Incorporation of beef heart cytochrome c oxidase as a proton-motive force-generating mechanism in bacterial membrane vesicles.
Membrane vesicles derived from the strictly fermentative lactic acid bacterium Streptococcus cremoris have been fused with proteoliposomes containing the beef heart mitochondrial cytochrome c oxidase by means of a freeze/thaw-sonication technique. Evidence that fusion has taken place was obtained by freeze-etch electron microscopy, showing a less-dense intramembranous particle distribution in the fused membranes than in the bacterial membranes, and by sucrose gradient centrifugation, indicating a buoyant density of the majority of the membranes after fusion that was between the buoyant densities of the starting membrane preparations. In the fused membranes, 55-60% of the cytochrome c oxidase molecules are oriented with the cytochrome c binding site at the outer surface of the membrane. With the electron-donor system ascorbate/N,N,N',N'-tetramethyl-p-phenylenediamine/cytochrome c, a high proton-motive force (greater than 130 mV), inside negative and alkaline, can be generated in the fused membrane, and this proton-motive force can drive secondary transport of several amino acids. The procedure described can be used for incorporating a proton-motive force-generating system in isolated membrane vesicles from bacterial or eukaryotic origin that lack a suitable primary proton pump.
D000818:Animals; D001419:Bacteria / Q000378:metabolism; D001692:Biological Transport; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism*; D003576:Electron Transport Complex IV / Q000378:metabolism*; D004734:Energy Metabolism; D006863:Hydrogen-Ion Concentration; D007930:Leucine / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism; D008561:Membrane Fusion; D009206:Myocardium / Q000201:enzymology; D013291:Streptococcus / Q000378:metabolism
D008081:Liposomes; D003576:Electron Transport Complex IV; D007930:Leucine