A sensitive radioimmunoassay for eucaryotic elongation factor Tu (eEF-Tu) was developed using radioiodinated elongation factor T (eEF-T) and goat anti-(rabbit eEF-T) immunoglobulins coupled to a solid support. eEF-T was iodinated with lZ5I to a specific activity of 7 x lo3 counts min-' ng-' using a system employing lactoperoxidase and glucose oxidase coupled to a solid support. The assay exhibits a limit of detection of about 1 ng eEF-Tu and an intraassay variability of < 10 %. By using the radioimmunoassay, it was found that eEF-Tu is a major non-hemoglobin protein of rabbit reticulocyte postribosomal supernatant proteins, comprising about 3 of the total hemoglobin and 10-13 % of the non-hemoglobin proteins. Similar results were found for a number of different tissues and cells, including rabbit heart, brain, liver and kidneys, as well as both induced and non induced Friend erythroleukemia cells. Values of eEF-Tu ranged from 1 % of supernatant proteins in heart to about 11 %, in noninduced erythroleukemic cells. The levels of eEF-Tu in these mammalian tissues were comparable to the level of the homologous factor EF-Tu in Escherichia coli. It has previously been found that EF-Tu constitutes about 6 -8 % of the supernatant proteins of E. coli [Furano, A. V. (1975) Proc. Natl Acad. Sci. USA, 72, 4780-47841. The level of eEF-Tu in reticulocytes was compared to the abundance of other components of protein synthesis in reticulocytes, such as translocase (eEF-G), tRNA, ribosomes and eIF-2. In all cases eEF-Tu was present in large excess over these other components.

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ABSTRACT60S ribosomes from encysted gastrulae of the brine shrimp Artemia salina contain two acidic proteins, which are homologous to the Escherichia coil proteins L7 and L12. The proteins were purified and characterized with respect to molecular weight, amino-acid composition, peptide maps, and their functional requirement in the elongatioh factor dependent binding of aminoacyl transfer RNA to the ribosome. for 48 hr against 0.1% formic acid followed by lyophilization. All isolation operations were performed at 20. In order to isolate EL7 and EL12, 300 mg of 60S A. saUna protein (7) were dialyzed against 50 ml of 0.01 M NH4OAc, pH 6.0, 1 mM 2-mercaptoethanol, 6 M urea followed by application to a carboxymethyl-cellulose column (Serva CM, grade A, 0.6 meq/g, 2.5 X 18 cm), equilibrated with the same buffer in 6 M urea. The column was washed with the equilibration buffer until the acidic protein fraction was completely eluted. The acidic protein fraction was dialyzed against 0.01 M NH4OAc, pH 5.7, 1 mM 2-mercaptoethanol, 6 M urea and applied to a DEAE-cellulose column (Whatman DEAE 32), equilibrated with the same buffer. The latter column (1.5 X 28 cm) was developed with a linear gradient of 600 ml each of 0.01 M NH4OAc, pH 5.7, 1 mM 2-mercaptoethanol, 6 M urea, and 0.2 M NH4OAc, pH 4.5, 1 mM 2-mercaptoethanol, 6 M urea. The molarity of the acetate buffer refers to acetic acid, which was adjusted with concentrated ammonia to pH 5.7 or pH 4.5.Protein EL12 eluted at pH 4.8 after about 480 ml of gradient buffer had passed through the column, followed by EL7 starting at an elution volume of about 640 ml. The two peak fractions, monitored at 230 nm, were desalted by extensive dialysis against 0.1% formic acid, and lyophilized. The yield from 300 mg of 60S ribosomal protein was 1.6 mg of EL7 and 0.4 mg of EL12. Estimates of the protein content of the dried samples are based on amino-acid analyses using a Beckman microanalyzer. Two-dimensional polyacrylamide gel electrophoresis was performed in 6 M urea (8) with the following modification. The first dimension contained 8% acrylamide on the anodic side and 4% acrylamide on the cathodic side. Sodium dodecyl sulfate gel electrophoresis was performed essentially according to Weber and Osborn (9). The method used for iscielectric focussing in polyacrylamide gels has been described (10). Peptide mapping was performed essentially according to Clegg et al. (11). In order to wash EL7 and EL12 selectively from the 60S ribosomes of A. salina cysts, we applied the method of Hamel et al. (12). Their salt-ethanol treatment selectively removes L7 and L12 from the 50S ribosomes of Escherichia coli. 60S ribosomes (22 mg) were suspended in 10 ml of 1 M NH4Cl, 20 mM Mg(OAc)2, 10 mM Tris1HCl, pH 7.4, 1 mM 2-mercaptoethanol. Two 5-ml portions of ice-cold ethanol were added dropwise within 15 min, while the mixture was kept at 00. The cloudy suspension was centrifuged at 00 for 20 min at 20,000 X g and the pellet was dissolved in 5 ml of standard buffer A (5), followed by a second ethanol ext...

Two forms of elongation factor 1 (EF‐1) have been purified from embryos of Artemia salina. A heavy form of the factor (EF‐1H) which is present exclusively in dried cysts, was found to contain three different polypeptide chains on the basis of electrophoresis in polyacrylamide gels containing dodecylsulphate: an A chain (Mr= 53000), a B chain (Mr= 51000) and a C chain (Mr= 26000). The holoenzyme elutes slightly ahead of beef liver catalase (Mr= 240000) on columns of Ultrogel ACA 34 and comigrates with catalase (s = 11 S) on sedimentation in a sucrose gradient. However, enzymically active more slowly sedimenting forms (8‐S and 5‐S) of EF‐1H, containing A, B and C chains were also observed. Gel filtration experiments confirm that the holoenzyme can readily dissociate into at least two lower‐molecular‐weight forms containing A, B and C chains. The C chain comprised approximately 30% of the mass of these different forms of EF‐1H. The light form of the factor, EF‐1L, which is exclusively present in hatched embryos (nauplii), was conveniently prepared from cysts which were developed for 6 h at 42°C. EF‐1L consists of a single basic polypeptide chain with a molecular weight of 53000 and a pl of about 8.3. EF‐1H also contained a basic polypeptide with an isoelectric point indistinguishable from EF‐IL. Further support for the presence of the EF‐1L polypeptide in EF‐1H was given by the weak but definite immunological crossreaction betweèn rabbit anti‐EF‐1L and EF‐1H. Based on the Mr of the subunits, gel filtration and sucrose density gradient behavior of the different forms of EF‐1H and the stoichiometry of the C polypeptide, a model of the enzyme is proposed in which the basic unit (Mr= 75000) consists of one A or B chain in combination with the C polypeptide. Although EF‐1L corresponds closely to a low‐molecular‐weight forni of EF‐1L prepared from pig liver, there is little similarity between Artemia EF‐1H and the heavy forms of EF‐1 from mammalian sources. The later have been found to consist of aggregates of a single polypeptide. On the other hand, there is a striking correspondence between Artemia EF‐1H and the heavy form of wheat embryo EF‐1 which also contains three different polypeptides. These similarities lead us to suggest that the additional polypeptides play an important role in maintaining the activity of EF‐1 during periods of biological dormancy.