The respiratory enzyme systems of Azotobacter vinelandii

Abstract

1. 1. An extract from Azotobacter vinelandii was fractionated by centrifugation into a large particle fraction (WLP) and a small particle fraction (WSP), isolated respectively in a field of 22,000 g for 30 min and 145,000 g for 60–120 min, and the supernatant (S 2) from the latter centrifugation. 2. 2. The small particles have a greater respiratory activity than the activity than the large particles, and also a higher P:O ratio with both succinate and DPNH as substrate. If prepared by centrifugation for 120 min at 145,000 g, S 2 has no respiratory activity. 3. 3. The washed small particles do not oxidize a-ketoglutarate unless S 2 is added. Under these conditions, oxidative phosphorylation was demonstrated with both substrates. 4. 4. The effects of added S 2 on the rate of oxidation of succinate can be explained in terms of inhibition by oxaloacetate formed during the oxidation. 5. 5. Treating the large particles with supersonic vibration for 0.5 to 5 min raised the specific activity of the DPNH oxidase or the succinic oxidase systems to the value characteristic of the small particles. 6. 6. The respiratory chain in extracts of Azotobacter vinelandii is localized in the small particles. It is possible that these small particles are identical with granules of about the same size which can be seen in the cytoplasm in electron micrographs of the whole cell. It cannot be excluded, however, that they might be derived by the disintegration of the cell membrane. In any case, these granules differ in several respects from mitochondria, which do not appear to be present in bacterial cells. 7. 7. The fraction WLP probably consists of a mixture of large particles (possibly volutin granules) and aggregated small particles or large pieces of the cell membrane. The small particles are liberated by the action of supersonic vibration.