/ . Biochem. 82, 1633-1646 (1977)
Purification and Properties of Polypeptide Chain Elongation Factor-la from Pig Liver Shigekazu NAGATA, Kentaro IWASAKI, and Yoshito KAZIRO Institute of Medical Science, University of Tokyo, Takanawa, Minato-ku, Tokyo 108 Received for publication, July 18, 1977
Eukaryotic polypeptide chain elongation factor-la (EF-la) has been purified from pig liver by steps including aqueous two-phase separation, ammonium sulfate fractionation, and three successive column chromatographies on CM-Sephadex, DEAE-Sephadex, and CM-Sephadex. On the last column chromatography on CM-Sephadex, EF-1 a activity was eluted as four peaks. These peaks except the first one appear to be homogeneous as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and isoelectric focusing in polyacrylamide gel. There was no difference between these four peaks in the activity to promote the binding of Phe-tRNA to ribosomes in the presence of guanyl-5'-yl methylenediphosphonate. They had similar molecular weights and similar amino acid compositions but different isoelectric points which varied from 9.3 to 9.9. The molecular weight of EF-lar was estimated to be 53,000 and 61,000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and by gel filtration on Sephadex G-150, respectively, indicating that EF-la is composed of a single polypeptide. EF-la appears to contain 6 half-cystine residues per molecule, at least one of which is essential for its activity. The effect of the concentration of NH4CI on the dissociation constant of the binary complex containing EF-la and guanine nucleotides (EF-la-GDP or EF-la-GTP) was extensively investigated, and it was found that the logarithm of the dissociation constant of EF-la-GDP increased proportionally to the square root of the concentration of NH4C1, while that of EF-1 a-GTP decreased. Similar relationships were observed between the rate constant of dissociation of EF-la-GDP or EF-la-GTP and the concentration of NH4CI. When the rate constant of association was calculated from these values, the logarithm of the constant of EF-la-GDP and that of EF-1 aGTP found to decrease proportionally to the square root of the NHjCl concentration.
In the eukaryotic system, the polypeptide chain elongation reaction requires two complementary factors, EF-11 and EF-2, of which the former
catalyzes the GTP-dependent binding of aminoacyltRNA to the aminoactyl site of ribosomes and the latter promotes the GTP-depcndent translocation
1 A uniform nomenclature for elongation factors, EF-1, EF-2, EF-Tu, EF-Ts, and EF-G, is used in this paper (sec Ref. /). Abbreviations: GMP-P(CH,)P, guanyl-5'-yl methylenediphosphonate; GMP-P(NH)P, guanyl-5'-yl imidodiphosphate; Na-DodSO4, sodium dodecyl sulfate; DTT, dithiothreitol; pCMBp-chloromercuribenzoate.
Vol. 82, No. 6, 1977
1633
1634 of peptidyl-tRNA from the aminoacyl to the peptidyl site on the same ribosomes (2). In spite of many attempts with various tissues, the molecular form of EF-1 has not been well elucidated because of its heterogeneity (5-7). Schneir and Moldave (3) found three forms of EF-1 with different molecular weights ranging from 100,000 to 400,000 in rat liver, while McKeehan and Hardesty (4) reported that EF-1 from rabbit reticulocytes had a molecular weight of 186,000. It has been further suggested that the high molecular weight form of EF-1 (EF-1H) isolated from calf brain and Krebs ascites cells was an aggregated form of the light species (EF-1L) having a molecular weight of 50,000 to 60,000 (6, 7). On the other hand, EF-1 H purified from wheat germ and Artemia salina cysts has been reported to consist of three different polypeptides (8, 9). In the course of the purification of EF-1 from pig liver we found that the factor could be resolved into two complementary factors, EF-1 a and EF-1/3;- (JO). Furthermore, as reported in the previous papers (77, 12), it was found that in a variety of tissues including liver, reticulocytes and Artemia salina cysts, there were two species of EF-1 a differing in their molecular weights, of which the low molecular weight form was found to be the major one, when it was stabilized by the addition of glycerol to the solution (77, 12). Thus, extensive purification of the low molecular weight form of EF-1 a has been attempted to obtain a homogeneous preparation (77). Purified EF-1 a has been found to form not only a binary complex of EF-1 a-GDP or EF-la-GTP, but also a ternary complex consisting of EF-1 a, Phe-tRNA, and GTP (13). These results clearly indicate that the low molecular weight form of EF-1 a is functionally equivalent to EF-Tu in the bacterial system. Recently, we were able to purify EF-1 fiy from pig liver to a homogeneous state and showed that it stimulates three reactions, namely, polyphenylalanine synthesis in the presence of EF-1 a and EF-2, the EF-lar-dependent binding of Phe-tRNA to ribosomes and the exchange of bound GDP to EF-1 a with exogenous GTP (14-16). From these observations we have concluded that EF-10;functions similarly to bacterial EF-Ts. Since the previous report (77) on the purification of the low molecular weight form of EF-lcr from pig liver was preliminary, the detailed pro-
