Functional incorporation of beef-heart cytochrome c oxidase into membranes of Streptococcus cremoris.
Beef heart mitochondrial cytochrome c oxidase has been incorporated into membrane vesicles derived from the homofermentative lactic acid bacterium Streptococcus cremoris. Proteoliposomes containing cytochrome c oxidase were fused with the bacterial membrane vesicles by means of a freeze/thaw sonication technique. Evidence that membrane fusion has taken place is presented by the demonstration that nonexchangeable fluorescent phospholipid probes, originally present only in the bacterial membrane or only in the liposomal membrane, are diluted in the membrane after fusion and, by sucrose gradient centrifugation, indicating a buoyant density of the membranes after fusion in between those of the starting membrane preparations. The fused membranes are endowed with a relatively low ion permeability which makes it possible to generate a high proton motive force (100 mV, inside negative and alkaline) by cytochrome-c-oxidase-mediated oxidation of the electron donor system ascorbate/N,N,N',N'-tetramethyl-p-phenylenediamine/cytochrome c. In the fused membranes this proton motive force can drive the uptake of several amino acids via secondary transport systems. The incorporation procedure described for primary proton pumps in biological membranes opens attractive possibilities for studies of proton-motive-force-dependent processes in isolated membrane vesicles from bacterial or eukaryotic origin which lack a suitable proton-motive-force-generating system.
D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D001693:Biological Transport, Active; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D003574:Cytochrome c Group / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008081:Liposomes; D008561:Membrane Fusion; D008564:Membrane Potentials; D013291:Streptococcus / Q000648:ultrastructure*
D000596:Amino Acids; D003574:Cytochrome c Group; D008081:Liposomes; D003576:Electron Transport Complex IV