TEMPERATURE AND IONIC EFFECTS ON THE INTERACTION OF ERYTHROID SPECTRIN WITH PHOSPHATIDYLSERINE MEMBRANES

被引:28
作者
MACDONALD, RI
机构
[1] Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston
关键词
D O I
10.1021/bi00078a021
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 K(d) of 0.31 muM and an apparent maximum spectrin binding of 33 nM/mM PS at 25-degrees-C, an apparent K(d) of 0.35 muM and an apparent maximum spectrin binding of 40 nM/mM PS at 31-degrees-C, and an apparent K(d) of 3.4 muM and an apparent maximum spectrin binding of 113 nM/0.1 mM PS at 37-degrees-C. Resembling the extraction of spectrin from red cell ghosts, dissociation of the spectrin-PS complex was enhanced when the salt content of the buffer was decreased to millimolar concentrations at 25-degrees-C but not at 4-degrees-C.
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页码:6957 / 6964
页数:8
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