Investigations of spectrin-lipid interactions using fluoresceinphosphatidylethanolamine as a membrane probe.
The binding of human erythrocyte spectrin to large unilamellar vesicles (LUVET) formed by the extrusion technique has been studied using fluoresceinphosphatidylethanolamine (FPE) as a reporter of electrostatic membrane potential. Spectrin aliquots were added to a suspension of FPE-labelled LUVETs to elucidate both the type of charge involved and the dissociation constants for spectrin binding to various lipids. All binding experiments showed serial increases in FPE fluorescence intensity upon serial additions of spectrin, indicative of increasing positive charge at the membrane surface. This proves for the first time that although exhibiting an overall net negative charge, spectrin binds to lipid surfaces by presenting positive charges to the lipid surface. Binding curves were obtained from the change in fluorescence intensity upon each spectrin addition and analysed to determine dissociation constants. A K(d) of 0.14+/-0.12 microM was found for spectrin binding to FPE-labelled phosphatidylcholine/phosphatidylserine (PC/PS) LUVETs at 22 degrees C in high salt conditions. A similar K(d) of 0.17+/-0.11 microM was obtained for spectrin binding to neutral LUVETs composed of PC. However, binding was found to be much weaker for PC/PS LUVETs under low salt conditions with a K(d) of 1.22+/-0.48 microM.
O'Toole
P J
PJ
Department of Biological Sciences, University of Essex, Wivenhoe Park, Essex CO4 3SQ, Colchester, UK. pjotoo@essex.ac.uk
Morrison
I E
IE
Cherry
R J
RJ
D002021:Buffers; D005456:Fluorescent Dyes / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D010712:Phosphatidic Acids / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000378:metabolism*; D010718:Phosphatidylserines / Q000378:metabolism*; D011189:Potassium Chloride / Q000737:chemistry; D011485:Protein Binding; D013049:Spectrin / Q000378:metabolism*
D002021:Buffers; D005456:Fluorescent Dyes; D010712:Phosphatidic Acids; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; C063389:fluorescein phosphatidylethanolamine thiocarbamide; D013049:Spectrin; D011189:Potassium Chloride