Abstract
In the previous communication, it was demonstrated that the ribosome fraction was obtained from the cell-free extract of Mycobacterium 607 by preparative ultracentrifugation.
This paper deals with isolation procedure and characterization of the ribosome of BCG.
BCG was cultured with a Tween synthetic medium at 37°C for 14 days. The cells were harvested by centrifugation, and washed with and resuspended in the 0.01M tris buffer containing 0.01M of MgCl2(tris-Mg buffer). The cell-free extract of the washed cells was prepared by passing through the French pressure cell at 400Kg/cm2 followed by centrifugation at 5, 000g for 20minutes.
Particulate fractions were isolated from the cell-free extract by a successive ultracentrifugation at 20, 000g for 60 minutes (20p60 fraction), 60, 000g for 90 minutes (60p90 fraction) and 105, 000g for 120 minutes (105p120 (I) fraction). Final supernatant fraction (105s120 (I) fraction) was used as a soluble enzyme. In some experiments, the ribosome fraction was directly obtained by supercentrifugation at 105, 000g for 120 minutes (105p120 (II) fraction) after removal of 20p60 fraction from the cell-free extract by centrifugation. Each particulate fraction was suspended in the tris-Mg buffer, and analyzed physicochemically and biologically. Results obtained were as follows:
1) All of the 60p90, 105p120 (I) and 105p120 (II) fractions were found to contain a higher amount of nucleic acid than 105s120 fraction U-V spectrophotometorically.
2) The RNA and protein ratios in the 60p90, 105p120 (I) and 105p120 (II) fractions were 58, 8: 41, 2, 51, 5: 48, 5 and 61, 3: 38, 7, respectively.
3) As observed by analytical centrifugation patterns, the 60p90, 105p120 (I) and 105p120 (II) fractions consisted of 71, 4 S and 48, 3 S particles.
4) An electronmicrograph of the 105p120 (II) fraction demonstrated globular granules with an average diameter of 30-40mμ.
5) The 105p120 (II) fraction was capable to incorporate14C-amino acids into its protein fraction in the presence of ATP and its generator, 105s120 fraction, GTP and GSH.
These results may indicate that the 60p90, 105p120 (I) and 105p120 (II) fractions are the ribosome fractions of BCG.
