scholarly journals The organization of formate dehydrogenase in the cytoplasmic membrane of Escherichia coli

1981 ◽  
Vol 195 (3) ◽  
pp. 627-637 ◽  
Author(s):  
A Graham ◽  
D H Boxer
Keyword(s):  
Escherichia Coli ◽  
Formate Dehydrogenase ◽  
Cytoplasmic Membrane ◽  
Membrane Vesicles ◽  
Covalent Modification ◽  
Opposite Orientation ◽  
Specific Antibodies ◽  
F1 Atpase ◽  
Cytoplasmic Face ◽  
Two Populations

The arrangement of the proton-translocating formate dehydrogenase of the anaerobic respiratory chain of Escherichia coli within the cytoplasmic membrane was examined by direct covalent modification with non-membrane-permeant reagents. Three methods were employed, lactoperoxidase-catalysed radioiodination, labelling with diazotized [125I] di-iodosulphanilic acid and labelling with diazobenzene [35S] sulphonate. All three procedures yield consistent with the view that the two larger subunits of the enzyme, Mr 110000 and 32000, both occupy transmembranous locations within the membrane. In each case the modification of the Ca2+ or Mg2+-activated F1-ATPase was monitored, and all reagents employed correctly located this enzyme at the cytoplasmic face of the membrane. A procedure involving agglutination with specific antibodies is described which appears to fractionate membrane vesicles of mixed orientation into two populations, one with the same membrane orientation as that of spheroplasts and the other opposite orientation.

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 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
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