Biological characterization of various forms of elongation factor 1 from rabbit reticulocytes

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 234, No. 2, November 1, pp. 603-611, 1964

Biological Characterization of Various Forms of Elongation from Rabbit Reticulocytes MARIA

DA GLORIA

Deparkent

of Biochemistry,

DA COSTA CARVALHO; JOSE FRANCISCO AND WILLIAM C. MERRICK’

Received

Case Western April

Reserve

University,

1’7, 1934, and in revised

scrod form

of Medicine, June

Factor 1 CARVALHO,l

Cleveland,

Ohio

4.4106

20, 1984

Two forms of elongation factor 1 (EF-1) have been tested for a variety of biological functions. One form, EF-lH, is a high-molecular-weight aggregate (M, > 500,000) containing four distinct polypeptides (a, /3, y, 6). The other form, EF-la, consists of a single polypeptide which is the same as the (Y subunit of EF-1H. Both EF-la and EF-1H function catalytically in binding Phe-tRNA to ribosomes, and in poly(U)directed polyphenylalanine synthesis. The activity of EF-la is enhanced in polyphenylalanine synthesis by a complementary component, EF-l/36. It is also shown that EF-l/36 can facilitate an exchange of EF-la-bound GDP for GTP. The EF-la dissociation constants for GDP and GTP were 0.47 and 0.55 PM respectively, while the EF-1H dissociation constants for GDP and GTP were 2.0 and 1.6 /IM, respectively. Thus, while EF-la and EF-1H had approximately the same affinities for GDP and GTP, the EF-la! dissociation constants were about fourfold lower than the EF-1H dissociation constants. Attempts to isolate complexes of EF-lcu or EF-1H with GTP and PhetRNA or with GTP, Phe-tRNA, and ribosomes were unsuccessful using either Millipore filters, gel filtration, or sucrose density gradients. The results presented in this report, along with studies from other laboratories, strengthen the hypothesis that the general mechanism of the elongation cycle is similar in eucaryotes and procaryotes. 0 1984 Academic

Press, Inc

Elongation factor 1 (EF-1): which catalyzes the GTP-dependent binding of aminoacyl-tRNA to ribosomes, has been purified from various eucaryotic sources, occurring in multiple forms and ranging in molecular weight from 50,000 to over l,OOO,OOO(1-15). The low-molecular-weight form (EF-la) has been purified from pig liver (8) and Artemia salina (13), and has been considered to be functionally analor Present address: Universidade Federal do Rio de Janeiro, Centro de Ciencias de Saude, Instituto de Biofisica, Ilha doFundao-Cidade Universitaria, Rio de Janeiro-R.J., Brasil. e To whom correspondence should be addressed. * Abbreviations used: EF, elongation factor; Hepes, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; GMP-P(NH)P, guanosine 5’-(&T-imino)triphosphate.

gous to bacterial EF-Tu. A complementary factor containing the other polypeptides (p, y, 6 or mixture) has been purified from pig liver (16), silk worm (17), A. salina (18), Krebs II mouse ascites tumor cells (19), and rabbit reticulocytes (20), and the activity has been compared to bacterial EF-Ts. As shown previously in this laboratory (12), the high-molecular-weight form (EF-1H) purified from rabbit reticulocytes contains four major polypeptides with molecular weights of 53,000 (ar), 50,000 (p), 38,000 (y), and 33,000 (6). This form is equivalent to bacterial EF-Tu * Ts. In analogy to bacterial EF-Tu, it has been proposed that eucaryotic EF-la is released from the ribosomes in the form of an EF-la . GDP complex subsequent to continued aminoacyl-tRNA binding (15). 603

0003-9861/&t Copyright All rights

$3.00

8 1984 by Academic Press. Inc. of reproduction in any form reserved.

