Methods of cell disintegration and the sedimentation patterns of ribosomes inEscherichia coli

A number of methods for the disintegration ofEscherichia coli cells was followed to investigate their suitability for the isolation of ribosomes. The decrease of viability and the conditions for fine disruption of the cells for all methods were established. The sedimentation coefficients of ribosomes and their sizedistribution in the cell-free extracts in dependence upon the method employed and the concentration of Mg⁺⁺ ions were determined by analytical ultracentrifugation. The sedimentation coefficients and the sizedistribution of ribosomes did not differ in most methods and the results obtained were basically in agreement with the data given by other authors. The method of grinding with alumina seemed to be too vigorous and the results obtained were less reproducible. Ribosomes from extracts prepared by osmotical shocking of lysozyme spheroplasts showed ultracentrifugation patterns differing from those found with other methods: In extracts with low (0.00025m) and high (0.01m) concentration of Mg⁺⁺ almost the whole range of ribosomes was found whereas in the buffer with 0.001m Mg⁺⁺ only 30 and 50s ribosomes were present. alumina Particles with a sedimentation constant higher than 100s (polysomes) were not found in any of our extracts. We attempted to discuss our results and compare them with the data given in other reports. The method of osmotical lysis of spheroplast seems to give results most closely related to thein vivo situation in the cell and enables us to study the functional relationship of ribosomes to other cell components namely the cytoplasmic membrane.