Studies on the role of the morphological constituents of the microsome fraction from rat liver in protein synthesis

Abstract

Microsomal and submicrosomal fractions were characterized by electron microscopy or analysis for ribonucleic acid (RNA) and protein, or both. The whole microsome pellet from rat liver is believed to contain ribosomes attached to endoplasmic reticulum, free ribosomes and reticulum not associated with ribosomes; the RNA:protein ratio is about 0. 25. Ribonucleoprotein particles were obtained by treatment of the whole microsome pellet with deoxycholate; the RNA:protein ratio is about 1.0. An interface fraction was obtained from the whole microsome pellet by treatment with iso-octane, and contains a modified phospholipid membrane intermixed with free ribosomes; the RNA: protein ratio is 0.13-0. 20. A pellet fraction also obtained from the whole microsome pellet by treatment with iso-octane appears to contain ribosomes most of which were originally attached to endoplasmic reticulum; the RNA:protein ratio is 0. 22-0. 29. A fraction obtained from the whole microsome pellet by sucrose-density-gradient centrifu-gation contains endoplasmic reticulum and free ribosomes but practically no attached ribosomes; the RNA:protein ratio is about 0.05. The effect of polyuridylic acid at various concentrations of Mg2+ ions on the incorporation of C14-phenylalanine into protein was studied by incubating each fraction with an energy source. This optimum Mg2+ ion concentration for fractions containing ribosomes and endoplasmic reticulum was 10 mM. For fractions containing ribonucleoprotein particles detached from the endoplasmic reticulum by either deoxycholate or iso-octane the optimum Mg2+ ion concentration was 15 mM. The results were the same whether the preparations were made from normal or regenerating liver. The optimum conditions for stimulation of C14-phenylalanine incorporation by whole microsomes involved incubation of the preparations washed free of cell sap in the presence of small amounts of pH 5 fraction. Under these conditions there was no significant difference in degree of stimulation by the addition of polyuridylic acid with preprations from either normal or regenerating liver. Whole microsomes were stimulated to a greater extent than ribonucleoprotein particles. The endoplasmic reticulum and free ribosome fraction had very low activity for the incorporation of C14-phenylalanine compared with the whole microsome fraction, but was more markedly stimulated by polyuridylic acid than any of the other fractions. The major protein-synthesizing activity of the liver cell is associated with the ribosomes attached to endoplasmic reticulum. The free ribosomes seem to be comparatively inactive but may be stimulated to synthesize protein by combination with an RNA fraction possessing messenger-like characteristics (ie polyuridylic acid.).