Submicroscopic particles in extracts of Azotobacter agilis

Attempts to describe the location of enzymes in a bacterial cell have been generally unsuccessful because the methods used have been indirect. Alexander (1956) has recently reviewed the literature pertaining to this subject. The discovery that certain enzymes in bacterial extracts are easily sedimented by centrifugation has resulted in these enzymes being correctly designated as particulate. Most investigators have inferred that submicroscopic particles found in extracts exist as such in the cytoplasm of the cell. It is equally possible that these sub-microscopic particles may be derived from a larger structure. The work described in this report has shown that a particulate fraction of Azotobacter agilis (Azotobacter vinelandii) contains two types of particles which differ in chemical composition, enzymatic function, physical structure, and origin. One type contains hydrogenase, cyto-chrome, and phospholipid and is produced by fragmentation of the cell envelope; a second type is a ribonucleoprotein which exists as discrete particles in the cytoplasm. METHODS Cultures. Seven L cultures of Azotobacter agilis (Azotobacter vinelandii strain 0) were grown in Burk's medium for 24 to 30 hr at 30 C. The cells were recovered by centrifugation and washed 3 times by centrifuging from distilled water at 2000 x G for 10 min. The packed cells were stored at-10 C. The details of the methods of cultivation are given by Marr and Cota-Robles (1957). Sonic disruption. Forty ml of a suspension of cells in 0.05 M phosphate buffer, pH 6.8, con-taining 200 mg wet weight of cells per ml was treated in a 10 kc Raytheon sonic oscillator at 1.20 to 1.33 amp, audio frequency (50 to 70 acous