604

CARVALHO,

CARVALHO,

This model is not in agreement with the conclusions of Drews and co-workers (1922). They propose that EF-lcu in ascites tumor cells becomes permanently bound to the ribosome during the elongation cycle and, as such, catalyzes multiple aminoacyl-tRNA bindings (21, 22). We have recently defined the conditions for the purification of EF-la and EF-l&3 from rabbit reticulocyte lysate. This has permitted us to study the mechanisms of action of these factors. This paper presents evidence that EF-la is released from the ribosome after each aminoacyl-tRNA is bound to the ribosome, and that the functional characteristics of EF-la and EFl/36 are similar to procaryotic EF-Tu and EF-Ts, respectively. MATERIALS

AND

METHODS

The radiolabeled compounds r’C]GTP (sp act, 320 mCi/mmol), [&“H]GDP (sp act, 11 Ci/mmol), [“Clphenylalanine (sp act, 450 mCi/mmol), and r’C]formaldehyde (sp act, 45.8 mCi/mmol) were purchased from New England Nuclear Corporation; unlabeled GTP and ATP were obtained from Calbiochem; cellulose nitrate filters (typo HA, 0.45-am pore size, 25 mm diameter) from Miihpore Corporation; sucrose (ultrapure, ribonuclease free) from Schwarz Bioresearch, Inc.; and rabbit reticulocyte lysate from Clinical Convenience, Madison, Wisconsin. All other chemicals were of the highest purity available. Elm&ion factors The low-molecular-weight form of elongation factor 1 (EF-lcu) and the elongation factor (EF-lj3G) from rabbit reticulocytes lysate were purified to homogeneity as described in the accompanying paper (23). The high-molecular-weight form of elongation factor 1 (EF-1H) and EF-2 were purified as previously described (12, 24). Poly(U)-directed polyphen&danine synthesis Reaction mixtures for the synthesis of polyphenylalanine contained, in 50 al, 20 mM Tris-HCl (pH 7.5), 10 mrd Mg(CH&Oe)p, 100 mM KCl, 1 mM dithiothreitol, 0.3 Am unit poly(U), 1 mrd GTP, 2 mM phosphoenolpyruvate, 0.2 IU pyruvate kinase, 0.2 Am unit ribosomes, 20 pmol [I’CJPhe-tRNA (sp act, 450 mCi/ mmol), 0.8 pg EF-2, and various levels of the different forms of EF-1 as indicated in the figure legends (12). Incubations were at 37’C for various lengths of time. Polyphenylalanine was determined as hot trichloroacetic acid-precipitable radioactivity, which was collected by vacuum filtration onto Millipore filters (type HA). Radioactivity was determined by liquid scintillation spectrometry.

AND

MERRICK

Enzyme binding of [%‘JPhe-tRNA to ribosmmx The reaction mixture contained, in a total volume of 50 pl, 20 rnrd Tris-HCl (pH 7.5). 10 mre Mg(CH&Oe)a, 100 mM KCl, 1 rnrd dithiothreitol, 30% glycerol, 1 mM GTP, 0.2 Am unit poly(U), 1 mM phosphoenolpyruvate, 0.1 IU pyruvate kinase, 1.5 Am units ribosomes, 20 pmol [“CjPhe-tRNA (sp act, 450 mCi/mmol) and either EF-1H (0.21 pg) or EF-la (0.05 pg). The solution was incubated at 37°C for various times as indicated. The binding of [‘C]Phe-tRNA to rihosomes was determined by retention on Millipore filters using an ice-cold wash buffer of 100 rnrc KCl, 20 rnbi Tris-HCl (pH ‘7.5). and 10 rnrd Mg(CH&Oa)c. The filters were dissolved in Bray’s scintillation fluid (New England Nuclear), and radioactivity was determined by liquid scintillation spectroscopy. Exchange of EF-la bmd GDP for GTP. The binary complex containing EF-la and [8H]GDP was formed in a 400-pl reaction mixture containing 20 mM Tris-HCl (pH 7.5), 10 mM Mg(CH&O&. 100 mM KCl, 1 mM dithiothreitol, 2 PM [8IXJGDP (2.5 Ci/ mmol), glycerol as indicated, and 15 pg EF-la. The reaction mixtures were incubated for 30 min on ice, and then EF-l@G (44 pg) and/or excess GTP (500 CM) were added. Continued incubation on ice was as indicated in the figure. A 50-111 aliquot of the reaction mixture was taken at different times to measure m]GDP bound to EF-la by retention on Millipore filters. The filters were washed with 1 ml ice-cold buffer containing 100 mre KCI, 20 mM Tris-HCl (pH 7.5), and 10 rnrc Mg(CH,CO.&. Filters were dissolved in Bray’s scintillation fluid, and radioactivity was determined by liquid scintillation spectrometry. Imlatim of 80 S. Phe-tRNA complexes b?/ sucrose density gradients The 80 S. Phe-tRNA complexes were formed in 200-pl reaction mixtures which contained 100 mM KCI, 25 mM Hepes-KOH (pH 7.5), 10 mM Mg(CH&Os)a, 1 mM dithiothreitol, 1.5 Azso units ribosomes, 1.8 mM phosphoenolpyruvate, 0.8 IU pyruvate kinase, 1 mM GTP, 2.88 pg [“C]EF-FLU (280 cpm/pmol) or 18 pg [“C]EF-1H (1600 cpm/pmol), 53 pmol [SHIPhe-tRNA (1000 cpm/pmol), and 6% glycerol. The reaction mixtures were incubated for 10 min on ice. The reaction mixtures were then layered on lo-40% (w/v) linear sucrose gradients containing 25 mM Hepes-KOH, (pH 7.5), 10 mM Mg(CH&O,),, 100 mM KCl, and 1 mM dithiothreitol. The tubes were centrifuged for 2.5 hr at 35,000 rpm at 4°C using a Spinco SW41 rotor. The sucrose gradients were unloaded by pumping out 1 ml/min through a flow cell in a Gilford spectrophotometer to record absorbance at 260 nm. Half-milliliter fractions were collected. Two hundred microliters of each gradient fraction was added to 10 ml of Bray’s scintillation fluid in the presence of 1 ml H,O. Radioactivity in the fractions was determined by liquid scintillation spectroscopy.

