This thesis concerns the mechanism of oxidative phosphorylation, with special reference to the bacterium Paracoccus denitrificans. A new method using affinity chromatography is described for the separation of vesicles of different membrane orientation. The working hypothesis is the chemisosmotic theory of Mitchell with its emphasis on the transmembrane proton motive force made up of a membrane potential and a transmembrane pH gradient. The 3 experimental systems used ape bovine heart submitochondrial particles, chromatophores from the photosynthetic bacterium Rhodospirillum rubrum and phosphorylating vesicles from P. denitrificans. From the uncoupling obtained with various ions added to the P. denitrificans particles it is concluded that the transmembrane distribution of weak bases and permeant anions permits a determination of the protonmotive force. A flow-dialysis method has been developed to measure the steady-state transmembrane distribution of weak bases and anions in a determination of the protonmotive force. This method successfully overcomes problems which arise with the techniques previously used for these measurements. Under the experimental conditions used to determine the extent of oxidative phosphorylation (the phosphorylation potential) no transmembrane pH gradient was detectable, and the protonmotive force could be accounted for completely by the membrane potential. A method using ion-selective electrodes was developed to provide a continuous determination of this membrane potential. In all 3 systems the proton motive force determined by the above methods was found to be significantly lower than that required by the chemiosmotic hypothesis. A simple modification of the chemiosmotic hypothesis is proposed. This new concept is based on electrodic theory. It is shown to be useful in explaining results reported in the literature (from both photosynthetic and respiratory systems) which appeared to be at odds with the chemiosmotic theory in its usual form.

Universiẗat, Diss., 2007--Frankfurt (Main). / Zsfassung in dt. und engl. Sprache.

Frankfurt (Main), Universiẗat, Diss., 2006.

Universiẗat, Diss., 2004--Frankfurt (Main).

University, Diss., 2004--Frankfurt (Main).

Universiẗat, Diss., 2007--Frankfurt (Main). / Zsfassung in dt. und engl. Sprache.

Frankfurt (Main), Univ., Diss., 2009

Universiẗat, Diss., 2008--Frankfurt (Main). / Engl. Übers. des Hauptsacht.: Studies on the structure-function relationship of the cytochrome bc1 complex from Paracoccus denitrificans.

A ninhydrin-positive, phosphorus-negative lipid from Paracoccus denitrificans ATCC 13543 has been isolated and purified by mild alkaline methanolysis followed by silicic acid column chromatography and preparative thin-layer chromatography. The lipid was identified as an ornithine-containing lipid. The major ester-linked fatty acid was cis vaccenic acid. Major amide-linked fatty acids were 3-OH-20:1 and 3-OH-18:0. Ornithine-containing lipid was a major lipid component of P. denitrificans. Phospholipids made up about 57% and ornithine-containing lipid about 14% of the weight of the total lipid of the organism. The ratios of lipid ornithine: lipid phosphorus were 0.23, 0.65 and 0.58 in cytoplasmic membrane, outer membrane, and an NaCl extract, which is thought to represent chiefly outer membrane, respectively. Thus ornithine-containing lipid appears to be present in larger amounts in outer membrane than cytoplasmic membrane. No substantial variations in lipid ornithine levels were noted in stationary phase versus exposnential phase organisms, organisms grown in complex medium versus organisms grown in minimal medium with and without amino acid supplements, or in organisms grown in low phosphate-containing medium.

科学研究費補助金 研究種目:一般研究(C) 課題番号:05650796 研究代表者:山根 恒夫 研究期間:1993-1994年度