EF-1 Gamma Research Articles

Construction of a subtractive library from hexavalent chromium treated winter flounder ( Pseudopleuronectes americanus) reveals alterations in non-selenium glutathione peroxidases

Chromium is released during several industrial processes and has accumulated in some estuarine areas. Its effects on mammals have been widely studied, but relatively little information is available on its effects on fish. Gene expression changes are useful biomarkers that can provide information about toxicant exposure and effects, as well as the health of an organism and its ability to adapt to its surroundings. Therefore, we investigated the effects of Cr(VI) on gene expression in the sediment dwelling fish, winter flounder ( Pseudopleuronectes americanus). Winter flounder ranging from 300 to 360 g were injected i.p. with Cr(VI) as chromium oxide at 25 μg/kg chromium in 0.15N KCl. Twenty-four hours following injections, winter flounder were euthanized with MS-222 and the livers were excised. Half of the livers were used to make cytosol and the other half were used to isolate mRNA for subtractive hybridization. Subtractive clones obtained were spotted onto nylon filters, which revealed several genes with potentially altered expression due to Cr(VI), including an α class GST, 1-Cys peroxiredoxin (a non-selenium glutathione peroxidase), a P-450 2X subfamily member, two elongation factors (EF-1 gamma and EF-2), and complement component C3. Semi-quantitative RT-PCR was performed and confirmed that Cr(VI) down-regulated complement component C3, an EST, and two potential glutathione peroxidases, GSTA3 and 1-Cys peroxiredoxin. In addition, cytosolic GSH peroxidase activity was reduced, and silver stained SDS-PAGE gels from glutathione-affinity purified cytosol demonstrated that a 27.1 kDa GSH-binding protein was down-regulated greater than 50%. Taken together, Cr(VI) significantly altered the expression of several genes including two potential glutathione peroxidases in winter flounder.

Regulated interaction between polypeptide chain elongation factor-1 complex with the 26S proteasome during Xenopus oocyte maturation

BackgroundDuring Xenopus oocyte maturation, the amount of a 48 kDa protein detected in the 26S proteasome fraction (p48) decreased markedly during oocyte maturation to the low levels seen in unfertilized eggs. The results indicate that the interaction of at least one protein with the 26S proteasome changes during oocyte maturation and early development. An alteration in proteasome function may be important for the regulation of developmental events, such as the rapid cell cycle, in the early embryo. In this study, we identified p48.Resultsp48 was purified by conventional column chromatography. The resulting purified fraction contained two other proteins with molecular masses of 30 (p30) and 37 (p37) kDa. cDNAs encode elongation factor-1γ and δ were obtained by an immuno-screening method using polyclonal antibodies against purified p48 complex, which recognized p48 and p37. N-terminal amino acid sequence analysis of p30 revealed that it was identical to EF-1β. To identify the p48 complex bound to the 26S proteasome as EF-1βγδ, antibodies were raised against the components of purified p48 complex. Recombinant EF-1 β,γ and δ were expressed in Escherichia coli, and an antibody was raised against purified recombinant EF-1γ. Cross-reactivity of the antibodies toward the p48 complex and recombinant proteins showed it to be specific for each component. These results indicate that the p48 complex bound to the 26S proteasome is the EF-1 complex. MPF phosphorylated EF-1γ was shown to bind to the 26S proteasome. When EF-1γ is phosphorylated by MPF, the association is stabilized.Conclusionp48 bound to the 26S proteasome is identified as the EF-1γ. EF-1 complex is associated with the 26S proteasome in Xenopus oocytes and the interaction is stabilized by MPF-mediated phosphorylation.

A major substrate for MPF: cDNA cloning and expression of polypeptide chain elongation factor 1 gamma from goldfish (Carassius auratus).

One of the eukaryotic polypeptide chain elongation factors, EF-1 beta gamma delta complex, is involved in polypeptide chain elongation via the GDP/GTP exchange activity of EF-1 alpha. In the complex, EF-1 gamma has been reported to be a major substrate for maturation promoting factor (MPF). Here, we present the cloning, sequencing and expression analysis of goldfish, Carassius auratus, EF-1 gamma from the goldfish ovary. The cloned cDNA was 1490 bp in length and encoded 442 amino acids. The deduced amino acid sequence was highly homologous to EF-1 gamma from other species. Although, the phosphorylation site identified in Xenopus EF-1 gamma was not conserved in the goldfish homologue, phosphorylation analysis showed that the goldfish EF-1 gamma was phosphorylated by MPF. We concluded that EF-1 gamma is a substrate for MPF during oocyte maturation in goldfish.