CHARACTERIZATION

OF

RABBIT

RETICULOCYTE

Labeling of EF-1H and EF-la bg reductive m&hEF-la and EF-1H were labeled by reductive methylation with [“Cjformaldehyde (sp act, 45.8 mCi/mmol) and sodium cyanoborohydride (25). About 1 mg EF-la or EF-1H was incubated in a reaction mixture containing, in 1.5 ml, 100 mM KCl, 10 rnre Hepes-KOH (pH 7.5), 1 mrd dithiothreitol, 2 mre r’Cjformaldehyde, 20 rnre sodium cyanoborohydride, and 20% glycerol. The samples were incubated on ice. Aliquots of 5 pl were taken and assayed for polyphenylalanine synthesis and for trichloroacetic acid-precipitated protein in order to measure the incorporation of radioactivity into protein. The reaction was continued until about 5 to 10% of the total lysyl residues were modified (30 min). The reaction was stopped by the addition of 150 pl 1 M NH&l made 25% in glycerol, and then the reaction mixture was dialyzed against 25 mM Hepes-KOH (pH 7.5), 200 mM KCl, 1 mM dithiothreitol, 25% glycerol, 0.1 mM EDTA. Under these conditions the labeled proteins were found to be fully active in polyphenylalanine synthesis. Dissooiatia constant for the binding of guanine nuchtides to EF-1. A binary complex containing [SH]GDP or [i”C]GTP and EF-lo or EF-1H was formed in a loo-cl reaction mixture containing 1.78 pg EF-la or 2.23 pg EF-lH, 20 mM Tris-HCl (pH 7.5). 10 mM Mg(CK&O& 100 mM KCl, 1 mM dithiothreitol, 5 pg ovalbumin, 15% glycerol, and guanine nucleotide from 0 to 10 pM. Incubation was for 10 min at 37°C. After incubation, 50 pl of the reaction mixture was poured onto a Millipore filter and washed once with cold buffer containing 100 mM KCl, 10 mM Mg(CHsCOp)2 and 20 my Tris-HCl (pH 7.5). The blank value without added factor was determined in each experiment and subtracted. The dissociation constants were determined by Scatchard plots.

