Ribosomes and Ribosomal Protein from Neurospora crassa I. Physical, Chemical, and Immunochemical Properties

Ribosomes from Neurospora crassa, initially characterized by ultracentrifugal and immunochemical analyses, have been used to prepare ribosomal protein for physical, chemical, and immunochemical study. The acrylamide gel disc electrophoretic profiles of Neurospora ribosomal protein exhibit a degree of heterogeneity comparable to what has been observed in other systems. Only by chemical modification or by aggregation of the protein do alterations in the profile become apparent. Disulfide-bond formation appears to play a role in the aggregation of ribosomal protein to complexes of S_20,w = 200. The aggregation can be prevented by alkylation of −SH groups, and protein treated in this fashion has a subunit molecular weight of about 20,000 as determined by equilibrium centrifugation. Finger-printing of tryptic peptides indicates that more than one unique sequence of amino acids must be present in ribosomal protein, although gross primary structural heterogeneity is questioned. Antigenic heterogeneity is much less apparent; only a few precipitin bands are resolved by immunodiffusion tests, although complete reactivity of total ribosomal protein is suggested by quantitative precipitin analysis. The antigenically active ribosomal protein components appear to reside in at least two fractions; one is removed readily from the ribosome by CsC1 treatment. Ribosomal protein of N. crassa possesses antigenic determinants present in E. coli ribosomal protein as judged by spur formation in immunodiffusion tests.     

Isolation and characterization of proteolytic ruminal bacteria from sheep and goats fed the tannin-containing shrub legume Calliandra calothyrsus.

Tannins in forages complex with protein and reduce the availability of nitrogen to ruminants. Ruminal bacteria that ferment protein or peptides in the presence of tannins may benefit digestion of these diets. Bacteria from the rumina of sheep and goats fed Calliandra calothyrsus (3.6% N and 6% condensed tannin) were isolated on proteinaceous agar medium overlaid with either condensed (calliandra tannin) or hydrolyzable (tannic acid) tannin. Fifteen genotypes were identified, based on 16S ribosomal DNA-restriction fragment length polymorphism analysis, and all were proteolytic and fermented peptides to ammonia. Ten of the isolates grew to high optical density (OD) on carbohydrates (glucose, cellobiose, xylose, xylan, starch, and maltose), while the other isolates did not utilize or had low growth on these substrates. In pure culture, representative isolates were unable to ferment protein that was present in calliandra or had been complexed with tannin. One isolate, Lp1284, had high protease activity (80 U), a high specific growth rate (0.28), and a high rate of ammonia production (734 nmol/min/ml/OD unit) on Casamino Acids and Trypticase Peptone. Phylogenetic analysis of the 16S ribosomal DNA sequence showed that Lp1284 was related (97. 6%) to Clostridium botulinum NCTC 7273. Purified plant protein and casein also supported growth of Lp1284 and were fermented to ammonia. This is the first report of a proteolytic, ammonia-hyperproducing bacterium from the rumen. In conclusion, a diverse group of proteolytic and peptidolytic bacteria were present in the rumen, but the isolates could not digest protein that was complexed with condensed tannin.     

N(α)-Acetylation of yeast ribosomal proteins and its effect on protein synthesis

N(α)-Acetyltransferases (NATs) cause the N(α)-acetylation of the majority of eukaryotic proteins during their translation, although the functions of this modification have been largely unexplored. In yeast (Saccharomyces cerevisiae), four NATs have been identified: NatA, NatB, NatC, and NatD. In this study, the N(α)-acetylation status of ribosomal protein was analyzed using NAT mutants combined with two-dimensional difference gel electrophoresis (2D-DIGE) and mass spectrometry (MS). A total of 60 ribosomal proteins were identified, of which 17 were N(α)-acetylated by NatA, and two by NatB. The N(α)-acetylation of two of these, S17 and L23, by NatA was not previously observed. Furthermore, we tested the effect of ribosomal protein N(α)-acetylation on protein synthesis using the purified ribosomes from each NAT mutant. It was found that the protein synthesis activities of ribosomes from NatA and NatB mutants were decreased by 27% and 23%, respectively, as compared to that of the normal strain. Furthermore, we have shown that ribosomal protein N(α)-acetylation by NatA influences translational fidelity in the presence of paromomycin. These results suggest that ribosomal protein N(α)-acetylation is necessary to maintain the ribosome's protein synthesis function.     

