Structural characterization of polysomal poly(A)-protein particles in rat liver.
Poly(A)-protein particles were prepared from rat liver polyribosomes, washed with 0.5 M KCl or unwashed, after digestion with pancreatic ribonuclease and ribonuclease T1 by two 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 ribonuclease 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 six polypeptides with molecular weights 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-molecular-weight 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 ribonuclease 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 electron micrographs 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.
D000818:Animals; D002499:Centrifugation, Density Gradient; D004720:Endonucleases; D004722:Endoribonucleases; D008099:Liver / Q000032:analysis*; D046911:Macromolecular Substances; D008854:Microscopy, Electron; D011061:Poly A / Q000032:analysis*; D011132:Polyribosomes / Q000032:analysis*; D051381:Rats; D012260:Ribonucleases; D012269:Ribosomal Proteins / Q000032:analysis*
D046911:Macromolecular Substances; D012269:Ribosomal Proteins; D011061:Poly A; D004720:Endonucleases; D004722:Endoribonucleases; D012260:Ribonucleases; C022543:ribonuclease T(2)