Succinoxidase system of Pasteurella tularensis.

Preparation of washed particles. Cell-free extracts of highly virulent P. tularensis strain Sm were prepared by sonic disintegration of cells as described by Rendina and AMills (1957). Twenty ml of undialyzed centrifuged (3000 x G for 30 min) sonic extract, containing 3.8 to 4.2 mg SN per ml, were centrifuged for 3 hr at 105,000 X G at 5 C. The supernatant was decanted and thc reddish precipitate was suspended in 20 ml 0.1 M potassium phosphate buffer, pH 7.5, and recentrifuged 1 hr at 2000 X G. The precipitate, after an additional washing with phosphate buffer, was called "washed particles." Alcohol fractionation of sonic extract. Fifty ml of sonic extract, after dialysis overnight against I LJ distilled water at 4 C, were adjusted to pH 5.4 with acetate buffer. The precipitate was separated by centrifuging at 3000 X G, restispended in distilled water, and the pH adjusted to 6.5 with bicarbonate buffer. Ethanol, 95 per cent, was added slowly to a final concentration of 20 per cent, while maintaining the temperature at or below -2 C. The precipitate was centrifuged off at 3000 X G at -5 C, taken up in water, and lyophilized. This material contained all of the cytochrome b as well as the succinoxi(lase activity of the sonic extract. Oxygen consumption was meastred by conventional techniques in the Warburg respirometer at 37 C, with air as gas phase. Final reaction volume was 3.0 ml, at pH 7.4. Succinoxidase activity was also determined by measuring ac-