An efficient proteomics based strategy for the functional characterization of a novel halophilic enzyme from Halobacterium salinarum

The extremely halophilic archaeon, Halobacterium salinarum grows in environments containing over 25% NaCl. The enzymes of this organism have thus been adapted to be active and stable in hypersaline conditions, which makes them strong candidates as robust industrial enzymes. In this study, the proteomics approach was applied to screen novel halophilic enzymes. We focused initially on proteins that are differentially expressed under different salt concentrations in culture media. After two-dimensional gel electrophoresis over a pH 3.5-4.5 range, 29 differentially expressed protein spots were identified by tandem mass spectrometry and six of these had no similarity to preexisting genes of known function. To predict the function of them, we used various bioinformatic methods. Among other proteins, we selected Vng0487h, which showed a high similarity to acetyltransferases. As a step toward assaying the enzymatic activity of this protein, we cloned the Vng0487h gene of H. salinarum and expressed and purified the recombinant protein with a glutathione-S-transferase (GST) tag in Escherichia coli. Using a GST-pulldown assay, a protein fragment derived from E. coli could interact with recombinant Vng0487h, and was identified to be the ribosomal protein L3. This protein showed high sequence homology with ribosomal protein L7/12 from E. coli and ribosomal protein L13p from H. salinarum. This suggests that Vng0487h acetylates a subunit of ribosomal protein, possibly L13p, in H. salinarum. During the present study, an efficient procedure was established to screen novel halophilic enzymes, and to predict and assess their functions.     

Isolation and Characterization of Proteolytic Ruminal Bacteria from Sheep and Goats Fed the Tannin-Containing Shrub Legume Calliandra calothyrsus

Tannins in forages complex with protein and reduce the availability of nitrogen to ruminants. Ruminal bacteria that ferment protein or peptides in the presence of tannins may benefit digestion of these diets. Bacteria from the rumina of sheep and goats fed Calliandra calothyrsus (3.6% N and 6% condensed tannin) were isolated on proteinaceous agar medium overlaid with either condensed (calliandra tannin) or hydrolyzable (tannic acid) tannin. Fifteen genotypes were identified, based on 16S ribosomal DNA-restriction fragment length polymorphism analysis, and all were proteolytic and fermented peptides to ammonia. Ten of the isolates grew to high optical density (OD) on carbohydrates (glucose, cellobiose, xylose, xylan, starch, and maltose), while the other isolates did not utilize or had low growth on these substrates. In pure culture, representative isolates were unable to ferment protein that was present in calliandra or had been complexed with tannin. One isolate, Lp1284, had high protease activity (80 U), a high specific growth rate (0.28), and a high rate of ammonia production (734 nmol/min/ml/OD unit) on Casamino Acids and Trypticase Peptone. Phylogenetic analysis of the 16S ribosomal DNA sequence showed that Lp1284 was related (97.6%) to Clostridium botulinum NCTC 7273. Purified plant protein and casein also supported growth of Lp1284 and were fermented to ammonia. This is the first report of a proteolytic, ammonia-hyperproducing bacterium from the rumen. In conclusion, a diverse group of proteolytic and peptidolytic bacteria were present in the rumen, but the isolates could not digest protein that was complexed with condensed tannin.     

Substrate preference and phosphatidylinositol monophosphate inhibition of the catalytic domain of the Per-Arnt-Sim domain kinase PASKIN

The Per-Arnt-Sim (PAS) domain serine/threonine kinase PASKIN, or PAS kinase, links energy flux and protein synthesis in yeast, regulates glycogen synthesis and protein translation in mammals, and might be involved in insulin regulation in the pancreas. According to the current model, binding of a putative ligand to the PAS domain disinhibits the kinase domain, leading to PASKIN autophosphorylation and increased kinase activity. To date, only synthetic but no endogenous PASKIN ligands have been reported. In the present study, we identified a number of novel PASKIN kinase targets, including ribosomal protein S6. Together with our previous identification of eukaryotic elongation factor 1A1, this suggests a role for PASKIN in the regulation of mammalian protein translation. When searching for endogenous PASKIN ligands, we found that various phospholipids can bind PASKIN and stimulate its autophosphorylation. Interestingly, the strongest binding and autophosphorylation was achieved with monophosphorylated phosphatidylinositols. However, stimulated PASKIN autophosphorylation did not correlate with ribosomal protein S6 and eukaryotic elongation factor 1A1 target phosphorylation. Although autophosphorylation was enhanced by monophosphorylated phosphatidylinositols, di- and tri-phosphorylated phosphatidylinositols inhibited autophosphorylation. By contrast, target phosphorylation was always inhibited, with the highest efficiency for di- and tri-phosphorylated phosphatidylinositols. Because phosphatidylinositol monophosphates were found to interact with the kinase rather than with the PAS domain, these data suggest a multiligand regulation of PASKIN activity, including a still unknown PAS domain binding/activating ligand and kinase domain binding modulatory phosphatidylinositol phosphates.     