ELONGATION

FACTOR

1

605

&dim

RESULTS

Catalytic Function of EF-1H and EF-la in Polyphenylalanine Synthesis In the eucaryotic system, EF-1H has been reported to show catalytic activity in polyphenylalanine synthesis. However, there have been differing reports on whether EF-la requires (26, 27) or does not require (28,29) a complementary factor to function catalytically. To address this question, a kinetic study was conducted to compare the biological function of rabbit reticulocyte EF-la and EF-1H in polyphenylalanine synthesis. As shown in Fig. 1, with a limiting amount of both elongation factors (approximately 2 pmol)

B

14+I.4

2

4

pmol

6

EF-IH

8 IO I2 TIME (mini

14

I6

I6

FIG. 1. Catalytic functioning of EF-1H and EF-la in polyphenylalanine synthesis. Polyphenylalanine synthesis was performed as described under Materials and Methods. The length of incubation is given in the figure. The picomoles of added EF-1H and EF-la were based on the molecular weights for each species as 150,000 and 50,000, respectively, which would correspond to equal inputs of EF-la.

in the reaetlon mixture, catalytic activity could be observed with each elongation factor. In this experiment both factors were at least fivefold more catalytically active. However, on a molar basis, EF-1H was twice as active as EF-la. The catalytic functioning of EF-la in the polymerization of polyphenylalanine was not dependent on a complementary factor, contrary to previous reports in bacteria and mammalian systems (15, 26, 27). The difference found in the activity between EF-lcu and EF-1H could be attributed to the association of EF-lcu with EF-l@. These factors could combine to form the complex EF-la - EF-l@G (EFlH), which would correspond to elongation factor Tu-Ts in bacteria. To test this possibility, a reconstitution experiment using EF-la and EF-l@ was performed. As shown in Fig. 2, protein synthesis did not occur in the presence of EF-l/36 (3.3 pmol) alone. However, the amount of polyphenylalanine synthesized in the presence of EF-la! (2 pmol) and EF-l@G (3.3 pmol) was comparable on a molar

CARVALHO,

CARVALHO,

AND

MERRICK

tivity was observed after 6 min of incubation. ll.OII.0

Catalytic Functioning of EF-1H and EF-la in the Binding of Phe-tRNA to Ribosm

20 pm, EF-la + 3 3 pmol EF-I,36

lO.OIO.0

/‘O

\ /’ /

9.0 i 6.0 -

1’0

Stoichiometric functioning for reticulocyte and mouse ascite cells EF-1 in the attachment of aminoacyl-tRNA to ribosomes has been reported (12, 19). Other studies with Artemia saline EF-l(28) and ascites tumor cell EF-1 (30) indicated multiple rounds of aminoacyl-tRNA binding to ribosomes in the absence of polymerization. In order to test the activity of EF-1H and EF-la, a kinetic study was performed in the presence of limiting amounts (1.4 and 1.0 pmol) of these elongation factors. As shown in Fig. 3, under these conditions both factors function catalytically in the binding of Phe-tRNA to ribosome. However, stoichiometric binding was observed when the reaction mixture was incubated on ice or when GMP-P(NH)P was substituted for GTP (data not shown). The effect of EF-l@ on the EF-la-dependent binding of PhetRNA to ribosomes was tested as described in Fig. 3. Under these conditions, a very slight stimulation of the binding reaction was observed. No activity was found for EF-l@ alone (data not shown).

/’

----a 0

2

4

6 TIME

8 (mini

IO

I2

I4

FIG. 2. Synergistic effect of EF-la and EF-l/36 on polyphenylalanine synthesis. The conditions for polyphenylalanine synthesis were described under Materials and Methods, except that EF-l,% was added as indicated in the figure; 0, 2 pmol EF-lq 0,2 pmol EF-la in the presence of 3.3 pmol EF-la& control (-), 3.3 pmol EF-lj3b in the absence of EF-la.

basis to the amount formed in the presence of EF-1H alone. Under these conditions, a 2.3-fold stimulation of EF-la! ac-

7

2

ii -3

-EF-IH

&I

-EF-la

d B 0

4

8

I2

4 TIME

8

12

16

20

24

(mid

FIG. 3. Factor-dependent binding of [l’C)Phe-tRNA to ribosomes. The EF-l-dependent binding of Phe-tRNA to ribosomes was performed as described under Materials and Methods. The amounts of EF-1H and EF-la added (as pmol of M, 150,000 and 50,000), and the lengths of incubation are given in the figure. Omission of EF-1 is as indicated.