Inhibition of Endothelial Cell Thromboresistance by Homocysteine

Homocysteine (HC) is a highly reactive thiol intermediate in amino acid metabolism, which can modify the function of endothelial cells in a myriad of ways. In vitro, homocysteine can inhibit the thromboresistance properties of the endothelial cell by induction of procoagulant factors, inactivation of natural anticoagulant systems, and suppression of vasodilatory and platelet-modulating factors. HC also inhibits the fibrinolytic system by impairing the ability of the endothelial cell to bind tissue plasminogen activator (t-PA), by interacting directly with the t-PA binding "tail" domain of its endothelial cell receptor, annexin II. Moreover, HC influences endothelial cell gene expression as exemplified by induction of the elongation factor-1 family of polypeptides, which promote polypeptide chain elongation during mRNA translation. Induction of EF-1 subunits alpha, beta, gamma and delta by homocysteine is associated with increased turnover of at least one free thiol-containing protein, suggesting that up-regulation of these subunits may represent a mechanism for replacement of damaged or modified proteins. A more complete understanding of the diverse effects of homocysteine on endothelial cell function may provide important clues to the precise role homocysteine may play in the initiation and progression of vascular disease.

DNA binding activity of the mammalian translation elongation complex: recognition of chromium- and transplatin-damaged DNA.

The elongation factor complex, EF-1H, serves an essential function in protein biosynthesis in eukaryotic cells, although the role of EF-1H in other physiological processes is unknown. In this report, we demonstrate that three components of EF-1H (EF-1 beta, EF-1 delta, EF-1 gamma) bind to DNA modified with chromium (Cr), a potent DNA-damaging agent and an established human carcinogen. The EF-1H complex also binds to transplatin modified DNA but not to cisplatin-modified DNA. These results demonstrate that the EF-1H complex has functional DNA binding activity and is capable of recognizing the distortions in DNA structure resulting from the covalent binding of Cr and transplatin to DNA.

Elongation factor 1 gamma mRNA expression in oesophageal carcinoma.

Elongation factor 1 gamma (EF1 gamma) is known to be a subunit of EF1, one of the G proteins that mediate the transport of aminoacyl tRNA to 80S ribosomes during translation. As little is known regarding the expression of EF1 gamma in human oesophageal carcinoma, this study looked at its expression using a northern blot analysis. Thirty six cases of oesophageal carcinoma and 15 oesophageal carcinoma cell lines were studied. The EF1 gamma mRNA overexpression at a level of twofold or more was seen in five (14%) of 36 carcinomatous tissues compared with the normal counterparts. All five overexpressed cases showed severe lymph node metastases compared with the non-overexpressed cases, and the difference was significant (p = 0.028). The stage of the disease of these five cases was far advanced compared with the nonoverexpressed cases (p = 0.012). All 15 oesophageal carcinoma cells expressed EF1 gamma mRNA relatively lower than the gastric or pancreatic carcinoma cell lines, in which EF1 gamma was originally isolated. As the expression of EF1 gamma mRNA could be detected even in the biopsy specimens, its overexpression in tumour tissue may provide preoperative useful information for predicting the aggressiveness of tumours.

