[125I]calmodulin binding to synaptic plasma membrane from rat brain: kinetic and Arrhenius analysis.
Binding of [125I]calmodulin was characterized in highly purified synaptic plasma membrane (SPM) prepared from rat brain. By Scatchard analysis, the Ca(2+)-dependent membrane binding of [125I]calmodulin was found to have a Bmax of 284 pmol/mg protein and an apparent affinity with a Kd of 131 nM. Kinetic analysis indicates that at 37 degrees C, the dissociation of [125I]calmodulin-membrane complexes follows first-order reaction and consists of two components: a dissociation constant (k) of 3.7 x 10(-1) min-1 and a half-time (t1/2) of 1.8 min for the fast component, and a k of 4.8 x 10(-2) min-1 and a t1/2 of 14.5 min for the slow component. At 0 degrees C, substantial dissociation still occurred, with a k of 4.5 x 10(-2) min-1 and a t1/2 of 15.3 min for the fast component, and a k of 5.5 x 10(-3) min-1 and a t1/2 of 125.5 min for the slow component. These data on binding affinity and dissociation kinetics are consistent with the notion that SPM can readily and rapidly associate and dissociate calmodulin. In Arrhenius analysis of temperature effects, [125I]calmodulin binding to SPM exhibits a biphasic function, with the transition temperature (Td) estimated to be 23.8 degrees C, suggesting that binding is influenced by lipid phase transition of the membrane. The binding of [125I]calmodulin to the synaptic membrane was found to be increased by corticosterone (10(-7)-10(-6) M), a steroid hormone, and decreased by ethanol (50-200 mM), a centrally acting drug.(ABSTRACT TRUNCATED AT 250 WORDS)
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Department of Pharmacology and Molecular Biology, Chicago Medical School, Illinois 60064.
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D000818:Animals; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D003345:Corticosterone / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D007457:Iodine Radioisotopes; D007700:Kinetics; D008297:Male; D051381:Rats; D017207:Rats, Sprague-Dawley; D013570:Synaptic Membranes / Q000378:metabolism*; D013816:Thermodynamics; D014268:Trifluoperazine / Q000494:pharmacology
D002147:Calmodulin; D007457:Iodine Radioisotopes; D014268:Trifluoperazine; D004533:Egtazic Acid; D002118:Calcium; D003345:Corticosterone