CHARACTERIZATION

OF RABBIT

Isolation of 80 Se Phe-tRNA Cm~ Sucrose Density Gradients

RETICULOCYTE

by

In an attempt to isolate EF-1 ribosome complexes, binding of Phe-tRNA to ribosomes was performed in the presence of radiolabeled EF-1H or EF-la. After incubation of the reaction mixture on ice, the binding of 14C-labeled elongation factors was analyzed by sucrose density gradients. As indicated in Fig. 4, only the radioactivity corresponding to the binding of [‘HIPhe-tRNA was found with the ribosomal peak (fraction 10); no stable ribosome +EF-1 complex was detected by this technique. These data reinforce the previous finding (Fig. 3) suggesting that EF-1 is bound and then released from the ribosome after the binding of each PhetRNA. Results similar to those described above were found when the nonhydrolyzable GTP analog, GMP-P(NH)P, was used (data not shown). This result is in contrast with the results observed with bacterial EF-Tu, where the use of GMP-P(NH)P “freezes” EF-Tu on the ‘70 S. PhetRNA . poly(U) complex (15). l

FRACTION

ELONGATION

FACTOR

1

In another series of experiments, the binding of Phe-tRNA to ribosomes was measured at different incubation times (2, 10, and 60 min), and complex formation was analyzed by Sepharose-6B column chromatography. Although the binding of Phe-tRNA to the ribosomes increased with the time of incubation, no corresponding increase in the level of the factor bound to ribosomes was observed (data not shown). Exchange of EF-la-Bound

GDP j&r GTP

Previous reports indicated that EF-l/3 catalyzed the exchange of free GTP with EF-lcu GDP, with a consequent stimulation of protein synthesis (31,32) analogous to the ability to EF-Ts to enhance the exchange of GTP for EF-Tu-bound GDP (15). To examine if the EF-166 purified in our laboratory had the same functional property, the exchange of EF-la-bound GDP for GTP was examined. The binary complex (EF-la . GDP) was formed in the absence and in the presence of glycerol. As can be seen from the data shown in l

NUMBER

4. Isolation of 30 S. Phe-tRNA complexes by sucrose density gradients. Reaction mixtures (0.2 ml) were incubated for 10 min at 0°C as described under Materials and Methods. After incubation, the samples were layered on a 11-ml 10-4096 (w/v) linear sucrose gradient in 25 mrd Hepes-KOH (pH 7.5), 10 rns! Mg(CH&O&, 100 mM KCl, 1 mM dithiothreitol. Centrifugation was for 2.45 h at 35,030 rpm in an SW 41 rotor at 4°C. Fractions were collected (0.5 ml), and 50 pl was used for PHjPhe-tRNA (0) and [“C]EF-1 (0) radioactivity determination. FIG.

607

608

CARVALHO,

CARVALHO,

Fig. 5, in the absence of glycerol GDP bound to EF-la was rapidly displaced by the addition of unlabeled GTP. On the other hand, the rate of this exchange was reduced in the presence of increasing concentrations of glycerol. Under these conditions, exchange of EF-la-bound GDP for GTP was stimulated by EF-168 (approximately 8- to lo-fold in the presence of 10% glycerol). The results also show that the level of binary complex formed was higher when 10% glycerol was present in the reaction mixture. This presumably reflects the instability of EF-lcu in the absence of glycerol, as originally described by Kaziro and co-workers (33). These results are consistent with the view that our EF-l@S has a functional property similar to bacterial EF-Ts. Ternarg

Cimplex

AND

MERRICK

capacity of EF-1H and EF-la! to form a ternary complex with GTP and Phe-tRNA was investigated in the presence and absence of glycerol. The results of these experiments (Table I) indicate that this complex was not stable enough to be isolated by filtration through Millipore filters (Type HA). Possible ternary complex was detected only in small amounts and only in the presence of EF-la and 25% glycerol. This would indicate that approximately 3.9% of the EF-lo was in ternary complex. Interact&n of &o&w with EF-1

