Purification and some properties of elongation factor 1 from wheat germ

Biochimica et Biophysica Acta, 324 (1973) 156-170

Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 97805

P U R I F I C A T I O N A N D SOME PROPERTIES OF E L O N G A T I O N FACTOR I FROM WHEAT GERM

BARBARA GOLII(ISKA and ANDRZEJ B. LEGOCKI Institute o f Biochemistry, University o f Agriculture, 60-637 Poznah, Woly~ska 35 (Poland)

(Received April 9th, 1973)

SUMMARY The elongation factor 1 (EF1) was extensively purified from wheat germ. The purification procedure consisted of (NH4)2SO4 precipitation, gel filtration on Sephadex G-150, double chromatography on hydroxylapatite and finally Sephadex G-150 gel filtration. A 70-fold purification of the factor was achieved with a recovery of 30 ~. The purified preparation contains three active forms of the factor differing markedly in molecular size. Determination of their molecular weights using gel filtration and gel electrophoresis techniques resulted in values of 250 000, 180 000 and 60 000. The larger EF1 forms dissociate to the single species of tool. wt 60 000, during electrophoresis in the presence of sodium dodecyl sulphate. The forms observed may represent a monomer, trimer and tetramer of a single protein unit. The most stable form of wheat germ EFI seems to be a trimer. Some properties of the factor have been tested. Optimum pH and temperature in phenylalanyl-tRNA binding assay are 8.1 and 30 °C, respectively. Heat inactivation studies of EF1 revealed that there is a direct interaction between GTP, aminoacylt R N A and the factor in the absence of ribosomes. The EFl-dependent binding of phenylalanyl-tRNA to ribosomes occurs in the presence of both guanosine triphosphate and 5'-guanylyl methylene diphosphonate.

INTRODUCTION It is now well established that the peptide chain elongation in eukaryotic systems requires two protein factors: elongation factor 1 - E F 1 (the factor required for binding of aminoacyl-tRNA to the ribosomal acceptor site) and elongation factor 2 - E F 2 (the factor involved in translocation). Three techniques are useful in elaborating the catalytic role of factor EF1. The first of these is the stimulation at low Mg 2 ÷ concentration of the binding ofaminoacyltRNA to ribosomes using cellulose nitrate filter assay. The second technique is the EFl-dependent incorporation of labelled amino acid from aminoacyl-tRNA into growing peptide chain in the presence of EF2 which is the measure of overall peptide chain elongation. The third assay is based on the observation that EF1 specifically binds G T P and remains in complex form on a cellulose nitrate filter. This reaction

PURIFICATION AND PROPERTIES OF WHEAT GERM EFI

157

occurs without ribosomes and may supply information about involvement of EFI in pre-ribosomal interactions with G T P and aminoacyl-tRNA. For characterization of the catalytic properties of the factor the first two methods are usually applied. It was previously reported that two complementary elongation factors from wheat germ may be resolved and partially purified ~- 3. This paper presents an extension of the above investigations concerning further purification and some of the characteristics of the pure factor EF1, and demonstrates its catalytic role in the peptide chain elongation on 80-S ribosomes. MATERIALS AND METHODS Uniformly labelled L-[14C]phenylalanine (spec. act. 220 Ci/mole) was purchased from Amersham; poly(U), GTP, hydroxylapatite-SC, dithiothreitol were obtained from Serva; GDP, GMP-methylene diphosphonate were obtained from Miles Laboratories.

