Characterization of calcium binding to brain spectrin.
Brain spectrin alpha and beta chains bind 45Ca2+, as shown by the calcium overlay method. Flow dialysis measurements revealed eight high affinity binding sites/tetramer that comprise two binding components (determined by nonlinear regression analysis). The first component has one or two sites (kd = 2-30 x 10(-8) M), depending on the ionic strength of the binding buffer, with the remaining high affinity sites in the second component (kd = 1-3 x 10(-6) M). In addition, there is a variable, low affinity binding component (n = 100-400, kd = 1-2 x 10(-4) M). Magnesium inhibits calcium binding to the low affinity sites with a K1 = 1.21 mM. Proteolytic fragments from trypsin or chymotrypsin digests of brain spectrin bind 45Ca2+ if they include alpha domain IV, alpha domain III, or the amino-terminal half of the beta chain (but more than 25 kDa from the amino-terminal). These data suggest that calcium ions bind with high affinity to the putative EF-hands in alpha domain IV and to one site in the amino-terminal half of the beta chain that is associated with alpha domain IV in the native dimer. The localization is consistent with a direct calcium modulation of the spectrin-actin-protein 4.1 interaction. In addition, there appears to be one high affinity site near the hypersensitive region of alpha brain spectrin. All four proposed binding sites occur near probable calmodulin-binding or calcium-dependent protease cleavage sites.
Wallis
C J
CJ
Department of Chemistry, Texas Christian University, Fort Worth 76129.
Wenegieme
E F
EF
Babitch
J A
JA
D000818:Animals; D001345:Autoradiography; D001665:Binding Sites; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism; D002413:Cations, Divalent; D002417:Cattle; D002918:Chymotrypsin / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006736:Horses; D006868:Hydrolysis; D009994:Osmolar Concentration; D013049:Spectrin / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism
D002147:Calmodulin; D002413:Cations, Divalent; D013049:Spectrin; D002918:Chymotrypsin; D014357:Trypsin; D002118:Calcium