ABSTRACT
We demonstrate that phospholipid vesicles affect the intrinsic fluorescence of isolated brain spectrin. In the present studies we tested the effects of vesicles prepared from phosphatidylcholine (PtdCho) alone, in addition to vesicles containing PtdCho mixed with other phospholipids [phosphatidylethanolamine (PtdEtn) and phosphatidylserine] as well as from total lipid mixture extracted from brain membrane. The largest effect was observed with PtdEtn/PtdCho (3:2 molar ratio) vesicles; the effect was markedly smaller when vesicles were prepared from egg yolk PtdCho alone. Brain spectrin injected into a subphase induced a substantial increase in the surface pressure of monolayers prepared from phospholipids. Results obtained with this technique indicated that the largest effect is again observed with monolayers prepared from a PtdEtn/PtdCho mixture. The greatest effect was observed when the monolayer contained 50-60% PtdEtn in a PtdEtn/PtdCho mixture. This interaction occurred at salt and pH optima close to physiological conditions (0.15 M NaCl, pH7.5). Experiments with isolated spectrin subunits indicated that the effect of the beta subunit on the monolayer surface pressure resembled that measured with the whole molecule. Similarly to erythrocyte spectrin-membrane interactions, brain spectrin interactions with PtdEtn/PtdCho monolayer were competitively inhibited by isolated erythrocyte ankyrin. This also suggests that the major phospholipid-binding site is located in the beta subunit and indicates the possible physiological significance of this interaction.
Subject(s)
Brain/metabolism , Carrier Proteins/metabolism , Microfilament Proteins/metabolism , Nerve Tissue Proteins/metabolism , Phospholipids/metabolism , Animals , Ankyrins/metabolism , Binding Sites , Buffers , Carrier Proteins/chemistry , Cattle , Fluorescence Polarization , Hydrogen-Ion Concentration , Liposomes/chemistry , Microfilament Proteins/chemistry , Nerve Tissue Proteins/chemistry , Phosphatidylcholines/metabolism , Phosphatidylethanolamines/metabolism , Phospholipids/chemistry , Sodium Chloride/metabolism , Spectrin/chemistry , Spectrin/metabolism , Spectrometry, FluorescenceABSTRACT
Binding of brain spectrin to frozen and thawed liposomes was studied, using a pelleting assay, as a function of lipid composition. Saturable binding was observed for all lipid mixtures that included aminophospholipids, as well as for the total lipid of synaptic plasma membranes. Binding was strong and saturable, with dissociation constants in the nanomolar range. There were two pH optima at ca. 6.0 and 7.5 and a sharp ionic strength optimum, corresponding to physiological solvent conditions. No competition could be detected with extraneous globular proteins, serum albumin, and hemoglobin. The results imply a strong, direct interaction between brain spectrin and the neuronal plasma membrane in the cell.