The overexpression of elongation factor 1 gamma mRNA in gastric carcinoma

Elongation factor 1 gamma (EF1 gamma) has been isolated from a pancreatic cancer cell line, while the overexpression of EF1 gamma has also been observed in colorectal carcinomas and adenomas. There is, however, still little information on the expression of EF1 gamma in gastric carcinoma. The authors examined the expression of EF1 gamma mRNA in 30 gastric carcinomas and their corresponding paired adjacent normal mucosae obtained during surgery while also examining 10 human digestive tract carcinoma cell lines using Northern blot hybridization. An overexpression was observed in 22 (73%) of 30 gastric carcinomas, relative to the corresponding normal tissue. In a clinicopathologic study, no relationship was observed between the overexpression of EF1 gamma mRNA and the histologic grading or depth of tumor invasion. However, 15 (68%) of 22 overexpressed cases were positive for vascular permeation, whereas only 1 of 8 nonoverexpressed cases was positive for vascular permeation, and the difference was significant (P < 0.05). Nine (90%) of 10 with more than a 10-fold overexpression relative to the corresponding normal tissue showed severe vascular permeation in the histologic specimens. Conversely, the expression of EF1 gamma was observed not only in the pancreatic and gastric carcinoma cell lines, but also in the hepatic, ileocecal, duodenal, and colon carcinoma cell lines. The overexpression of EF1 gamma mRNA was observed in both the gastric carcinoma tissue and in all the other cell lines examined. This probable correlation with vascular permeation in gastric carcinoma is thus considered to provide another piece of useful pathologic information.

Eukaryotic translation elongation factor 1 gamma contains a glutathione transferase domain--study of a diverse, ancient protein superfamily using motif search and structural modeling.

Using computer methods for multiple alignment, sequence motif search, and tertiary structure modeling, we show that eukaryotic translation elongation factor 1 gamma (EF1 gamma) contains an N-terminal domain related to class theta glutathione S-transferases (GST). GST-like proteins related to class theta comprise a large group including, in addition to typical GSTs and EF1 gamma, stress-induced proteins from bacteria and plants, bacterial reductive dehalogenases and beta-etherases, and several uncharacterized proteins. These proteins share 2 conserved sequence motifs with GSTs of other classes (alpha, mu, and pi). Tertiary structure modeling showed that in spite of the relatively low sequence similarity, the GST-related domain of EF1 gamma is likely to form a fold very similar to that in the known structures of class alpha, mu, and pi GSTs. One of the conserved motifs is implicated in glutathione binding, whereas the other motif probably is involved in maintaining the proper conformation of the GST domain. We predict that the GST-like domain in EF1 gamma is enzymatically active and that to exhibit GST activity, EF1 gamma has to form homodimers. The GST activity may be involved in the regulation of the assembly of multisubunit complexes containing EF1 and aminoacyl-tRNA synthetases by shifting the balance between glutathione, disulfide glutathione, thiol groups of cysteines, and protein disulfide bonds. The GST domain is a widespread, conserved enzymatic module that may be covalently or noncovalently complexed with other proteins. Regulation of protein assembly and folding may be 1 of the functions of GST.

Reconstitution in vitro of the valyl-tRNA synthetase-elongation factor (EF) 1 beta gamma delta complex. Essential roles of the NH2-terminal extension of valyl-tRNA synthetase and of the EF-1 delta subunit in complex formation.

Valyl-tRNA synthetase from mammalian cells is isolated exclusively as a complex with elongation factor (EF) 1H (the "heavy" form of eukaryotic EF-1, composed of subunits alpha, beta, gamma, and delta). In a previous study, the 140-kDa valyl-tRNA synthetase subunit dissociated from the purified rabbit liver complex was shown to display hydrophobic properties, unlike the corresponding yeast cytoplasmic enzyme of 125 kDa (Bec, G., and Waller, J.-P. (1989) J. Biol. Chem. 264, 21138-21143). Compared to the sequence of yeast cytoplasmic valyl-tRNA synthetase, that of the human enzyme displays an NH2-terminal extension of approximately 200 amino acid residues that bears strong sequence similarity to the NH2-terminal moiety of EF-1 gamma (Hsieh, S. L., and Campbell, R. D. (1991) Biochem. J. 278, 809-816). We now show that this NH2-terminal extension can be selectively excised by elastase treatment of the isolated rabbit valyl-tRNA synthetase, without impairing catalytic activity. To examine the role of the NH2-terminal extension of mammalian valyl-tRNA synthetase in complex formation and to identify the subunit(s) of EF-1H responsible for binding the enzyme, reconstitution experiments were undertaken. Native or truncated valyl-tRNA synthetases were incubated with the isolated EF-1 subunits beta gamma and delta, either separately or in combination, and the ensuing products were analyzed by chromatography on DEAE-Sepharose FF and Superose 6. The results demonstrate that the NH2-terminal extension of valyl-tRNA synthetase is required for complex formation and that the enzyme-binding site(s) resides on the EF-1 delta subunit. Moreover, although the EF-1 beta gamma binary complex does not bind valyl-tRNA synthetase, it is nevertheless required for assembly of a complex of defined quaternary structure by preventing the formation of high molecular weight aggregates generated in the presence of EF-1 delta alone.

