Characterization of a Protein Species Isolated from HeLa Cell Cytoplasm by Affinity Chromatography on Polyadenylate-Sepharose

Abstract

Chromatography of different soluble extracts from HeLa cells on poly(A)-Sepharose columns has allowed the isolation of a protein fraction eluted by 0.2 M NaCl and localized predominantly in the cytoplasmic supernatant and in the 0.5 M KCI ribosomal wash. This fraction is present in large amounts (around 3% of total cytosolic proteins) and appears to contain a major protein species that is acidic on electrofocusing (pI around 4.5) and phosphorylated. It runs on glycerol gradients and Sephadex G-200 chromatography close to the aldolase marker (158,000 daltons) and dissociates into apparently identical subunits of 38,000 i 2,000 daltons on sodium dodecyl sulfate-acrylamide gels, suggesting a tetrameric structure.

Interaction of poly(A) and mRNA with eukaryotic initiator met-tRNA-f binding factor: identification of this activity on reticulocyte ribonucleic acid protein particles.

“…The same RNP could be identified by sedimentation in a sucrose gradient, and a similar complex of lower sedimentation value was obtained by incubating synthetic [sH]poly(A) with crude cytoplasmic protein. These results suggested a role for the 3'-poly(A) segment of mRNA as a specific protein binding site (34), and other investigators have reported poly(A)-protein complexes with various partially purified cell fractions (35,36).…”

RNA-binding properties of a translational activator, the adenovirus L4 100-kilodalton protein

“…class of cellular RNA-binding proteins exhibit strong preferences for specific ribohomopolymers. Poly(A)-binding proteins can, for example, be purified by virtue of their selective binding to poly(A) (8,12), the heteronuclear ribonucleoprotein (hnRNP) Cl and C2 proteins bind preferentially to poly(U), and hnRNPs K and J bind very tightly to poly(C) (8,12,38,53). Therefore, we employed the immunoprecipitation assay to determine whether the 100K protein bound preferentially to one or more ribohomopolymers.…”

Developmental changes in the protein and ribonucleic acid components of rat brain messenger ribonucleic acid-protein particles isolated from free polyribosomes by oligo(dT)-cellulose chromatography

“…Fractions containing mRNAprotein particles isolated without a prior 0.5 M-KCI/salt wash of polyribosomes displayed decreased buoyant densities on CsCl gradients and more complex protein composition of the E1 and E2 mRNA-protein fractions (results not shown). A number of the 0.5 M-KCl-extracted polyribosomeassociated proteins have also been shown to bind to poly(A)-Sepharose columns (Blanchard et al, 1974) and to poly(A)-mRNA (Rosenfeld & Barrieux, 1977). However, it is not clear whether in these specific cases the 'mRNA-protein particles' represent reconstituted mRNA-protein particles or merely a fortuitous association of these proteins with mRNA molecules.…”