COUPLED OXIDATIVE PHOSPHORYLATION IN CRUDE EXTRACTS OF AZOTOBACTER'

Two outstanding characteristics of azoto-bacter, their abilitv to fix nitrogen and unusually high rates of oxidation, have stimulated interest in obtaining cell free extracts suitable for bio-chemical analysis of these processes. Many difficulties have been encountered in obtaining such extracts. Attempts to obtain cell free ex-tracts capable of nitrogen fixation have given extremely inconsistent results (reviewed by Burris, 1956, and Magee and Burris, 1956). The methods used for demonstrating coupled oxida-tive phosphorylation in crude and fractionated extracts of mycobacteria and corynebacteria (Brodie and Gray, 1955a, 1955b, 1956a, 1956b, 1956c) did not yield satisfactory results with azotobacter. Recently coupled oxidative phos-phorylation has been demonstrated in fraction-ated extracts of azotobacter (Slater, 1955; Tissieres and Slater, 1955; Rose and Ochoa, 1956). Reinvestigation of the crude azotobacter svstem has revealed that the initial erratic results were largely due to the extremely labile nature of the crude system. The results achieved with extracts stabilized during preparation were reproducible. The nature and properties of this labile system are described and compared with other azotobacter and mycobacterial systems. MATERIALS AND METHODS Azotobacter agile (A. vinelandii), ATCC strain 9104, was grown in 2 L shake flasks containing 600 ml of nitrogen free medium. The composition of the medium was similar to that described b