Preparation of tRNA and ribosomes from wheat germ Wheat germ tRNA was isolated in a similar manner as tRNA from lupin seeds 4. The preparation of t R N A enriched in tRNA Phe was made according to Dudock et aL 5 by double BD-cellulose chromatography at pH values of 7.3 and 4.3 (ref. 5). t R N A ph¢ was charged using homologous synthetase partially purified in a similar manner to lupin synthetases 6. For the kinetic measurements nucleotide-free [14C]Phe-tRNA was used and was obtained by filtration through Sephadex G-50. Ribosomes were prepared from the wheat germ microsomal pellet resulting from the 175 000 × g centrifugation during the purification of factor EF1. The pellet was dissolved in a buffer solution composed of 50 mM Tris-HCl, pH 8.05; 100 mM KCI; 5 mM magnesium acetate; 3 m M mercaptoethanol by gentle homogenization. This solution was layered over 2.5 ml of the above buffer containing 17 % sucrose and was centrifuged for 55 min at 175 000 × g. This procedure was repeated twice. The ribosomal pellet was suspended in a buffer containing 50 mM Tris-HC1, pH 8.05; 50 m M KC1; 5 m M magnesium acetate; 1 mM dithiothreitol and 20 % (v/v) glycerol, clarified by centrifugation for 8 min at 10 000 × g and stored at --25 °C at a concentration of 5 mg RNA/ml. Binding of [14C]Phe.tRNA to ribosomes Enzymatic binding activity was measured by the cellulose nitrate filter technique according to Nirenberg and Leder 7. The values of nonenzymatic binding were determined for each set of experiments and subtracted from the total amount of [14C]Phe-tRNA bound to ribosomes. One unit of factor EF1 activity was defined as the net enzymatic binding of 1 pmole of [14C]Phe_tRNA to ribosomes in the conditions described below. In all these assays, a constant amount of ribosomes, optimal for binding 25 pmoles of Phe-tRNA, was used (130 #g). The standard enzymatic binding reaction mixtures contained in the final volume of 0.3 ml: 50 mM Tris-HCl buffer, pH 8.0; 5 mM magnesium acetate; 70 mM KCI; 3 mM dithiothreitol; 130/zM GTP; 10 #g ofpoly(U); 25 pmoles [14C]Phe_tRNA (11 000 cpm); 130 #g of ribosomes and 2-6 #g of factor EF1. The incubation was carried out at 30 °C for 10 min, unless otherwise indicated after which 3.5 ml of cold wash medium containing 10 mM Tris-

158

B. GOLI~SKA, A. B. LEGOCKI

HCI buffer, pH 7.7; 80 mM KC1; 10 mM magnesium acetate was added to stop the reaction. The mixtures were then immediately poured over cellulose nitrate filters under gentle suction, followed by washing three times with a wash medium. The filters were dried and the radioactivity determined in a toluene scintillator with 60 efficiency for [14C]carbon in scintillation counter SE 2 (BUTJ, Poland). For kinetic data requiring initial rates of the reaction, the incubation mixtures contained 10-15 #g of EF1 and the reaction time was reduced to 8 min.

Phenylalanine polymer&ation assay The EFl-dependent polyphenylalanine synthesis was measured as the incorporation of [14C]phenylalanine into peptides insoluble in hot 5 ~ trichloroacetic acid in the presence of saturating amounts of factor EF2 ( 1 4 #g). The reaction mixture was as described for enzymatic binding except that 20 #g of poly(U); 250 #M GTP; 5 mM dithiothreitol and 4/~g of factor EF2 were substituted for the amounts of these materials used in the reaction mixtures for enzymatic binding. Incubation was carried out for 15 min at 30 °C. Factor EF2 was prepared as described in the accompanying paper 8.

Polyacrylamide gel electrophoresis Purity of EF1 preparations was tested by electrophoresis on 4.1 ~ acrylamide gels, at pH 8.9, at 20 °C, according to the procedure of Weber and Osborn 9. Protein was layered on gel in 30 ~o glycerol and electrophoresis was continued until the bromophenol blue marker dye migrated to about 5 mm from the end of the gel (at 2 mA per tube during the first 20 min and 4 mA during a further 50 min). The gels were stained with 0.12 ~ Coomassie brilliant blue in 20 ~ methanol and 7 ~o acetic acid, and destaining in 3 ~ acetic acid.