A comparison of ribosomal proteins from rabbit reticulocytes phosphorylated in situ and in vitro.

A comparison has been made between the ribosomal proteins phosphorylated in intact cells and proteins isolated from ribosomal subunits after modification in vitro by purified protein kinases and [gamma-32P]ATP. When intact reticulocytes were incubated for 2 h in a nutritional medium containing radioactive inorganic phosphate, one phosphorylated protein was identified as a 40S ribosomal component using two-dimensional polyacrylamide gel electrophoresis followed by electrophoresis in a third step containing sodium dodecyl sulfate. This protein, containing 99% of the total radioactivity associated with ribosomal proteins as observed by two-dimensional electrophoresis, is found in a nonphosphorylated form in addition to several phosphorylated states. These states differ by the number of phosphoryl group attached to the protein. The same 40S protein is modified in vitro by the three cAMP-regulated protein kinases from rabbit reticulocytes. Two additional proteins associated with the 40S subunit are phosphorylated in situ. These proteins migrate as a symmetrical doublet, and contain less than 1% of the radioactive phosphate in the 40S subunit. A number of phosphorylated proteins associated with 60S subunits are observed by disc gel electrophoresis after incubation of whole cells with labeled phosphate. These proteins do not migrate with previously identified ribosomal proteins and are not present in sufficient amounts to be identified as ribosomal structural proteins. Proteins in the large subunit are modified in vitro by cAMP-regulated protein kinases and ATP, and these modified proteins migrate with known ribosomal proteins. However, this phosphorylation has not been shown to occur in intact cells.     

A mitochondrial protein from Neurospora crassa detected both on ribosomes and in membrane fractions. Analysis of the gene, the message, and the protein

We have isolated clones representing at least three nuclear genes for mitochondrial ribosomal proteins from Neurospora crassa by screening a lambda gt11 cDNA library with an antiserum against a mixture of these proteins. The cDNA and genomic DNA sequence for one of these genes, mrp-3, was determined. The MRP3 protein was purified by immune-affinity chromatography, using a monoclonal antibody probe, and subjected to amino acid sequence analysis to identify the mature amino terminus and a prospective mitochondrial-targeting presequence. MRP3 was identified as the largest, least basic protein detected from the small subunit of ribosomes which had been salt-washed and fractionated on sucrose gradients. However, the mRNA and protein products of mrp-3 were found to be present in excess over those of other N. crassa mitoribosomal protein genes. Using a solution hybridization/S1 nuclease assay, we found three-fold- more mRNA for mrp-3 than for another mito-ribosomal protein gene. In addition, a 30- to 50-fold excess of non-ribosomal MRP3 protein was discovered. The additional protein was localized in mitochondrial membrane fractions; none was detected in matrix fractions after removal of the ribosomes. An immunologically related protein was detected in ribosome and membrane fractions of mitochondria from Saccharomyces cerevisiae. The functional significance of this dual localization remains an enigma.     

Amino acid incorporation by ribosomes and polyribosomes from wheat chloroplasts

Sucrose-gradient and analytical ultracentrifugation showed that chloroplast polyribosomes from 4-day-old seedlings had mono-, di-, tri-, tetra- and traces of penta-ribosomes, in contrast with those from 7-day-old seedlings in which only the mono-, di- and traces of tri-ribosomes were present. Without Mg(2+) the polyribosomes dissociated into ribosomal subunits. The rate of l-[U-(14)C]phenylalanine incorporation was threefold greater for preparations from 4- than from 7-day-old seedlings. Incorporation by the latter was stimulated by polyuridylic acid. The rates of incorporation were similar whether the reaction mixture contained chloroplast or wheat-germ transfer RNA and amino acid synthetases purified on methylated albumin-on-kieselguhr and Sephadex G-75 columns respectively. The cofactor requirement was the same as for isolated intact chloroplasts. Osmotic rupture of chloroplasts with and without Triton X-100 revealed the presence of free and bound ribosomes. Free single ribosomes isolated by osmotic shrinkage or prepared by pancreatic ribonuclease digestion of chloroplast polyribosomes had negligible incorporation activity. This activity was increased by washing or by polyuridylic acid, but was still only a fraction of that given by polyribosomes. A comparison of incorporation activity of chloroplast polyribosomes with those from the surrounding cytoplasm showed the former to be 20 times more active.     

