Polyribosomes in Azotobacter vinelandii: I. Isolation, characterization and distribution of ribosomes, polyribosomes and subunits in logarithmically growing Azotobacter

Abstract

1. 1. This report details the procedural requirements for preparing cell-free extracts rich in polyribosomes from Azotobacter vinelandii. This enabled us to demonstrate the occurrence of polyribosomes in Azotobacter and to devise methods for their resolution and isolation. Azotobacter was used in this study since it is readily disrupted by osmotic shock; the gentlest mode of cell breakage available. When certain parameters are followed (the use of log-phase cells, rapidly halting cell metabolism, gentle handling of lysates, sedimentation through exponential sucrose density gradients, the use of flow cells in which remixing is minimized and the maintenance of low temperatures through all procedures) cell extracts containing up to 80–90 % polyribosomes of the total ribosomal population result. Individual polyribosome size classes ranging up to the octamers can be fractionated and separated from their nearest neighbors. Larger size classes are resolved partially among themselves. This was confirmed by extensive electron microscopic studies of material from the various fractions obtained upon density-gradient centrifugation of Azotobacter extracts. 2. 2. The physical and chemical properties of these isolated ribosomes and polyribosomes were then studied. The polyribosomes are ribonuclease sensitive, deoxyribonuclease insensitive, exhibit Mg 2+ dependency for structural integrity, are extremely fragile and have sedimentation coefficients similar to those of other bacteria.