Reddish supernatant thus obtained was total membrane proteins and subjected to a first ion-exchange chromatography (Biologic LP System, BIO-RAD) on a DEAE-Sephadex column( 4.0 by 16.0 cm) equilibrated with buffer A. This suspension was then ultra- centrifuged at 45000 rpm and 4☌ for 45 min. The resulting mixture was stirred using a magnetic stirrer for 1 hour to solubilize the total cell membrane proteins. Then cell membrane pellet was suspended in 25ml of 50mM Tris-HCl buffer (pH 8.4) with 2.5ml of 50mM EDTA and 4.2 ml of 20% (wt/vol) Triton-X 100 (with final concentration of detergent is 3%). The reddish pellet obtained was of cell membrane whereas the supernatant was of cytoplasm. The supernatant then ultra centrifuged at 35000 rpm and 4☌ for 60 min. Supernatant contained membrane fraction and cytoplasmic fraction where as pellets were of unbroken cells. After sonication the suspension was subjected to centrifuge by using centrifuge machine (HITACHI-CP 80 MX) at 15000 rpm at 4 oC for 15 min. The suspension was sonicated with a sonic oscillator (Soniprep 150 SANYO UK) at 12-14 KHz for total period of 15 min with intervals of 1 min at 4 oC. Purification of membrane bound complex- IIįrozen cells (about 25gm in a centrifugally packed state) were suspended in 100ml of 50mM Tris HCl buffer (pH: 8.4) and 20ml of 50mM EDTA (buffer A). In present study we report the isolation and purification of complex II found in the plasmic membrane of Proteus mirabilis strain ATCC 29245 and its activity in vitro. denitrificans (15) contains one molecule of heme b per FAD which showed that cytochrome b was generally present with the association of succinate: quinine oxidoreductase.
The purified succinate quinine oxidoreductase from Bacillus subtilis contains two hemes b and the other gram negative bacteria such as E. Iron sulfur clusters and flavin adenine dinucleotide (FAD) are located in the water soluble moiety, while cytochrome b is present in membrane-bound moiety. SQR (respiratory complex II) is involved in aerobic metabolism as part of the citric acid cycle and of the aerobic respiratory chain (11). Membrane-bound succinate dehydrogenases (succinate:quinone reductases, SQR) couples the oxidation of succinate to fumarate to the reduction of quinone to quinol and also catalyzes the reverse reaction. It serves as the only direct link between activity in the citric acid cycle and electron transport in the membrane (1). Complex II (succinate:quinone oxidoreductase) of aerobic respiratory chain oxidizes succinate to fumarate and passes electrons directly into the quinone pool. This membrane associated complex catalyses the oxidation of succinate to fumarate and reduce ubiquinone to ubiquinol, and has been characterized in bacteria and heterophilic eukaryotes (19). It functions as a dehydrogenase in the respiratory system and plays an important role in the tricarboxylic acid cycle (TAC) (17). However the presence of the succinate: quinine oxidoreductase (complex II) of Proteus mirabilis has not so far been completely studied.Ĭomplex II of the bacterial electron transport chain is of special interest. Thus a few types of respiratory components of Proteus mirabilis have been studied with their enzymatic and structural features. The function of cytochrome b was investigated in Proteus mirabilis (21) revealed the presence of cytochrome b in Q or b-cycle between the two HQNO inhibition sites. also (20) characterized the presence of cytochrome b and c in plasmic membranes of Proteus mirabilis by means of 77K spectra in both aerobic and anaerobic conditions.
(22) observed the presence of cytochrome b, a1 and d in spectral investigation of the plasmic membrane of Proteus mirabilis. The respiratory chain of bacteria usually composed of enzyme complexes I to IV, ubiquinone, cytochromes, and ATP synthase (complex V), transfers electrons from NADH and succinate at one end to molecular oxygen at the other (2).
In bacterial cells, the generation of energy in the form of adenosine triphosphate (ATP) is mainly motivated by the activity of respiratory chain enzymes of inner plasma membrane (5). It also causes pneumonia, chills, fever, cough and chest pain (4, 12, 27). It infects and persists for a long period of time (26). It is mainly found in GI tract, soil, infections of bladder, lung, urinary tract and wounds. Proteus mirabilis belongs to family Enterobacteriaceae and is facultative anaerobic, rod shaped, and gram negative bacterium (14).