Identification of antigenic proteins from Neospora caninum recognized by bovine immunoglobulins M, E, A and G using immunoproteomics

Antigenic proteins of Neospora caninum (N. caninum) against bovine immunoglobulins M, E, A, and G were investigated by using immunoproteomics. Proteins of N. caninum (KBA-2) tachyzoite lysates separated by two-dimensional gel electrophoresis were transferred to polyvinylidene difluoride (PVDF) membranes, probed with different bovine immunoglobulin class and classified. Antigenic spots recognized were also identified by matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis. 132, 84, 4, and 40 antigenic protein spots were recognized on N. caninum immunoblot profiles against bovine IgM, IgE, IgA, and IgG, respectively. Of these protein spots, the antigenic proteins recognized by either IgM, IgE, and IgG, or IgM and IgG were HSP70, pyruvate kinase, actin, NCDG-1, tubulin alpha-chain, and putative ribosomal protein S2. On the other hand, IgM, IgE, and IgA reacted with NTPase, HSP60, tubulin beta-chain, putative protein disulfide isomerase, enolase, lactate dehydrogenase, serine-threonine phosphatase, 14-3-3 protein homologue, and GRA2 protein. Most of the antigenic proteins identified were associated with the process of invasion, proliferation, and egression of apicomplexans. In our study, HSP70, actin, NTPase, HSP60, pyruvate kinase, enolase, putative ribosomal protein S2, NCDG-1, and GRA2 proteins were found to be immunodominant proteins, which may contribute to the development of diagnostic markers and vaccine.     

Purification and properties of a ribosomal protein methylase from Eschericha coli Q13.

Ribosomal protein methylase has been purified from Escherichia coli strain Q13 using methyl-deficient 50S subunits as substrates. The purified enzyme (or enzyme complex) which is devoid of rRNA methylating activity is quite stable and has a pH optimum around 8.0. The Km for S-adenosyl-L-methionine is 3.2 muM. The molecular weight of the enzyme is 3.1 X 10(4); minor methylating activity was also detected for protein peaks with molecular weights of 1.7 X 10(4) and 5.6 X 10(4). Protein L11 is the major protein methylated by the purified enzyme. Product analysis revealed the presence of N epislon-trimethyllysine, a methylated neutral amino acid(s) previously observed in protein L11 and N epislon-monomethyllysine. Free ribosomal proteins were much better substrates for the methylation, indicating that methylation of 50S ribosomal proteins can occur before the complete assembly of the 50S ribosomal subunit.     

High heterogeneity within the ribosomal proteins of the <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> 80S ribosome

Proteomic studies have addressed the composition of plant chloroplast ribosomes and 70S ribosomes from the unicellular organism Chlamydomonas reinhardtii But comprehensive characterization of cytoplasmic 80S ribosomes from higher plants has been lacking. We have used two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) to analyse the cytoplasmic 80S ribosomes from the model flowering plant Arabidopsis thaliana. Of the 80 ribosomal protein families predicted to comprise the cytoplasmic 80S ribosome, we have confirmed the presence of 61; specifically, 27 (84%) of the small 40S subunit and 34 (71%) of the large 60S subunit. Nearly half (45%) of the ribosomal proteins identified are represented by two or more distinct spots in the 2-DE gel indicating that these proteins are either post-translationally modified or present as different isoforms. Consistently, MS-based protein identification revealed that at least one-third (34%) of the identified ribosomal protein families showed expression of two or more family members. In addition, we have identified a number of non-ribosomal proteins that co-migrate with the plant 80S ribosomes during gradient centrifugation suggesting their possible association with the 80S ribosomes. Among them, RACK1 has recently been proposed to be a ribosome-associated protein that promotes efficient translation in yeast. The study, thus provides the basis for further investigation into the function of the other identified non-ribosomal proteins as well as the biological meaning of the various ribosomal protein isoforms.Patrick Giavalisco, Daniel Wilson are contributed equally to this work.     

