Abstract
Respiratory NADH oxidation in the rumen bacterium Prevotella bryantii is catalyzed by the Na+-translocating NADH:quinone oxidoreductase (NQR). A method for cell disruption and membrane isolation of P. bryantii under anoxic conditions using the EmulisFlex-C3 homogenizer is described. We compared NQR activity and protein yield after oxic and anoxic cell disruption by the EmulsiFlex, by ultrasonication, and by glass beads treatment. With an overall membrane protein yield of 50 mg L–1 culture and a NADH oxidation activity of 0.8 µmol min−1 mg−1, the EmulsiFlex was the most efficient method. Anoxic preparation yielded fourfold higher NQR activity compared to oxic preparation. P. bryantii lacks genes coding for superoxide dismutases and cell extracts do not exhibit superoxide dismutase activity. We propose that inactivation of NQR during oxic cell rupture is caused by superoxide, which accumulates in P. bryantii extracts exposed to air. Anoxic cell rupture is indispensable for the preparation of redox-active proteins and enzymes such as NQR from P. bryantii.
Highlights
Prevotella bryantii is a Gram-negative obligate anaerobe, which is found in anoxic zones of the intestine, such as the rumen of cows (Deusch and Seifert 2015)
During the fermentation of sugar (Hackmann et al 2017), NADH oxidation is catalyzed by a single enzyme, the N a+-NADH:quinone oxidoreductase (NQR) (Deusch et al 2019)
Ultrasonication or compulsion followed by rapid relieve of pressure can be performed. The latter is achieved with the help of the EmulsiFlex-C3 homogenizer (Avestin Inc., Ottawa, Canada) (Tong 2011)
Summary
Prevotella bryantii is a Gram-negative obligate anaerobe, which is found in anoxic zones of the intestine, such as the rumen of cows (Deusch and Seifert 2015). Keywords EmulsiFlex-C3 homogenizer · Prevotella bryantii · Cell rupture · Na+-translocating NADH:quinone oxidoreductase (NQR) · Superoxide We addressed the putative inactivation of P. bryantii NQR by oxidative damage by comparing its activity in oxically and anoxically prepared membranes.
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