Structural Characterization of Polysomal Poly(A)‐Protein Particles in Rat Liver

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Abstract
Poly(A)-protein particles were prepared from rat liver polyribosomes, washed with 0.5 M KCl or unwashed, after digestion with pancreatic RNase and RNase T1 by 2 successive rounds of sucrose gradient centrifugation. The particles were sedimented in a range of 5-13 S with a peak at about 9 S. The KCl wash of polysomes had no effect on the sedimentation properties of the particles. The particles isolated in this manner were 99% resistant to further pancreatic RNase treatment and contained about 96% adenylic acid. The length of the poly(A) molecules prepared from the poly(A)-protein particles showed a broad distribution of about 70-290 nucleotides with a peak around 130 nucleotides, as measured by polyacrylamide gel electrophoresis. In CsCl density gradient the poly(A)-protein particles banded in a density range of 1.30-1.42 g/cm3 with a peak at 1.36 g/cm3, which amounts to about 80% of the protein content. Sodium dodecyl sulfate/polyacrylamide and urea/sodium dodecyl sulfate/polyacrylamide gel electrophoresis demonstrated 6 polypeptides with MW of 50,000, 54,000, 58,000, 63,000, 76,000 and 90,000 in the poly(A)-protein particles but the main components were dependent on the method. The treatment of polysomes with KCl resulted in a loss of the 90,000 MW component. Amino acid analysis of the polypeptides bound to poly(A) revealed that they contained a relatively large amount of aspartic plus glutamic acid (21.6%) as well as hydrophobic amino acids (41.4%). Digestion of glutaraldehyde-fixed particles with RNase T2 showed that about 50% of poly(A) was accessible to the enzyme, thus this part of poly(A) was located on the surface of the particles. In the EM the shadowed poly(A)-protein particles appeared in a globular, somewhat elongated form and were mostly 14-18 nm in diameter. On the basis of the results a model for the average 9-S particles was constructed.