Genetics of ribosomal protein methylation in Escherichia coli. II. A mutant lacking a new type of methylated amino acid, N5-methylglutamine, in protein L3.

The ribosomes of an Escherichia coli mutant, designated prm-2, can be methylated in vitro by an enzymatic fraction from wild-type. This enzyme is inactive on the ribosomes from another mutant, prm-1, is reported previously to be methyl group-deficient in protein L11. In vitro methylation of prm-2 ribosomes resulted in the incorporation of about one methyl group per molecule of protein L3. After acid hydrolysis, all the methyl groups were found in a very basic compound which was identified as methylamine. This compound could have been generated by acid hydrolysis of N-methylated amide-groups from glutamine or asparagine. Therefore, chemically-synthesized N4-methyl-asparagine and N5-methylglutamine were chromatographed together with an enzymatic hydrolysate of methylated prm-2 proteins. In all the chromatogrphic systems studied the methylated amino acid was found in the same position as N5'-methylglutamine. These results indicate that mutant prm-2 lacks one residue of N5-methylglutamine present in ribosomal protein L3 of wild type E. coli.     

Evidence for an additional archaebacterial gene cluster in Halobacterium marismortui encoding ribosomal proteins HL46e and HL30

A small and extremely basic ribosomal protein (HL46e) has been purified from Halobacterium marismortui using reversed-phase high-performance liquid chromatography (HPLC). The amino acid sequence of the protein was determined by automated N-terminal and internal sequence analysis. Comparison of this sequence with other ribosomal protein sequences from eubacteria, archaebacteria and eukaryotes revealed a strong homology to SL46e from Sulfolobus solfataricus, YeaL46 from yeast and RL39 from rat. No significant sequence similarly was found to any eubacterial ribosomal protein so far known. Using a specific oligonucleotide probe the HL46e gene was identified, cloned and the nucleotide sequence including the 5'- and 3'-flanking regions were analysed. The HL46e gene is followed by the gene coding for HL30. A putative halobacterial promoter sequence with the motive 'TTTAAA' has been localized 32 bp upstream of the HL46e gene and a putative terminator sequence localized downstream from the HL30 gene. An equivalent to this HL46e/HL30 operon is apparently not present in Escherichia coli.     

Application of high-performance liquid chromatography to the purification and characterization of ribosomal protein L3 from trichodermin-resistant yeast mutants.

A new h.p.l.c. cation-exchange method has been used to separate proteins from 60S ribosomal subunits prepared from strains of Saccharomyces cerevisiae sensitive or resistant to trichodermin. Ribosomal protein L3 was identified in column eluates by one-dimensional and two-dimensional gel electrophoresis and purified further by reverse-phase h.p.l.c. The protein was cleaved with CNBr and the products were analysed, again by reverse-phase h.p.l.c. A marked difference was observed in the peptide profiles between preparations from trichodermin-sensitive and trichodermin-resistant yeast strains. These results provide the first direct demonstration that, in yeast, mutationally induced resistance to trichodermin can alter the covalent structure of ribosomal protein L3. They convincingly demonstrate the potential of the experimental technique for the rapid and preparative separation of a selected yeast ribosomal protein and its subsequent characterization.     

The ribosomal proteins phosphorylated in vitro by protein kinase activities from Krebs II ascites cells

Studies were performed to identify in cytoplasmic extracts of Krebs II ascites cells protein kinase activities that might be responsible for the phosphorylation of the ribosomal proteins previously identified as phosphoproteins in these cells in vivo. Column chromatography resolved a casein kinase activity that could use ATP or GTP as a phosphoryl donor to phosphorylate, in ribosomes, exclusively the acidic 60S phosphoprotein(s) phosphorylated in vivo. A second casein kinase fraction could use ATP, only, in a similar reaction, but also contained protein kinase activity with respect to other ribosomal proteins, including the basic ribosomal protein phosphorylated in vivo, ribosomal protein S6. This latter was also among several proteins phosphorylated by an activity in the cyclic AMP-independent histone kinase fraction.     