Molecular weight determination o.1"EF1 The molecular weight of purified EF1 preparation was determined by gel filtration according to the method of Andrews xo and also by disc electrophoresis in the presence of 1 ~o sodium dodecyl sulphate 1x. Using bovine serum albumin monomer (mol. wt 62 000) and dimer (tool. wt 124 000), human 7-globulin (mol. wt 160 000) and EF1, a linear relationship was found to exist between the elution volume and electrophoretic mobility.

Protein determination Protein was measured by the method of Lowry et al. 12 using crystalline bovine serum albumin as standard. The standards and blanks contained mercaptoethanol or dithiothreitol at the same concentration as the protein samples. Since both the reagents strongly interfere in this assay, all the samples were diluted at least 10-fold.

Purification of the elongation factor 1 All steps were carried out at 4 °C and all buffer solutions used in the factor purification contained 3 m M mercaptoethanol or 2 mM dithiothreitol. A summary of the purification procedure is given in Table I.

PURIFICATION AND PROPERTIES OF WHEAT G E R M EFI

159

TABLE I PURIFICATION OF FACTOR EF1 F R O M WHEAT G E R M The average of the results from 63 g of wheat germ are presented through the 1st Sephadex G-t50 gel filtration stage. Subsequent steps were carried out in portions; hence the results of further purification are related to the initial homogenate. With regard to specific activity, one unit corresponds to 1 pmole of [t4C]Phe-tRNA bound to ribosomes per 10 rain at 30 °C.

Step of pur([ication

Total protein (my)

Specific activity (units]my protein)

Cumulative purification (-fold)

Recovery (%)

1 2 3 4 5 6

1470 865 283 54 13 6

117 200 534 2409 7338 8963

1 1.7 4.6 20.6 62.7 76.6

100 100 88 76 56 31

High speed supernatant (NH4)2SO4 precipitation (65 % satn) Sephadex G-150 1st Hydroxylapatite 2nd Hydroxylapatite Sephadex G-150

Preparation of the crude extract and high speed supernatant 63 g of locally purchased wheat germs were (after soaking for 2 min) homogenized in a Waring blendor at top speed (5 × 10 s with 30 s intervals), with 500 ml of 50raM Tris-HCl buffer, pH 8.05, containing 5 mM magnesium acetate; 50 mM KCI; and 4 mM CaC12. The slurry was filtered through a double cheese cloth and centrifuged twice for 15 min at 21 000 × g. After the first centrifugation the supernatant was adjusted to pH 7.7 with 1 M untreated Tris. The resulting supernatant was then centrifuged for 75 min at 175 000 x g. The high speed supernatant fluid was removed and could be stored at --25 °C in the presence of 2 mM dithiothreitol for several months.

( NH4 )2SO * precipitation Solid ( N H 4 ) 2 S O , up to 65 % saturation was added, with constant stirring, to the high speed supernatant. The pH was kept at 7.7 by carefully adding 1 M NH3. The resulting precipitate was collected by centrifugation for 15 min at 21 0 0 0 × g , dissolved in 20 mM Tris-HC1, pH 7.7, containing 10 mM KCI, 5 % (v/v) glycerol and dialysed against 80 vol. of the same buffer overnight with two buffer changes. After dialysis the insoluble material was removed by centrifugation.

Sephadex G-150 #el filtration The dialysed solution was subjected to gel filtration on Sephadex G-150 equilibrated with the dialysed buffer. 860 mg of protein in the volume of 50 ml were applied on the column (5 cm x 90 cm and developed at a flow rate of 50-60 ml/h. Most of the activity of factor EF1 emerges in the first peak of protein, well separated from the later eluted factor EF2. A filtration pattern obtained from the Sephadex G-i 50 column is shown in Fig. 1. Fractions with a specific activity of over 400 units/mg protein were pooled and stored at --25 °C. Since EFI was relatively stable at this stage of purification, it may be kept for 1-3 months before further purification.

