Reconstitution of membranes by recombining proteins and lipids derived from erythrocyte stroma

Abstract

Reconstitution of membranes of the red blood cell has been obtained by recombining the membrane proteins with the membrane lipids. The native membranes have been solubilized in 90% 2-chloroethanol, and membrane proteins and lipids have been separated by gel filtration on Sephadex LH-20. The two components were combined in 2-chloroethanol and then dialyzed against aqueous buffer whereby precipitation of aggregated lipoprotein took place. Equilibrium density gradient centrifugation of original stroma and reconstituted stromal lipoproteins proved the binding of lipid to about 60% of the protein after recombination. Chemical analysis of the isolated zones of stroma and recombined material showed similarity in the composition of the proteins and the main lipid classes. Electron microscopy of fixed and sectioned material revealed identical structure of original and recombined membranes; both were trilaminar, had an overall thickness of 70–80 Å and were found to have an identical granular substructure of the dense layers. The recombined membranes formed stacks or concentric shells which could be clearly differentiated from “myelin figures”. The two subfractions of proteins and lipids did not form membrane-like structures.