylphosphatidylcholine; D005260:Female; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives*; D005456:Fluorescent Dyes; D005614:Freeze Fracturing; D005732:Gangliosides / Q000378:metabolism; D006046:Gold; D006801:Humans; D007328:Insulin / Q000031:analogs & derivatives / Q000378:metabolism*; D008027:Light; D008051:Lipid Bilayers / Q000378:metabolism*; D008081:Liposomes / Q000378:metabolism*; D008854:Microscopy, Electron; D010920:Placenta / Q000032:analysis; D011972:Receptor, Insulin / Q000378:metabolism*; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D005456:Fluorescent Dyes; D005732:Gangliosides; D007328:Insulin; D008051:Lipid Bilayers; D008081:Liposomes; C031573:insulin, fluorescein-isothiocyanated-; D003208:Concanavalin A; C016481:trisialoganglioside GT1; D006046:Gold; D011972:Receptor, Insulin; D016650:Fluorescein-5-isothiocyanate; D004134:Dimyristoylphosphatidylcholine