DRS1 to DRS7, novel genes required for ribosome assembly and function in Saccharomyces cerevisiae.

To identify Saccharomyces cerevisiae mutants defective in assembly or function of ribosomes, a collection of cold-sensitive strains generated by treatment with ethyl methanesulfonate was screened by sucrose gradient analysis for altered ratios of free 40S to 60S ribosomal subunits or qualitative changes in polyribosome profiles. Mutations defining seven complementation groups deficient in ribosomal subunits, drs1 to drs7, were identified. We have previously shown that DRS1 encodes a putative ATP-dependent RNA helicase necessary for assembly of 60S ribosomal subunits (T. L. Ripmaster, G. P. Vaughn, and J. L. Woolford, Jr., Proc. Natl. Acad. Sci. USA 89:11131-11135, 1992). Strains bearing the drs2 mutation process the 20S precursor of the mature 18S rRNA slowly and are deficient in 40S ribosomal subunits. Cloning and sequencing of the DRS2 gene revealed that it encodes a protein similar to membrane-spanning Ca2+ ATPases. The predicted amino acid sequence encoded by DRS2 contains seven transmembrane domains, a phosphate-binding loop found in ATP- or GTP-binding proteins, and a seven-amino-acid sequence detected in all classes of P-type ATPases. The cold-sensitive phenotype of drs2 is suppressed by extra copies of the TEF3 gene, which encodes a yeast homolog of eukaryotic translation elongation factor EF-1 gamma. Identification of gene products affecting ribosome assembly and function among the DNAs complementing the drs mutations validates the feasibility of this approach.

DRS1 to DRS7, novel genes required for ribosome assembly and function in Saccharomyces cerevisiae.

To identify Saccharomyces cerevisiae mutants defective in assembly or function of ribosomes, a collection of cold-sensitive strains generated by treatment with ethyl methanesulfonate was screened by sucrose gradient analysis for altered ratios of free 40S to 60S ribosomal subunits or qualitative changes in polyribosome profiles. Mutations defining seven complementation groups deficient in ribosomal subunits, drs1 to drs7, were identified. We have previously shown that DRS1 encodes a putative ATP-dependent RNA helicase necessary for assembly of 60S ribosomal subunits (T. L. Ripmaster, G. P. Vaughn, and J. L. Woolford, Jr., Proc. Natl. Acad. Sci. USA 89:11131-11135, 1992). Strains bearing the drs2 mutation process the 20S precursor of the mature 18S rRNA slowly and are deficient in 40S ribosomal subunits. Cloning and sequencing of the DRS2 gene revealed that it encodes a protein similar to membrane-spanning Ca2+ ATPases. The predicted amino acid sequence encoded by DRS2 contains seven transmembrane domains, a phosphate-binding loop found in ATP- or GTP-binding proteins, and a seven-amino-acid sequence detected in all classes of P-type ATPases. The cold-sensitive phenotype of drs2 is suppressed by extra copies of the TEF3 gene, which encodes a yeast homolog of eukaryotic translation elongation factor EF-1 gamma. Identification of gene products affecting ribosome assembly and function among the DNAs complementing the drs mutations validates the feasibility of this approach.

Cloning and genetic characterization of a calcium‐ and phospholipid‐binding protein from Saccharomyces cerevisiae that is homologous to translation elongation factor‐1γ

We have isolated a gene (CAM1) from the yeast Saccharomyces cerevisiae that encodes a protein homologous to the translational cofactor elongation factor-1 gamma (EF-1 gamma) first identified in the brine shrimp Artemia salina. The predicted Cam1 amino acid sequence consists of 415 residues that share 32% identity with the Artemia protein, increasing to 72% when conservative substitutions are included. The calculated M(r) of Cam1p (47,092 Da) is in close agreement with that of EF-1 gamma (M(r) = 49,200 Da), and hydropathy plots of each protein exhibit strikingly similar profiles. Disruption of the CAM1 locus yields four viable meiotic progeny, indicating that under normal growth conditions the Cam1 protein is non-essential. Attempts to elicit a translational phenotype have been unsuccessful. Since EF-1 gamma participates in the regulation of a GTP-binding protein (EF-1 alpha), double mutants with cam1 disruptions and various mutant alleles of known GTP-binding proteins were constructed and examined. No evidence was found for an interaction of CAM1 with TEF1, TEF2, SEC4, YPT1, RAS1, RAS2, CDC6, ARF1, ARF2 or CIN4. The possibility that Cam1p may play a redundant role in the regulation of protein synthesis or another GTP-dependent process is discussed.