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250

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Fig. 1. Gel filtration of crude wheat germ EFI preparation on Sephadex G-150. The details of the column operation are given in Materials and Methods. Aliquots of 10/4 from alternate fractions were assayed for the EFI activity in the binding of [t4CIPhe-tRNA to ribosomes.

1st hydroxylapatite chromatography A column (1.8 cm × 10 cm) of hydroxylapatite was equilibrated with the filtration buffer from the previous step. 50 ml of EF1 preparation recovered from Sephadex G-150 were passed through the column which was then washed with 30 ml of 10 m M potassium phosphate buffer, p H 7.3, containing 5 % glycerol. The factor was eluted with 0.25 M potassium phosphate buffer, pH 7.9, containing 5 % glycerol. The fractions containing enzymatic activity were pooled and immediately dialysed against 100 m M potassium phosphate buffer, pH 7.3, with glycerol for 3 h.

2nd hydroxylapatite chromatography The dialysed factor (23 mg in 30 ml) was applied to a column 1.6 cm ;,,"6 cm which had been packed under pressure with the aid of a peristaltic pump and equilibrated with dialysed buffer. After the sample was loaded, the column was washed with 30 ml of the initial buffer and the protein eluted with a linear gradient of increasing potassium phosphate buffer from 100 m M to 300 mM and increasing pH from 7.3 to 8.0. Flow rate was maintained at 15 ml/h and 2.5-ml fractions were collected. The bulk of the protein is eluted early, while EFI appears later in the gradient as a discrete peak. The three to five fractions of highest specific activity were pooled and concentrated in a dialysis bag with dry Sephadex G-200, to a concentration of 1.52.5 mg protein/ml.

Sephadex G-150filtration The final purification of factor EF1 was carried out by gel filtration on Sephadex G-150. The concentrated EFI solution was applied to a column (0.9 cm × 48 cm) equilibrated with 0.15 M potassium phosphate buffer, pH 7.6, with 5 % glycerol. EF1 was eluted in the same buffer at the flow rate of 15 ml/h. Aliquots of 2-ml fractions were dialysed against 100 vol. of 20 m M Tris-HC1 buffer, pH 7.7, with glycerol and assayed for binding activity. An elution pattern obtained from gel filtration is shown

PURIFICATION AND PROPERTIES OF WHEAT GERM EFI

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in Fig. 2. Fractions 8-12 were pooled concentrated as before and kept at --25 °C. This EF1 preparation was used for further experiments involving the properties of the factor.

Storage of purified EF1 preparations The purified factor EF1 was highly unstable. The greatest stability (about 10 days) was found when the preparation was stored at - 25 °C at a concentration of no less than 1 mg/ml, in the presence of 0.15 M potassium phosphate, in the pH range 7.2-8.0. Storage of the EF1 at 0 °C at low protein concentration (0.2 mg/ml) resulted in a loss of 80 ~o of the activity overnight. There was a considerable loss of the activity after preincubation below pH 7.0. The addition of 10 % glycerol during purification procedure appeared to enhance the stability of EF1. The factor lost its activity when precipitated with (NH4)2SO4 (except the initial step of purification). Mercaptoethanol, dithiothreitol and EDTA had little, if any, effect on the stability. Since freezing and thawing appear to decrease the activity, these treatments were avoided where possible. RESULTS

Purity and molecular weight of EF1 As shown in Fig. 2, the gel filtration pattern of the factor when purified on hydroxylapatite demonstrates two protein components of EF1, each of which has

162

B. GOLII{ISKA, A. B. LEGOCKI

a similar specific activity. Calibration of the Sephadex G-150 column with the protein standards, made it possible to determine the molecular weight of EF1 forms. From these experiments a molecular weight of 180 000 can be derived for a large EF1 form and 60 000 for a small one (Fig. 3). Taking into account the observed deviations for protein standards, the maximum uncertainty will be about 4 % of the above values. This finding indicates the existence of multiple forms of wheat germ E F I.

