Temperature and ionic effects on the interaction of erythroid spectrin with phosphatidylserine membranes.
Specific binding of human erythroid spectrin to large, unilamellar vesicles of bovine brain phosphatidylserine, made by an extrusion technique (LUVETs), has been measured and characterized by a new gel filtration assay. Vesicle-bound spectrin was separated from free spectrin by Sepharose CL-2B chromatography and detected by its intrinsic (tryptophan) or extrinsic (carboxyfluorescein) fluorescence. That the bound spectrin was not an aberrant, adhesive form was shown by the ability of a portion of free spectrin, which had not bound to PS LUVETs during a previous incubation, to bind during a subsequent incubation. Spectrin binding reached a plateau by 30 min of incubation at room temperature and at 37 degrees C. Binding increased from a low level below 31 degrees C to about twice as much as 37 degrees C and to 4-7 times as much between 40 and 43 degrees C. Similar results were obtained with LUVETs composed of DOPS but not PC. Triton treatment of PS LUVETs and spectrin after incubation of spectrin and vesicles at 40 and 43 degrees C but prior to chromatography on Sepharose CL-2B eliminated the bound spectrin peak, which thus did not consist of large aggregates of covalently associated spectrin. Binding isotherms fit by nonlinear regression gave an apparent Kd of 0.31 microM and an apparent maximum spectrin binding of 33 nM/mM PS at 25 degrees C, an apparent Kd of 0.35 microM and an apparent maximum spectrin binding of 40 nM/mM PS at 31 degrees C, and an apparent Kd of 3.4 microM and an apparent maximum spectrin binding of 113 nM/0.1 mM PS at 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)
MacDonald
R I
RI
Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, Illinois 60208.
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002850:Chromatography, Gel; D005452:Fluoresceins; D006358:Hot Temperature; D007700:Kinetics; D008027:Light; D008567:Membranes, Artificial; D009994:Osmolar Concentration; D010718:Phosphatidylserines / Q000378:metabolism*; D012542:Scattering, Radiation; D013049:Spectrin / Q000378:metabolism*
D005452:Fluoresceins; D008567:Membranes, Artificial; D010718:Phosphatidylserines; D013049:Spectrin; C024098:6-carboxyfluorescein