title: Purified outer membranes of Serpulina hyodysenteriae contain cholesterol.
abstract: We have isolated outer and inner membranes of Serpulina hyodysenteriae by using discontinuous sucrose density gradients. The outer and inner membrane fractions contained less than 1 and 2%, respectively, of the total NADH oxidase activity (soluble marker) in the cell lysate. Various membrane markers including lipooligosaccharide (LOS), the 16-kDa outer membrane lipoprotein (SmpA), and the C subunit of the F1F0 ATPase indicated that the lowest-density membrane fraction contained outer membranes while the high-density membrane fraction contained inner membranes and that both are essentially free of contamination by the periplasmic flagella, a major contaminant of membranes isolated by other techniques. The outer membrane fractions (rho = 1.10 g/cm3) contained 0.25 mg of protein/mg (dry weight), while the inner membrane samples (rho = 1.16 g/cm3) contained significantly more protein (0.55 mg of protein/mg [dry weight]). Lipid analysis revealed that the purified outer membranes contained cholesterol as a major component of the membrane lipids. Treatment of intact S. hyodysenteriae with different concentrations of digitonin, a steroid glycoside that interacts with cholesterol, indicated that the outer membrane could be selectively removed at concentrations as low as 0.125%.
authors.affiliation: Department of Microbiology, University of Georgia, Athens 30602, USA.
authors.forename: H
authors.initials: H
authors.lastname: Plaza
authors.forename: T R
authors.initials: TR
authors.lastname: Whelchel
authors.forename: S F
authors.initials: SF
authors.lastname: Garczynski
authors.forename: E W
authors.initials: EW
authors.lastname: Howerth
authors.forename: F C
authors.initials: FC
authors.lastname: Gherardini
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000032:analysis; D001426:Bacterial Proteins / Q000032:analysis; D017401:Brachyspira hyodysenteriae / Q000737:chemistry*; D002462:Cell Membrane / Q000737:chemistry*; D002784:Cholesterol / Q000032:analysis*; D004072:Digitonin / Q000494:pharmacology; D005408:Flagellin / Q000032:analysis; D007202:Indicators and Reagents / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000032:analysis; D008074:Lipoproteins; D008563:Membrane Lipids / Q000032:analysis; D006180:Proton-Translocating ATPases / Q000032:analysis
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D007202:Indicators and Reagents; D008070:Lipopolysaccharides; D008074:Lipoproteins; D008563:Membrane Lipids; C095510:SmpA protein, Treponema hyodysenteriae; C023023:lipid-linked oligosaccharides; D005408:Flagellin; C416532:flaB flagellin; D002784:Cholesterol; D006180:Proton-Translocating ATPases; D004072:Digitonin