Measurement of proton pump activity of the thermophilic bacterium PS3 and Nitrobacter agilis at the cytochrome oxidase level using total membrane and heptyl thioglucoside.
It is possible to prepare liposomal vesicles by solubilization of total bacterial membranes with n-heptyl beta-D-thioglucoside followed by reconstitution into proteoliposomes by a freeze-thaw-sonication procedure with soybean phospholipids. The resulting proteoliposomes from total membrane fraction of sufficiently aerated cells of the thermophilic bacterium PS3 containing cytochrome aa3 showed a reasonable H+ pumping activity upon addition of reduced cytochrome c. On the other hand, the proteoliposomes reconstituted from air-limited PS3 cells containing cytochrome o and those from Nitrobacter agilis cells containing cytochrome aa3 did not show H+ pumping upon addition of reduced cytochrome c, although the vesicles showed "respiratory control"; 3-4-fold stimulation of oxygen consumption took place upon addition of an uncoupler. In proteoliposomes prepared from PS3 membranes by this method, H+-translocating ATPase (F0 X F1) was successfully reconstituted as well, suggesting that this method has wide applicability for investigation of enzymes catalyzing transmembrane processes.
D001420:Bacteria, Aerobic / Q000378:metabolism*; D001693:Biological Transport, Active; D002384:Catalysis; D002462:Cell Membrane / Q000378:metabolism; D003576:Electron Transport Complex IV / Q000378:metabolism*; D005615:Freezing; D008564:Membrane Potentials; D009576:Nitrobacter / Q000378:metabolism*; D010101:Oxygen Consumption; D011510:Proteolipids / Q000378:metabolism; D006180:Proton-Translocating ATPases / Q000378:metabolism; D011522:Protons; D012995:Solubility; D013010:Sonication; D013865:Thioglycosides
D011510:Proteolipids; D011522:Protons; D013865:Thioglycosides; C020485:proteoliposomes; C046542:heptyl 1-thioglucoside; D003576:Electron Transport Complex IV; D006180:Proton-Translocating ATPases