title: Measurement of proton pump activity of the thermophilic bacterium PS3 and Nitrobacter agilis at the cytochrome oxidase level using total membrane and heptyl thioglucoside.
abstract: 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.
mesh_terms: 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
chemical_list: D011510:Proteolipids; D011522:Protons; D013865:Thioglycosides; C020485:proteoliposomes; C046542:heptyl 1-thioglucoside; D003576:Electron Transport Complex IV; D006180:Proton-Translocating ATPases