Studies on the Amino Acid Acceptor RNA in Washed Liver Microsomes^*

Abstract

The amount of amino acid acceptor RNA found associated with the microsomes (or ribosomes) depends upon the nature of the medium used in their isolation and upon the time used for their sedimentation. When homogenates of mouse liver were sedimented for varying times at 105,000 X g and the supernatant fraction assayed for RNA and protein content and for its ability to bind amino acids to RNA, it was found that the amino acid acceptor RNA's were not "soluble" but were sedimented out of solution and at different rates. The post-microsomal particles and microsomes can be aggregated with increasing concentrations of MgCl2 up to about 0.015 M and then added MgCl2 causes disaggregation and loss of sedimentation properties. At 0.015 M MgCl2 about half of the RNA normally found in the soluble fraction is sedimented by one hour at 105,000 X g. The ability of the remaining RNA to accept amino acids is increased by only about 20%, and this fact indicates that the sedimented RNA also contains a significant amount of amino acid acceptor activity. The amount of amino acid acceptor RNA associated with the microsomes due to the above conditions can be modified to some extent by the nature of subsequent washing steps. These have included multiple washings with 0.25 M sucrose-0.001 M MgCl2, and treatment with 0.3% deoxycholate or with 0.5 M NaCl. However, no conditions have been found for the complete removal of all of the amino acid acceptor activity. The amino acid acceptor RNA that cannot be washed free from the microsomes does not appear to be due to simple contamination by soluble RNA since the ratios of the acceptor activities of this RNA for leucine, valine, tyrosine, and methionine are different from those found for soluble RNA. Also, the amino acid acceptor RNA from the microsomes differs chromatographically from the majority of the soluble RNA and is more similar to a large portion of that found in the post-microsomal fraction. Microsomal RNA freed of amino acid acceptor RNA (by chromatography) still contains 7% of its uridylic acid as the 5-ribosyl isomer. It thus appears extremely difficult (if not impossible) to completely wash microsomes (or ribosomes) free of amino acid acceptor RNA.