Nuckot&a

The affinity of EF-1H and EF-la for GTP and GDP was determined by the Millipore filter technique. In contrast to the ternary complex, the binary complex between EF-1 and guanine nucleotides was stable enough to be measured by this method. As presented in Table II, the dissociation constants found for GTP and GDP for EF-1H were 1.6 and 2 X low6 M, respectively. For EF-la, the dissociation constants for GTP and GDP were 6.6 and 4.6 X lo-” M, respectively. Thus, the affinity

Forwmticrn

It has been suggested that EF-1H is disaggregated into a low-molecular-weight form (EF-la) in the presence of GTP, and that EF-1~ also forms a stable complex with aminoacyl-tRNA and GTP (15, 19). On the basis of these observations, the

14-

1

13-

0%

GLYCEROL

5 %

GLYCEROL

; \ \ \

12-LEVEL

MINUS

GTP

0 z

8-

2 m

7-

8

6-

FI!!5

5-

MINUS

MINUS GTP - 17.9 pm,

GTP

..

O

GLYCEROL

‘? -LEVEL

g-

LEVEL

\

a IIT $ IOe

10%

I I

I 2

o---o------c I I I 3 4 5

I 6

I

1

I

I

I

+GTP+EF-l/38

I

I23456

123456 TIME

(mid

FIG. 5. Exchange of EF-la-bound GDP for GTP. [8H]GDP bound to EF-la was exchanged for GTP (500 PM) in the presence or absence of EF-l@. Experimental conditions were as described under Materials and Methods. The amounts of EF-la and EF-lj% added were 15 and 40 pg, respectively. Glycerol concentrations used were as indicated in the figure.

CHARACTERIZATION TABLE TERNARY

COMPLEX

OF RABBIT

RETICULOCYTE

I FORMATION

[‘“CjPhe-tRNA retained on Millipore filter (pmol) 25% Glycerol

0% Glycerol

binding

0.26 0.15 0.11

0.14 0.25 0.00

binding

0.21 0.27 0.00

0.14 0.14 0.00

Additions EF-lc~ + GTP EF-la GTP-dependent EF-1H + GTP EF-1H GTP-dependent

Note. Reaction mixtures, 56 cl, contained 0.1 mg bovine serum albumin, 20 mM Tris-HCl, pH 7.4, 10 mM Mg(CH&O&, 196 mM KCl, 1 mM dithiothreitol, 2.9 pmol EF-lo or 4 pmol EF-lH, 0.65 mM GTP as indicated, 14 pmol [14CjPhe-tRNA, and glycerol as indicated. Incubation of the reaction mixture was for 5 min at 0°C. The ternary complex formation was determined by retention of [“C]Phe-tRNA on a Millipore filter using a wash buffer of 196 mM KCl, 20 mM Tris-HCl, pH 7.5, 10 mM Mg(CHsO&. The blank value without added factors was determined in each experiment and subtracted. Each value in the table represents the average of six experiments.

of EF-la for GTP was approximately four times that of EF-1H. However, it should be noted that the binding constants for GTP and GDP were the same for each form of elongation factor 1. This is in marked contrast to the loo-fold higher affinity of bacterial EF-Tu for GDP than the GTP (15). DISCUSSION

In this report, we have examined the biological properties of several eucaryotic elongation factors with the hope of advancing the understanding of their mode of action and their possible equivalency to the bacterial factors EF-Tu and EF-Ts (i.e., EF-la = EF-Tu, EF-l@y6 or EF-l/IS = EF-Ts, and EF-1H = EF-la/3y = EFTu - Ts). As a starting point, the catalytic functioning of EF-1H and EF-la in polyphenylalanine synthesis (Fig. 1) and in the binding of Phe-tRNA to ribosomes (Fig. 3) was examined. It was found that both forms of EF-1 could be demonstrated

