PURIFICATION AND CHARACTERIZATION OF A PROTEIN FACTOR FROM CALF LIVER EXTRACTS THAT PREVENTS REASSOCIATION OF EUKARYOTIC RIBOSOMAL SUBUNITS

Abstract

A protein factor that prevents the reassociation of eukaryotic 40S and 60S ribosomal subunits when the Mg('2+) concentration is raised from 1 mM to 5 mM has been purified to apparent electrophoretic homogeneity from post-ribosomal supernatant of calf liver extracts. Direct assay for ribosomal subunits antiassociation factor activity indicated that the majority (more than 90%) of such factor activity is located in the post-ribosomal supernatant.;The purified anti-association factor, designated AAF, is a single polypeptide chain protein of apparent M(,r)=25,500. AAF is much more efficient in preventing the reassociation of 40S and 60S ribosomal subunits than in directly dissociating 80S ribosomes. Two lines of evidence show that the factor interacts with the 60S subunits preventing reassociation of 60S with 40S subunits. First, preincubation of AAF with 60S subunits followed by addition of 40S subunits prevents reassociation of the subunits at 5 mM Mg('2+). In contrast, when AAF was first incubated with 40S subunits and then 60S subunits were added, the subunits associated at 5 mM Mg('2+) to form 80S ribosomes. Second, using AAF, labeled with ('14)C by reductive alkylation with ('14)C-HCHO, it was demonstrated that AAF forms stable complexes with 60S subunits that can be isolated by sucrose gradient centrifugation. 40S subunits did not significantly bind any labeled AAF.;The function of the factor in initiation of protein synthesis is indicated by the following observation. At 5 mM Mg('2+), the AUG-dependent transfer of Met-tRNA(,f) from ternary complex, (eIF-2.GTP.Met-tRNA(,f)) to 40S ribosomal subunits was prevented if 60S subunits were also included in initiation reaction mixtures presumably due to formation of 80S monosomes. This inhibition can be overcomed by prior incubation of ribosomal subunits with the anti-association factor indicating that the factor plays a role in initiation by ensuring an adequate supply of ribosomal subunits.