Expression of elongation factor 1 alpha (EF-1 alpha) and 1 beta gamma (EF-1 beta gamma) are uncoupled in early Xenopus embryos.

In the amphibian Xenopus laevis, the elongation factor 1 alpha proteins (EF-1 alpha) synthesised in oocytes and somatic cells correspond to distinct gene products. Furthermore, the somatic EF-1 alpha gene (EF-1 alpha S) produces one of the most highly expressed early zygotic transcripts in the embryo. The functional recycling of EF-1 alpha (conversion of EF-1 alpha-GDP to EF-1 alpha-GTP) is assured by the EF-1 beta gamma complex. We show here that in Xenopus laevis embryos, contrary to the situation for EF-1 alpha, EF-1 beta, and EF-1 gamma mRNAs are transcribed from the same genes in oocytes and somatic cells. In addition, the onset of transcription of the EF-1 beta and EF-1 gamma genes from the zygotic genome occurs several hours after that of the somatic EF-1 alpha S gene. Therefore, during early Xenopus development the expression of these three elongation factors is not co-ordinated at the transcriptional level. The consequences of this uncoupling on the efficiency of translational elongation in the early Xenopus embryo are discussed.

A major substrate of maturation promoting factor identified as elongation factor 1 beta gamma delta in Xenopus laevis

Protein synthesis is believed to be under control of the cell cycle during meiosis and mitosis. Any relationship between substrates for cdc2 kinase and components of the protein synthetic apparatus would therefore be of prime importance. During meiosis of Xenopus laevis oocytes one of the substrates for this kinase is a p47 protein, which is complexed to two other proteins, P36 and P30. Judged from partial amino acid sequence data on P47 and P30, the P30 and P47 proteins were reported to resemble the protein synthetic elongation factors (EF) 1 beta and 1 gamma from Artemia salina (Bellé, R., Derancourt, J., Poulhe, R., Capony, J.P., Ozon, R., and Mulner-Lorillon, O. (1989) FEBS Lett. 255, 101-104). This paper shows that the complex composed of P30, P47, and P36 from Xenopus is identical to the complex of EF-1 beta, EF-1 gamma, and EF-1 delta from Artemia according to two criteria. 1) Both stimulate elongation factor 1 alpha-mediated transfer RNA binding to ribosomes and exchange of guanine nucleotides on elongation factor 1 alpha to a comparable degree. 2) Each of the three subunits of the protein complex P30.P47.P36 from Xenopus shows a structural homology with one of the corresponding subunits of EF-1 beta gamma delta from Artemia. Presumably the phosphorylation of EF-1 gamma, which associates with tubulin at least in vitro, is important in processes following the onset of meiosis which is accompanied by a rise of protein synthesis.

Mapping the functional domains of the eukaryotic elongation factor 1βγ

The functional domains of the eukaryotic elongation factor (EF) 1 beta gamma have been delineated with the use of limited proteolysis, protein microsequencing, gel electrophoresis under non-denaturing conditions and antibodies against EF-1 beta and EF-1 gamma. By means of limited proteolysis, it was possible to obtain large fragments of EF-1 beta. In contrast to amino-terminal fragments, those derived from the carboxy-terminal part of EF-1 beta were still active in enhancing the guanine nucleotide exchange of GDP bound to EF-1 alpha. With the same technique of limited proteolysis, it was possible to isolate a trypsin-resistant core from EF-1 beta gamma containing polypeptide chain fragments derived from both subunits. A polyvalent antiserum against EF-1 beta and two monoclonal antibodies against EF-1 gamma were used to identify the protein fragments in this core. The monoclonal antibodies were shown to recognize different epitopes, one localized on the amino-terminal and another on the carboxy-terminal half of EF-1 gamma. The antiserum against EF-1 beta and one of the monoclonal antibodies (mAb 36E5), which recognized the amino-terminal half of EF-1 gamma, reacted with this trypsin-resistant core. We conclude that the amino-terminal halves of both EF-1 beta and EF-1 gamma are firmly attached to each other, and that the carboxy-terminal part of EF-1 beta interacts with EF-1 alpha.