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Analytical disc gel electrophoresis seems to confirm this observation. Two distinct protein bands were detected in the purified EF1 preparation when the electrophoresis was carried out in the presence of 1% sodium dodecyl sulphate. Their molecular weights closely corresponded to the values from gel filtration (180 000 and 60 000; Fig. 4). Moreover, electrophoresis of the EF1 preparation in the absence of dodecyl sulphate revealed the third protein factor with a molecular weight of above 200 000 (about 250 000). Assuming that the EF1 preparation does not contain other proteins, these three components can be related to the monomeric, trimeric and tetrameric forms of factor EFI. Such a suggestion would be confirmed by the fact that dodecyl sulphate gel electrophoresis of the preparation, after treatment with 1% dodecyl sulphate and 4 M urea, showed only one protein component the molecular weight of which was estimated to be 62 000~4000 (Fig. 5). This value coincides with the smallest EF1 component revealed by disc electrophoresis without sodium dodecyl sulphate. Although the final EF1 preparation is highly unstable, attempts at recovering the factor activity from the native analytical disc were undertaken. To this end the unstained gel 0.6 cmx 10 cm in size, containing pure factor preparation subjected

PURIFICATION AND PROPERTIES OF WHEAT GERM EF1

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Fig. 5. Polyacrylamide gel electrophoresis of purified EF1 preparation and the recovery of the factor activity from native gel. 30 pg of EFI were incubated with 4 M urea, 1 ~ sodium dodecyl sulphate and 1 ~o mercaptoethanol for 1 h at 45 °C, applied to the 4.1 ~ gel (0.4 cmx 6 cm) and electrophoresed in the Presence of I ~ sodium dodecyl sulphate (A). The anode and the front are indicated by a plus sign. For the recovery of EF1 activity from the native gels, 0.6 cmx I0 cm columns were used on which 100/zg of EF1 were applied. After electrophoresis one gel was stained for protein (B) and the other was sliced and eluted as described in the text. The resulting fluids were assayed for EFI activity in the ribosomal binding reaction. The obtained activities are shown as cpm [14C]Phe.tRNA bound to ribosomes (C). to electrophoresis at p H 8.9, was sliced into 30 sections o f 3 m m each. The sections were resuspended in 0.4 ml 20 m M T r i s - H C l buffer, p H 8.0, with 10 m M KCI, 5 % glycerol, 3 m M dithiothreitol, 1 mg/ml bovine serum albumin and left at 0 °C overnight. After centrifugation the supernatant fractions were assayed for EF1 activity as described in the text. L o w but distinct EF1 activity was detected in the sections corresponding to the protein bands (Fig. 5). The experiment together with the above data led us to the conclusion that factor EF1 from wheat germ occurs in several forms heterogeneous in size. It should be pointed out, however, that the relative proportions between the forms varied occasionally f r o m one commercial wheat g e r m batch to another. In some cases only two forms o f E F l were detected (mol. wt 180 000 and 60 000). Generally, the trimer f o r m was the main c o m p o n e n t o f the EF1 preparations, thus it m a y be concluded that this form of EF1 is the most stable one.

