Measurement of proton pump activity of the thermophilic bacterium PS3 and Nitrobacter agilis at the cytochrome oxidase level using total membrane and heptyl thioglucoside.

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Index: J. Biochem. 100(6) , 1465-70, (1986)

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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.