ELONGATION

FACTOR

1

609

to be catalytic. This result is in contrast to those reported by Grasmuk et al. (19, 22), where they proposed that EF-la remains bound to the ribosome during the entire peptide elongation cycle. If the recycling of either factor occurred “in situ” as they proposed, then catalytic binding of Phe-tRNA could only be explained by the synthesis of oligophenylalanine (promoted by nonenzymatic translocation or by a small contamination of EF-2 in the system). To check this, the binding reaction was stopped by the Millipore filter technique, followed by paper chromatography of the products extracted from the filter as described by Grasmuk et cd (19). It was found that all radioactivity was located in the area corresponding to phenylalanine (data not shown). Thus, the catalytic activity found in the binding assay was not due to the recycling of EFla or EF-1H “in situ” or to the synthesis of oligophenylalanine. Consistent with the catalytic nature of EF-la or EF-lH, we were unable to observe stable binding of either EF-la or EF-1H to ribosomes in the presence of GTP (or GMP-P(NH)P) and Phe-tRNA by either sucrose density gradient analysis (Fig. 4) or by gel filtration (data not shown) under near-physiological conditions. Grasmuk et al. have reported on the isolation of some complexes; however, only after formaldehyde fixation of the samples (22). Possibly either of our methods of isolation were too slow (2 h or 20 min, respectively) to allow isolation, or it is possible that, during its normal funcTABLE BINDING

CONSTANTS FOR GUANINE

II

OF EF-la AND EF-1H NUCLEOTIDES

Binding constants Nucleotide

EF-la (x10-7 M)

EF-1H (x10-6 M)

GDP GTP

4.6 6.6

2.0 1.6

Note. The binding of guanine nucleotides to either EF-la or EF-1H was determined as described under Materials and Methods using Millipore filters. The data were then analyzed by Scatchard plots.

610

CARVALHO,

CARVALHO,

tioning, EF-la is bound only transiently to the ribosome and thus little or no EFla would be observed in complex with the ribosome. While it is possible to explain our inability to find EF-la ribosome complexes, it is not clear why we could not detect a ternary complex between EF-la (or EFlH), GTP (or GMP-P(NH)P), and PhetRNA, as had been reported by others (23, 33). In spite of our ability to isolate binary complexes of EF-la or EF-1H with GTP (Table II) and the fact that the binding constant for Phe-tRNA was about lo-* M, we failed to observe any significant ternary complex formation using the rapid Millipore filter technique. In contrast, two laboratories have reported the detection of a ternary complex of EF-la, GTP, and aminoacyl-tRNA (28, 33). We have attempted to reproduce these experiments in detail and have been unsuccessful. This failure on our part may reflect species differences (rabbit vs. pig liver or A. SC& ina), differences in the preparation of factors, or a subtle difference in assay technique. With regard to this point, one laboratory reported that an EFla * GTP * aminoacyl-tRNA complex could be isolated by gel filtration using Sephadex G-75, but not Sephadex G-150 (33). In an alternate fashion, we have been able to demonstrate indirectly that EF-la or EF1H can form a ternary complex (data not shown). This was determined by the ability of EF-la or EF-1H to slow the nonenzymatic hydrolysis of the labile ester linkage in Phe-tRNA. In the presence of factor and GTP, the half-time of the hydrolysis reaction was increased two- to threefold. This protection was dependent on GTP, and did not occur with the initiator tRNA, Met-tRNAt, under identical conditions. Two functions have been proposed for EF-lj3S. First, this protein might increase the exchange of free GTP for GDP in EFla * GDP complexes, in analogy with bacterial EF-Ts (B-20). Second, EF-l@ might stimulate the recycling of EF-la by promoting its release from the ribosomes after the binding of Phe-tRNA and GTP hydrolysis (16). The data presented in Fig. 2 indicate that, although EF-la

