Reconstitution of a functional membrane enzyme system in a monomolecular film: I. Formation of a mixed monolayer of lipopolysaccharide and phospholipid

Abstract

The galactosyl transferase system of Salmonella typhimurium is a membranebound enzyme system which can be reconstituted in vitro from purified lipopoly-saccharide, phosphatidyl ethanolamine and enzyme protein (UDP-galactose-lipopolysaccharide-α, 3-galactosyl transferase). This report describes the first step in reconstitution of the system in a monomolecular film at an air-water interface. A monolayer of phosphatidyl ethanolamine was first formed on the surface of an aqueous subphase and lipopolysaccharide was then introduced beneath the surface. Formation of a binary film (lipopolysaccharide-phosphatidyl ethanolamine) was indicated by an increase in surface pressure and was confirmed by direct measurement of the components in the monolayer. Enzymic assay of galactosyl acceptor activity indicated that the arrangement of lipopolysaccharide and phosphatidyl ethanolamine molecules in the mixed film was similar to their arrangement in the native structure. The molecular surface areas and molar ratios of the two components in the monolayer suggested an arrangement in which lipopolysaccharide and phosphatidyl ethanolamine molecules lie side-by-side with their fatty acid residues directed perpendicular to the surface.