title: Role of fluidity of membranes on the guanyl nucleotide-dependent binding of cholecystokinin-8S to rat brain cortical membranes.
abstract: The binding of [3H]cholecystokinin octapeptide (sulphated) ([3H]CCK-8S), an agonist of the cholecystokinin receptors, to rat cortical membranes was fast, specific and saturable, with pH optimum at 6.5-7.0. The divalent cations Mg2+ and Ca2+ clearly enhanced [3H]CCK-8S binding, whereas the monovalent cations Na+ and K+ were inhibitors. Inactivation of the ligand binding ability of these membranes was dependent on the incubation temperature and corresponding tau1/2 values were 11 days at 4 degrees , 12 hr at 21 degrees , 154 min at 30 degrees and 51 min at 37 degrees , which revealed the apparent activation energy of this process to be 130+/-4 kJ/mol. Scatchard analysis of the saturation curves of [3H]CCK-8S binding was best described by a one site binding model with a Kd = 0.63+/-0.18 nM and a maximum binding of 32+/-2 fmol/mg protein. The stable GTP analogue guanosin-5'-O-(3-thiotriphosphate) (GTPgammaS) decreased the affinity of [3H]CCK-8S binding only up to 2-fold without significant influence on maximal binding. Modulation of membrane properties by different detergents revealed that only in the case of digitonin (0.03-0.04%) did the GTP-dependence of [3H]CCK-8S binding considerably increase without significant influence on the ligand binding properties in the absence of GTPgammaS. Other detergents studied (sodium cholate, sodium deoxycholate, 3-(3-cholamidopropyl)dimethylammonio-1-propanesulfonate (CHAPS), sucrose monolaurate, series Triton X and Tween) either had little influence on GTP-gammaS-dependence of [3H]CCK-8S binding or inactivated the receptor. Parallel studies of fluorescent polarization of diphenylhexatriene (DPH) in rat cortical membranes indicated that digitonin was the only detergent which at low concentrations caused a rapid increase in membrane fluidity and thereafter stabilized it at a certain level. Other detergents studied had only moderate influence on membrane fluidity (CHAPS, cholate, deoxycholate) or caused fast and continuous increase of membrane fluidity (Triton X-100, Tween 80). These data together point to the essential influence of the fluidity of membranes on the regulation of the interactions between G proteins and CCK receptors in rat cortical membranes. Under standard experimental conditions (temperature lower than 30 degrees), the CCK receptor-G protein complex is active for quantitative characterization of the receptors, but the membranes are too rigid for natural communication and regulation.
authors.affiliation: Institute of Chemical Physics, University of Tartu, Estonia. ago@ut.ee
authors.forename: A
authors.initials: A
authors.lastname: Rinken
authors.forename: J
authors.initials: J
authors.lastname: Harro
authors.forename: L
authors.initials: L
authors.lastname: Engström
authors.forename: L
authors.initials: L
authors.lastname: Oreland
mesh_terms: D000818:Animals; D002412:Cations / Q000494:pharmacology; D002540:Cerebral Cortex / Q000187:drug effects / Q000378:metabolism*; D003902:Detergents / Q000494:pharmacology; D004072:Digitonin / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism; D006150:Guanine Nucleotides / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008024:Ligands; D008560:Membrane Fluidity / Q000187:drug effects; D051381:Rats; D011949:Receptors, Cholecystokinin / Q000819:agonists; D012844:Sincalide / Q000031:analogs & derivatives* / Q000378:metabolism; D013816:Thermodynamics
chemical_list: C090019:8-sulfocholecystokinin octapeptide; D002412:Cations; D003902:Detergents; D006150:Guanine Nucleotides; D008024:Ligands; D011949:Receptors, Cholecystokinin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019204:GTP-Binding Proteins; D004072:Digitonin; D012844:Sincalide