Protein phosphorylation during meiotic maturation of Xenopus oocytes: cdc2 protein kinase targets.

M-Phase specific protein kinase or cdc2 protein kinase is a component of MPF (M-Phase promoting factor). During meiotic maturation of Xenopus oocytes, cdc2 protein kinase is activated in correlation with MPF activity. A protein phosphorylation cascade takes place involving several protein kinases, among which casein kinase II, and different changes associated with meiosis occur such as germinal vesicle breakdown, chromosome condensation, cytoskeletal reorganization and increase in protein synthesis. Our results provide a biochemical link between cdc2 protein kinase and protein synthesis since they show that the kinase phosphorylates in vitro a p47 protein identified as elongation factor EF1 (gamma subunit) and that the in vitro site of p47 corresponds to the site phosphorylated in vivo. Immunofluorescence showed that the elongation factor (EF1-beta gamma) is localized in the oocyte cortex. Furthermore, they show that cdc2 kinase phosphorylates and activates casein kinase II in vitro, strongly supporting the view that casein kinase II is involved in the phosphorylation cascade originated by cdc2 kinase.

Elongation factor 1 beta gamma from Artemia. Purification and properties of its subunits.

The guanine nucleotide exchange factor, elongation factor 1 beta gamma (EF-1 beta gamma) has been purified from Artemia cysts using an improved method. The protein consists of two distinct polypeptides with relative molecular masses of 26,000 (EF-1 beta) and 46,000 (EF-1 gamma). A nucleoside diphosphate phosphotransferase activity often found in EF-1 beta gamma preparations has been completely separated from the actual guanine nucleotide exchange stimulatory activity of EF-1 beta gamma, thus indicating that nucleotide diphosphate phosphotransferase is not an intrinsic property of EF-1 beta. Both EF-1 beta gamma and EF-1 beta have been shown to stimulate the following three reactions to a comparable degree: (a) exchange of GDP bound to EF-1 alpha with exogenous GDP; (b) EF-1 alpha-dependent binding of Phe-tRNA to ribosomes; (c) poly(U)-dependent poly(phenylalanine) synthesis. However, a significantly higher nucleotide exchange rate was observed in the presence of EF-1 beta gamma compared to EF-1 beta alone. Concerning elongation factor 1 gamma (EF-1 gamma) the following observations were made. In contrast to EF-1 beta, pure EF-1 gamma is rather insoluble in aqueous buffers, but the tendency to precipitate can be partially suppressed by the addition of detergents. In particular, EF-1 gamma partitions solely into the detergent phase of Triton X-114 solutions. EF-1 gamma is also more susceptible to spontaneous, specific fragmentation. It is remarkably that about 5% of the cellular pool of EF-1 beta gamma was found to be present in membrane fractions, under conditions where no EF-1 alpha was detectable in these fractions. Furthermore it was noted that EF-1 beta gamma copurified strongly with tubulin on DEAE-cellulose. Moreover, it was observed that from a mixture of EF-1 beta gamma and tubulin, EF-1 gamma coprecipitates with tubulin using a non-denaturating immunoprecipitation technique. These findings suggest that EF-1 gamma has a hydrophobic domain and interacts with membrane and cytoskeleton structures in the cell.

Studies on the high molecular weight form of polypeptide chain elongation factor-1 from pig liver. II. Interaction with guanine nucleotides and aminoacyl-tRNA.