AND

MERRICK

has catalytic activity in the polymerization of phenylalanine, the addition of EF-l@S results in an increase of the level of polymerization, a level equivalent to that observed for EF-1H. This increase could be attributed to either of the two functions described above for EF-l/38. The stimulation of the exchange of EF-la-bound GDP for GTP by EF-l/38 was examined, and was found to be dependent on the glycerol concentration in the assay mixture (Fig. 5). Under the conditions tested, an EFl/38 stimulation of the GDP-GTP exchange reaction was only evident at approximately 5 to 15% glycerol. At lower glycerol concentration no stimulation was observed and at higher glycerol concentrations, there was a very dramatic lack of GTP exchange for GDP, either in the presence or absence of EF-l@S (data not shown). The ability of EF-l/36 to stimulate the exchange of EF-la-bound GDP for GTP is analogous to the proposed role for EFTs (EF-Tu . GDP + GTP - EF-Tu . GTP + GDP) (36-38). However, there is one major difference. The EF-Tu binding constant for GDP is loo-fold lower than for GTP, whereas both EF-la and EF-1H exhibited similar binding constants for both GDP and GTP (Table II) (note: the affinity of EF-la for guanine nucleotides was approximately fourfold greater than that of EF-1H; the significance of this is not clear). Other laboratories have also reported comparable EF-la binding constants for GTP and GDP [pig liver (33), ascites tumor cells (39), and A. sulina (IS)]. The similarities in binding constants for GTP and GDP may explain why EFlj3S only stimulates the polymerization reaction several-fold, while a greater fold stimulation is observed with the corresponding bacterial component, EF-Ts. The results reported here are consistent with the growing evidence that the eucaryotic elongation cycle is similar to the procaryotic elongation cycle (15). The evidence presented in this report was obtained using elongation factors isolated by standard techniques, and no special treatments were required to isolate any component (23), whereas previous studies used forms of EF-l/3 that had been subjected to partially denaturing conditions

CHARACTERIZATION

OF

RABBIT

RETICULOCYTE

(16,20). Thus, hopefully these data reflect the function of native EF-la, EF-l/38, and EF-1H. In addition, the side by side comparison of EF-1~ and EF-1H has indicated a minimal difference between these two forms of EF-1, which probably reflects the somewhat minimal requirement for EF-l@ which usually yields only a twoto fourfold stimulation of EF-lar activity. In this regard, perhaps the greatest differences in the eucaryotic and procaryotic elongation cycle is the similarity in the binding constants for GDP and GTP (in contrast to the loo-fold difference in procaryotes) and the minimal requirement for EF-l@G (in place of procaryotic EF-Ts). ACKNOWLEDGMENTS This research was supported in part by fellowships from the Universidade Federal do Rio de Janeiro (M.G.C. and J.F.C.), from the Conselho National de Pesquiasa-Brasil (J.F.C.), and by Grant GM-26796 from the National Institute of General Medical Sciences (W.C.M.). The authors thank Yvonne Coleman for her excellent editorial assistance in the preparation of this manuscript. REFERENCES 1. DREWS, J., BEDNARIK, K., AND GRASMUK, H. (1974) Eur. J. Biochem 41,217-227. 2. MOON, H. M., REDFIELD, B., MILLARD, S., VANE, F., AND WEISSBACH, H. (1973) Proc NatL Ad Sti USA 70.3282-3286. 3. G~LINSKA, B., AND LEGOCKI, A. B. (1973) Biochim Biophys Acta 324,156-170. 4. LANZANI, G. A., BOLLINI, R., AND SOFFIENTINI, A. N. (1974) Biochim Biophya Actu 336, 2’75283. 5. LIU, C. K., LEGOCKI, A. B., AND WEISSBACH, H. (1974) in Lipmann Symposium (Richter, D., ed.), pp. 384-398, de Gruyter, Berlin. 6. SCHNEIR, M., AND MOLDAVE, K. (1968) B&him Biuphys. Acta 166,58-67. 7. COLLINS, J. F., MOON, H. M., AND MAXWELL, E. S. (1972) Biochemi&y 11,4187-4194. 8. IWASAKI, K., NAGATA, S., MIZUMOTO, K., AND KAZIRO, Y. (1974) J. Bid Chem 249, 50085010. 9. MCKEEHAN, W. L., AND HARDESTY, B. (1969) J. Biol Ckem 244,4330-4339. 10. B~LLINI, R., SOFFIENTINI, A. N., BERTANI, A., AND LANWNI, G. A. (1974) Biochemistry 13, 54215425. 11. TARRAGO, A., ALLENDE, J. E., REDFIELD, B., AND WEISSBACH, H. (1973) Arch. Biochem Biuphyx 159,353-361. 12. KEMPER, W. M., MERRICK, W. C., REDFIELD, B., LIU, C. K., AND WEISSBACH, H. (1976) Arch Biochem Biophgs. 174, 663-612.

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