Characterization of the 70S Ribosome from Rhodopseudomonas palustris using an integrated "top-down" and "bottom-up" mass spectrometric approach

We present a comprehensive mass spectrometric approach that integrates intact protein molecular mass measurement ("top-down") and proteolytic fragment identification ("bottom-up") to characterize the 70S ribosome from Rhodopseudomonas palustris. Forty-two intact protein identifications were obtained by the top-down approach and 53 out of the 54 orthologs to Escherichia coli ribosomal proteins were identified from bottom-up analysis. This integrated approach simplified the assignment of post-translational modifications by increasing the confidence of identifications, distinguishing between isoforms, and identifying the amino acid positions at which particular post-translational modifications occurred. Our combined mass spectrometry data also allowed us to check and validate the gene annotations for three ribosomal proteins predicted to possess extended C-termini. In particular, we identified a highly repetitive C-terminal "alanine tail" on L25. This type of low complexity sequence, common to eukaryotic proteins, has previously not been reported in prokaryotic proteins. To our knowledge, this is the most comprehensive protein complex analysis to date that integrates two MS techniques.     

The hemK gene in Escherichia coli encodes the N(5)-glutamine methyltransferase that modifies peptide release factors

Class 1 peptide release factors (RFs) in Escherichia coli are N(5)-methylated on the glutamine residue of the universally conserved GGQ motif. One other protein alone has been shown to contain N(5)-methylglutamine: E.coli ribosomal protein L3. We identify the L3 methyltransferase as YfcB and show that it methylates ribosomes from a yfcB strain in vitro, but not RF1 or RF2. HemK, a close orthologue of YfcB, is shown to methylate RF1 and RF2 in vitro. hemK is immediately downstream of and co-expressed with prfA. Its deletion in E.coli K12 leads to very poor growth on rich media and abolishes methylation of RF1. The activity of unmethylated RF2 from K12 strains is extremely low due to the cumulative effects of threonine at position 246, in place of alanine or serine present in all other bacterial RFs, and the lack of N(5)-methylation of Gln252. Fast-growing spontaneous revertants in hemK K12 strains contain the mutations Thr246Ala or Thr246Ser in RF2. HemK and YfcB are the first identified methyltransferases modifying glutamine, and are widely distributed in nature.     

Cross-linking of initiation factor IF3 to proteins of the Escherichia coli 30 S ribosomal subunit

Complexes of 30 S subunits and [¹⁴C]IF3 were allowed to react with the protein cross-linking reagents, N,N′-p-phenylenedimaleimide or dimethylsuberimidate. Non-cross-linked IF3 was removed from the complex by centrifugation in a buffer containing a high salt concentration, and the total protein was extracted from the pelleted particles. The mixture of cross-linked products was analyzed by radioimmunodiffusion with antisera prepared against all of the individual 30 S ribosomal proteins. Radioactivity was found in the precipitin bands formed with antisera against ribosomal proteins S1, S11, S12, S13, S19 and S21. The results show that IF3 was present in covalent cross-linked complexes containing those 30 S ribosomal proteins and imply that they comprise or are near the binding site for initiation factor IF3.     

Eukaryotic initiation factor 5 from calf liver is a single polypeptide chain protein of Mr = 62,000

Eukaryotic initiation factor 5 (eIF-5), which specifically catalyzes the joining of a 60 S ribosomal subunit to a 40 S initiation complex to form a functional 80 S initiation complex, has been purified from ribosomal salt wash proteins of calf liver. The purified factor exhibits only one polypeptide band of Mr = 62,000 following electrophoresis in 10% polyacrylamide gels in the presence of sodium dodecyl sulfate. The native protein has a sedimentation coefficient of 4.2 S and a Stokes radius of 33 A which is consistent with eIF-5 being a monomeric protein of Mr = 58,000-62,000. Less pure preparations of eIF-5 elute in gel filtration columns with an apparent Mr of 160,000-180,000 presumably due to association of eIF-5 with other high molecular weight proteins since eIF-5 activity present in such preparations can also be shown by gel electrophoretic separation under denaturing conditions to be associated with a 62,000-dalton protein. Furthermore, eIF-5 purified from calf liver extracts with or without a number of protease inhibitors is indistinguishable with regard to molecular weight and final specific activity of purified preparations. The purified factor catalyzes the hydrolysis of GTP present in 40 S initiation complexes in the absence of 60 S ribosomal subunits. The presence of 60 S ribosomal subunits neither stimulates nor inhibits the hydrolysis of GTP. However, the factor cannot mediate 40 S or 40 + 60 S ribosome-dependent hydrolysis of GTP in the absence of Met-tRNAf or other components required for 40 S initiation complex formation. It can be calculated that 1 pmol of eIF-5 protein can catalyze the formation of at least 10 pmol of 80 S initiation complex under the conditions of in vitro initiation reactions.