The high molecular weight form of polypeptide chain elongation factor-1, or EF-1H, has been purified from pig liver to apparent homogeneity and shown to be a 1:1:1 complex of EF-1 alpha, EF-1 beta, and EF-1 gamma, namely, EF-1 alpha beta gamma (Hattori, S. and Iwasaki, K. (1980) J. Biochem. 88, 725-736). Therefore, its enzymatic properties were investigated in detail. The effects of various components such as NH4Cl, Mg(CH3COO)2, guanine nucleotides and Phe-tRNA on its heat stability were investigated and it was found that its properties were very close to those of the aminoacyl-tRNA binding enzyme from rabbit reticulocytes (McKeehan, W.L. and Hardesty, B. (1969) J. Biol. Chem. 244, 4330-4339). Furthermore, it was reported that EF-1 alpha beta gamma seemed to dissociate into EF-1 alpha and EF-1 beta gamma when guanine nucleotides were present, and also to form a complex of EF-1 alpha . GTP . [14C]Phe-tRNA when both GTP and [14C]Phe-tRNA were present. On the contrary, however, when the interaction between EF-1 alpha beta gamma and guanine nucleotides was analyzed by gel filtration at 4 degrees C, the dissociation of EF-1 alpha beta gamma could not be detected, instead the formation of binary complexes containing EF-1 alpha beta gamma and guanine nucleotides was observed. The dissociation constants of the EF-1 alpha beta gamma . GDP and EF-1 alpha beta gamma . GTP complexes were estimated to be 6.8 x 10(-6) M and 7.3 x 10(-6) M, respectively, and the number of binding sites per molecule of EF-1 alpha beta gamma for these nucleotides was estimated to be one. Little, if any, interaction was detected between EF-1 alpha beta gamma and aminoacyl-tRNA even in the presence of GTP. The controversial results obtained by different methods as described above seemed to be compatible with each other, if one assumes that the dissociation constant for the conversion of EF-1 alpha beta gamma into EF-1 alpha and EF-1 beta gamma increased with the increase in temperature. When gel filtration of EF-1 alpha beta gamma was carried out with a solution containing both GTP and [14C]Phe-tRNA, the formation of a ternary complex containing EF-1 alpha, GTP and [14C]Phe-tRNA was detected at 4 degrees C, although the amount was very small. From these results, we assumed that EF-1 alpha beta gamma interacts with GTP and aminoacyl-tRNA (aa-tRNA) according to the following sequences; EF-1 alpha beta gamma + GTP in equilibrium with EF-1 alpha beta gamma . GTP EF-1 alpha beta gamma . GTP in equilibrium with EF-1 alpha . GTP + EF-1 beta gamma EF-1 alpha . GTP + aa-tRNA in equilibrium with EF-1 alpha . GTP . aa-tRNA.

Studies on the High Molecular Weight Form of Polypeptide Chain Elongation Factor-1 from Pig Liver

The high molecular weight form of the polypeptide chain elongation factor-1 (EF-1H) from pig liver was purified to an apparently homogeneous state. The procedure included aqueous two-phase separation, ammonium sulfate fractionation, four successive column chromatographies on Sephadex G-200, CM-Sephadex C-50, hydroxylapatite and octyl-Sepharose CL-4B, and glycerol density gradient centrifugation. By this procedure, the specific activity of EF-1H increased about 60-fold with a recovery of the total activity of 6%, as compared to the material obtained by aqueous two-phase separation followed by ammonium sulfate fractionation. Purified EF-1H was analyzed by polyacrylamide gel electrophoresis under non-denaturing conditions and glycerol gradient centrifugation, revealing that it is apparently homogeneous and has both EF-1 alpha and EF-1 beta gamma activities. The subunit structure of EF-1H was investigated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate as well as two-dimensional gel electrophoresis, which indicated that EF-1H consists of three different subunits, i.e., EF-1 alpha, EF-1 beta, and EF-1 gamma, in an equimolar ratio. The Stokes radius and the sedimentation coefficient of EF-1H were estimated to be 5.9 nm and 6.2S, respectively, from which the molecular weight of EF-1H was calculated to be about 157,000. This value is close to the sum of the molecular weights of EF-1 alpha (53,000), EF-1 beta (30,000), and EF-1 gamma (53,000). Thus, we concluded that EF-1H is a monomer of a 1:1:1 complex of EF-1 alpha, EF-1 beta, and EF-1 gamma, or EF-1 alpha beta gamma and its molecular weight is 136,000. This conclusion was further confirmed by measuring the amounts of EF-1 alpha and EF-1 beta gamma in EF-1H by enzymatic assays. The amino acid composition of EF-1H was determined, and the results revealed that there was a close resemblance between the values of EF-1H and those of EF-1 alpha beta gamma, which were calculated from the reported values for EF-1 alpha, EF-1 beta, and EF-1 gamma.