Steric immobilization of liposomes in chromatographic gel beads and incorporation of integral membrane proteins into their lipid bilayers.
Interactions between water-soluble substances and immobilized liposomes or proteoliposomes can be analyzed by chromatographic procedures. Methods were therefore developed to sterically immobilize large amounts of liposomes in gel beads. First, freeze-thawing of moist Sephacryl S-1000 beads mixed with egg yolk phospholipid liposomes of 200-300 mM lipid concentration immobilized liposomes representing 60-90 mumol lipid with an internal volume of 60-150 microliters per milliliter gel bed. The same procedure with freeze-dried beads immobilized liposomes representing more lipid but with lower internal volumes. Other lipid mixtures could also be used. Second, mixtures of Sephacryl S-1000 beads and liposomes were freeze-dried and rehydrated. Large amounts of liposomes became immobilized even with dilute liposome suspensions. Third, a mixture of diethylether-dried Sephacryl S-1000 beads and an emulsion of an aqueous solution in diethylether with lipids was subjected to reverse-phase evaporation. Liposomes of high specific internal volume became immobilized. Integral human red cell membrane proteins could be incorporated into the liposomal lipid bilayers. Upon mixing octyl glucoside-solubilized proteins with moist beads containing immobilized liposomes, up to 0.4 mg protein became incorporated per milliliter gel bed (yield 34%). Proteins could also be incorporated during immobilization by reverse-phase evaporation. Detergents could be removed efficiently from immobilized (proteo)liposomes in a gel bed.
Yang
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Q
Department of Biochemistry, Uppsala University, Sweden.
Lundahl
P
P
D019826:Cholic Acid; D002793:Cholic Acids / Q000032:analysis; D002850:Chromatography, Gel / Q000295:instrumentation / Q000379:methods*; D004563:Electrochemistry; D004910:Erythrocyte Membrane / Q000737:chemistry; D005612:Freeze Drying; D005615:Freezing; D005782:Gels; D008051:Lipid Bilayers / Q000737:chemistry*; D008081:Liposomes / Q000737:chemistry* / Q000528:radiation effects; D008565:Membrane Proteins / Q000737:chemistry*; D008863:Microspheres; D008968:Molecular Conformation; D011485:Protein Binding; D013499:Surface Properties; D014867:Water
D002793:Cholic Acids; D005782:Gels; D008051:Lipid Bilayers; D008081:Liposomes; D008565:Membrane Proteins; D014867:Water; D019826:Cholic Acid