Properties of the factor EF1 Heat stability. Factor EF1 was very unstable especially at higher stages o f its purification. One possible reason for the inactivation during purification and assay was the temperature factor. In order to examine this possibility the effect of heating the factor prior to the assay was studied. As shown in Fig. 6 the purified preparation was very heat labile losing more than 80 % of its activity when heated for 6 min at 40 °C in buffered salt solution. As was shown earlier, G T P and a m i n o a c y l - t R N A have a considerable effect on heat stability o f the binding factors f r o m higher organisms 13'14. Similar studies were performed on wheat germ E F I . A n addition o f P h e - t R N A has little effect on heat stability o f the factor, whereas an addition o f G T P causes a more rapid inactivation (after 6 min at 40 °C EF1 is practically inactive). However, in the presence o f both P h e - t R N A and G T P , the factor is better protected against heat inactivation (40 % activity after 6 min at 40 °C). These results indicate

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that there is a direct interaction between EF1, aminoacyl-tRNA and GTP. Fig. 7 shows the effect of Phe-tRNA concentration on the stability of the factor during 6 min heating at 38 °C. There is no significant protection in the presence of Phe-tRNA without GTP. In the presence of GTP, however, the factor is protected by relatively low concentrations of aminoacyl-tRNA. It is noteworthy that aminoacylated homol100

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20 40 Phenylal a n y l - t RNA (.ug) Fig. 7. Effect o f aminoacyl-tRNA on the heat stability o f EF1. 6 #g samples o f E F I were heated for 6 rain at 38 °C in buffered solutions containing indicated a m o u n t s o f either [14C]Phe-tRNA from wheat germ with ( 0 ) and without ( O ) G T P or [14C]Phe-tRNA from yeast with ( A ) and without ( ~ ) GTP. The ribosomal binding activity was measured as described in Materials and Methods.

PURIFICATION AND PROPERTIES OF WHEAT GERM EFI

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ogous t R N A protects E F I better than its counterpart from yeast. This could suggest some species specificity in the interaction of wheat germ EF1 with aminoacyl-tRNA. The heat lability of the EF1 during standard assay can be seen from the rate of the binding reaction at various temperatures (Fig. 8).

Effect of factor concentration. Figs 9 and 10 show an effect of EF1 concentration on the binding reaction and polyphenylalanine formation. In the first case, normal hyperbolic kinetics was observed with a linear relationship between the factor activity and its concentration up to 6 pg of EF1 per assay. However, the EFl-dependent phenylalanine polymerization curve has an obvious sigmoidal shape under standard assay conditions. It may be indicative of the more complicated kinetics of this reaction.

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Effect of inorganic ions. Fig. 11 shows a Mg 2+ requirement for enzymatic and nonenzymatic binding. Nonenzymatic binding was measured in the absence of EF1 in the incubation mixtures. Enzymatic binding was calculated as the difference between total binding and nonenzymatic binding. The values of nonenzymatic binding are proportional to the increased Mg 2 + concentrations, whereas the enzymatic binding reaches a m a x i m u m at about 6 m M magnesium acetate. To eliminate the effects o f nonenzymatic binding in standard assays, a concentration of 4.5-5 m M Mg 2+ was applied. O f three monovalent cations assayed K +, NH4 + and Na + the highest EF1 activity was observed with K ÷ (Fig. 12). NH4 + and Na + gave about 75 and 50 ~o of the EF1 activity observed for potassium, respectively.

