Purification of messenger ribonucleoprotein particles from rabbit reticulocytes by zonal centrifugation in metrizamide.

Abstract

A large-scale purification procedure for messenger ribonucleoprotein (mRNP) particles from rabbit reticulocyte polysomes is described. The mRNP particles were dissociated from polysomes by treatment with urea and separated by differential centrifugation under conditions of high ionic strength. Zonal centrifugation in a metrizamide buoyant density gradient was the final purification step. One major class of mRNA particle was observed. The RNA was defined as mRNA by polyacrylamide gel electrophoresis and by globin production in a cell-free protein-synthesizing system. The twenty-three different proteins associated with the particle were a discrete set of proteins, which ranged in molecular weight from 175,000 to 23,500. The relative amount of each peptide in the particle was determined from a gel scan of the stained protein by computer simulation. None of the polypeptides comigrated with proteins from the 40-S and 60-S ribosomal subunits when analyzed by two-dimensional polyacrylamide gel electrophoresis.