Functional lac carrier proteins in cytoplasmic membrane vesicles isolated from Escherichia coli. 1. Temperature dependence of dansyl galactoside binding and β-galactoside transport

Biochemistry ◽  
1977 ◽  
Vol 16 (17) ◽  
pp. 3772-3776 ◽  
Author(s):  
Helene Therisod ◽  
Lucienne Letellier ◽  
Rudolf Weil ◽  
Emanuel Shechter
Keyword(s):  
Escherichia Coli ◽  
Temperature Dependence ◽  
Cytoplasmic Membrane ◽  
Membrane Vesicles ◽  
Carrier Proteins

scholarly journals NorA Functions as a Multidrug Efflux Protein in both Cytoplasmic Membrane Vesicles and Reconstituted Proteoliposomes

2002 ◽  
Vol 184 (5) ◽  
pp. 1370-1377 ◽  
Author(s):  
Jian-Lin Yu ◽  
Leo Grinius ◽  
David C. Hooper
Keyword(s):  
Escherichia Coli ◽  
Expression Vector ◽  
Cytoplasmic Membrane ◽  
Membrane Vesicles ◽  
Aqueous Environment ◽  
Multidrug Transporter ◽  
Efflux Transporter ◽  
Multidrug Efflux ◽  
Hoechst 33342 ◽  
Experimental Systems

ABSTRACT Overexpression of NorA, an endogenous efflux transporter of Staphylococcus aureus, confers resistance to certain fluoroquinolone antimicrobials and diverse other substrates. The norA gene was amplified by PCR and cloned in the expression vector pTrcHis2. Histidine-tagged NorA (NorA-His) was overexpressed in Escherichia coli cells to prepare two experimental systems, everted membrane vesicles enriched with NorA-His and proteoliposomes reconstituted with purified NorA-His. In membrane vesicles, NorA-His actively transported Hoechst 33342, a dye that is strongly fluorescent in the membrane but has low fluorescence in an aqueous environment. Transport was activated by the addition of ATP or lactate and reversed by the addition of nigericin, with the addition of K+-valinomycin having little effect. Transport of Hoechst 33342 was inhibited competitively by verapamil, a known inhibitor of NorA, and by other NorA substrates, including tetraphenyl phosphonium and the fluoroquinolones norfloxacin and ciprofloxacin. In contrast, sparfloxacin, a fluoroquinolone whose antimicrobial activity is not affected by NorA expression, exhibited noncompetitive inhibition. NorA induction and overexpression yielded 0.5 to 1 mg of a largely homogeneous 40- to 43-kDa protein per liter of culture. NorA-His incorporated into proteoliposomes retained the ability to transport Hoechst 33342 in response to an artificial proton gradient, and transport was blocked by nigericin and verapamil. These data provide the first experimental evidence of NorA functioning as a self-sufficient multidrug transporter.

Cytoplasmic Membrane Vesicles of Escherichia coli

1978 ◽  
Vol 83 (1) ◽  
pp. 117-128 ◽  
Author(s):  
Ichiro YAMATO ◽  
Masamitsu FUTAI ◽  
Yasuhiro ANRAKU ◽  
Yoshiaki NONOMURA
Keyword(s):  
Escherichia Coli ◽  
Cytoplasmic Membrane ◽  
Membrane Vesicles

scholarly journals The organization of hydrogenase in the cytoplasmic membrane of Escherichia coli

1981 ◽  
Vol 197 (2) ◽  
pp. 283-291 ◽  
Author(s):  
A Graham
Keyword(s):  
Escherichia Coli ◽  
Cytoplasmic Membrane ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Labelling Pattern ◽  
F1 Atpase ◽  
Crossed Immunoelectrophoresis ◽  
Cytoplasmic Surface ◽  
Membrane Bound ◽  
Inside Out

The organization of the membrane-bound hydrogenase from Escherichia coli was studied by using two membrane-impermeant probes, diazotized [125I]di-iodosulphanilic acid and lactoperoxidase-catalysed radioiodination. The labelling pattern of the enzyme obtained from labelled spheroplasts was compared with that from predominantly inside-out membrane vesicles, after recovery of hydrogenase by immunoprecipitation. The labelling pattern of F1-ATPase was used as a control for labelling at the cytoplasmic surface throughout these experiments. Hydrogenase (mol.wt. approx. 63 000) is transmembranous. Crossed immunoelectrophoresis with anti-(membrane vesicle) immunoglobulins, coupled with successive immunoadsorption of the antiserum with spheroplasts, confirmed the location of hydrogenase at the periplasmic surface. Immunoadsorption with sonicated spheroplasts suggests that the enzyme is also exposed at the cytoplasmic surface. Inside-out vesicles were prepared by agglutination of sonicated spheroplasts, and the results of immunoadsorption using these vesicles confirms the location of hydrogenase at the cytoplasmic surface.

scholarly journals Sites and specificity of the reaction of bipyridylium compounds with anaerobic respiratory enzymes of Escherichia coli. Effects of permeability barriers imposed by the cytoplasmic membrane

1977 ◽  
Vol 164 (1) ◽  
pp. 199-211 ◽  
Author(s):  
Robert W. Jones ◽  
Peter B. Garland
Keyword(s):  
Escherichia Coli ◽  
Nitrate Reductase ◽  
Cytoplasmic Membrane ◽  
Reductase Activity ◽  
Membrane Vesicles ◽  
Respiratory Enzymes ◽  
Radical Species ◽  
Benzyl Viologen ◽  
E Coli ◽  
Nitrite Reductase Activity

The ability of the oxidized and singly reduced species of several bipyridylium cations to cross the cytoplasmic membrane of Escherichia coli was studied to locate the sites of reaction of the dyes with anaerobic respiratory enzymes. Benzyl Viologen radical crossed the membrane rapidly, whereas the oxidized species did not. The oxidized or radical species of Methyl Viologen, Morfamquat or Diquat did not rapidly cross the membrane. It was also shown that the dithionite anion does not cross the cytoplasmic membrane of E. coli. Diquat radical donates electrons to the nitrate reductase pathway at the periplasmic aspect of the membrane, whereas Benzyl Viologen radical reacted directly with nitrate reductase itself (EC 1.7.99.4) at the cytoplasmic aspect of the membrane. Thus the pathway of electron transfer in the nitrate reductase pathway is transmembranous. Formate hydrogenlyase (EC 1.2.1.2) and an uncharacterized nitrite reductase activity react with bipyridylium dyes at the periplasmic aspect of the membrane. Fumarate reductase (succinate dehydrogenase; EC 1.3.99.1) reacts with bipyridylium radicals, and formate dehydrogenase (cytochrome) (EC 1.2.2.1) with ferricyanide, at the cytoplasmic aspect of the membrane. The differing charge and membrane permeation of oxidized and radical species of bipyridylium dyes greatly complicate their use as potentiometric mediators in suspensions of cells or membrane vesicles.

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