PURIFICATION A N D PROPERTIES OF WHEAT G E R M EFI

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PURIFICATION AND PROPERTIES OF WHEAT GERM EFI

169

substituted for GTP. On the other hand it was found that the structural analogue of GTP, GMP-methylene diphosphonate (guanylyl-5'-methylene diphosphonate)can stimulate the binding reaction. Figs 14 and 15 show the effect of G T P and GMPmethylene diphosphonate concentration on the rate of enzymatic binding. From these data the K,, value for G T P and for GMP-methylene diphosphonate in the enzymatic binding are 2 • 1 0 - 6 M and 6.7 • 1 0 - 7 M , respectively. DISCUSSION The experiments described here were aimed at finding a convenient method of purifying elongation factor 1 from wheat germ and using it in the studies on the mechanism of peptide bond formation in plant systems. It seems to be particularly interesting since most of our information about the process of polypeptide chain elongation was elucidated from the studies on bacterial and mammalian systems. The properties of plant elongation factors have been described to a certain extent 1'2'15, but none of them was isolated in a homogeneous state. The procedure which has been developed here for factor EF1 appears to meet this requirement. Early attempts at purification of EF1 from wheat germ involved DEAE-cellulose chromatography 2. This step was very convenient since at low ionic strength the factor was not adsorbed to the anion exchanger, which enabled complete separation from strongly bound factor 2. However, the low yield of EF1, due to its partial inactivation during chromatography, does not allow a larger application of this operation. The results presented in the preceding section suggest that factor EFI from wheat germ contains several forms heterogeneous in size, containing potential enzymatic activity. The high purification degree of the factor reinforces such a conclusion. The separate forms dissociated in the presence of dodecyl sulphate into one species with a molecular weight of 60 000, which may suggest that the larger species represent the polymeric forms of one protein component. The separated forms of EF1 were detected by two techniques: gel filtration at pH 8.0 and disc electrophoresis at pH 8.9. Also a preliminary sucrose density gradient ultracentrifugation at pH 7.8 revealed two distinct EF1 peaks corresponding to monomeric (mol. wt 60 000 and trimeric mol. wt 180 000) forms. Since the intermediate component between these two forms was not detected by any of the above methods, it may be concluded that the dimeric form of EF1 is highly unstable. On the other hand, relative proportions of the forms observed indicate that the trimeric form with a molecular weight of 180 000 is the most stable. However, it is not certain yet whether the characteristic peculiarity of EF1, observed in this investigation to exist in the above forms, is not restricted by the isolation methods and factor assay conditions applied. The question of multiple forms of factor EF1 has often come up during recent investigations on the elongation in eukaryotic systems. They were first detected by Schneir and Moldave 16 in rat liver extract in 1968. The recent studies of Collins et al. 17 confirmed this finding and revealed that EF1 from this material is heterogeneous in size with a range of 60 000170 000 molecular weight. In 1969 McKeehan and Hardesty is described the purification to apparent homogeneity of factor EF1 from rabbit reticulocytes. The biological activity indicated a single protein species of molecular weight 186 000, which dissociated in the presence of dissociating agents into three subunits of molecular weight 62 000. Furthermore, two species of elongation factor 1 (EFIA and EF1B) from calf

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B. GOLINSKA, A. B. LEGOCKI

brain were recently observed as revealed by the gel filtration method 19. The structural alteration o f the factors incurred during the isolation procedure cannot be unequivocally eliminated. However, it is difficult to account for all o f the above findings. Thus, it seems very likely that the heterogeneity in molecular size represents a general feature o f eukaryotic factors E F I . An unsolved question remains whether such a p h e n o m e n o n has any biological significance. It would be then interesting to find out if the particular EF1 forms are involved in the same interactions and if they have the same catalytic properties. The purified factor EF1 from wheat germ shows several enzymatic properties similar to those reported for analogous factors obtained from other sources. The specific interactions o f EF1 with G T P and a m i n o a c y l - t R N A , as revealed by changes in heat stability o f EF1, are in agreement with the general conclusion that such interactions directly preceded the ribosomal reactions o f the elongation cycle 13'14. Although G T P is required for enzymatic binding, it m a y be replaced by the structural analogue G M P - m e t h y l e n e diphosphonate. A b o v e 40 % o f the original binding was demonstrated in the presence o f G M P - m e t h y l e n e diphosphonate. However, similarly to the observations made when using bacterial factors. P h e - t R N A b o u n d in the presence o f the analogue does not convert into peptide, which m a y be caused by the inability o f G M P - m e t h y l e n e diphosphonate to replace G T P in the course o f ribosomal binding 2°. ACKNOWLEDGMENTS The authors are grateful to Professor J. Pawelkiewicz for stimulating discussions during the course o f this investigation. The work was supported by the Polish A c a d e m y o f Sciences within project No. 09.3.-2.1. REFERENCES I 2 3 4 5

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