S. NAGATA, K. IWASAKI, and Y. KAZIRO cedure of its purification, together with some physicochemical properites of the purified EF-1 or are described in the present communication. MATERIALS AND METHODS Buffers—The following buffers were used. Buffer A, 50mM Tris-HCI (pH8.0),' 4 mM Mg(CHjCOO),, 5 mM 2-mercaptoethanol, 25 mM KCI, and 0.35 M sucrose; Buffer B, 20 mM Tris-HCI (pH 7.5), 0.1 min EDTA, 10 mM 2-mercaptoethanol, and 25% (v/v) glycerol; Buffer C, same as Buffer B except that the pH of the Tris-HCI buffer was 9.0; and Buffer D, 20 mM Tris-HCI (pH 7.5), 0.1 mM EDTA, 10 mM 2-mercaptoethanol, and 10% (v/v) glycerol. Ribosomes—Washed 80S ribosomes from Artemia salina cysts were prepared as described previously (77), which had no detectable EF-1 nor EF-2 activity. Assay—The EF-1 a activity was assayed by measuring the extent of the poly(U)-dependent binding of [ u C]Phe-tRNA to ribosomes in the presence of guanyl-5'-yl methylenediphosphonate (GMP-P(CH.)P) (13). The standard conditions used were as follows: the reaction mixture contained, in a final volume of 0.1 ml, 50 mM TrisHCI (pH7.5), 75 mM KCI, 0.2 mM dithiothreitol (DTT), 0.1 mM GMP-P(CH,)P, 10 /*g of bovine serum albumin, 16 pmol of [uC]Phe-tRNA (382 Ci/ mol), 1.0 Ait0 unit of ribosome-poly(U)-tRNA complex which was prepared as described previously (77), and an appropriate amount of EF-1 a. Incubation was carried out for 5 min at 37°C, and the reaction was stopped by the addition of 3 ml of chilled washing buffer containing 20 mM Tris-HCI (pH7.5), 5mM Mg(CHjCOO),, and 100 mM NH4CI. Then, the diluted sample was poured onto a nitrocellulose membrane filter (0.45 fim pore size, Sartorius-Membranfilter GmbH). The filter was washed twice with 3 ml of the washing buffer, dried and counted in a liquid scintillation spectrometer. Under these conditions, the amount of ["C]Phe-tRNA bound to ribosomes was proportional to that of EF-1 a added to the reaction mixture up to 10 pmol. One unit of EF-1 or is defined as the activity which promoted the binding 1
The pH of the buffer solution was always adjusted at 20°C. / . Biochem.
PURIFICATION AND PROPERTIES OF EF-la FROM PIG LIVER of 1.0 nmol [14C]Phe-tRNA to nbosomcs under the standard conditions. Because GMP-P(CH,)P in the standard assay mixture could be replaced by guanyl-5'-yl imidodiphosphate (GMP-P(NH)P) without affecting the results, 0.1 ITIM GMP-P(NH)P was used in place of GMP-P(CHi)P in some experiments as indicated. The activities of EF-2 and EF-Tu were measured according to Mizumoto et al. (17) and Arai et al. (18), respectively. Polyacrylamide Gel Electrophoresis—Gel electrophoresis in the presence of sodium dodecyl sulfate (Na-DodSO 4 ) was carried out on a slab gel (130x90 mm, 1.2 mm thick) according to Maizel (19). The molecular weight of E F - l a was estimated from its relative electrophoretic mobility to the standard proteins. The proteins used as standards and their sources were as follows: crystalline bovine serum albumin-, Armour; beef liver catalase [EC 1.11.1.6], Boehringer; lactate dehydrogenase [EC 1.1.1.27] from rabbit muscle, Boehringer; cytochrome c from horse heart, Sigma; purified EF-2 prepared from pig liver as described previously (75, 77); EF-Tu purified from E. coli MRE 600 (75); and EF-G purified from E. coli MRE 600 (20). Purified EF-Tu and EF-G were kindly supplied by Drs. K. Arai and N. Arai of this department, respectively. Isoelectric focusing of E F - l a was carried out at 0°C as described by Wrigley (27) with slight modifications. The gel contained 8 % acrylamide, 2% carrier ampholites (a mixture of equal parts of Ampholine of pH 7 to 9 and of pH 9 to 11; LKB Instruments, Inc.), 20% (v/v) glycerol and 0.1 ITIM DTT, and was photopolymerized in the presence of riboflavin. The sample containing 25% (v/v) glycerol was layered on the gel. over which was layered 100^1 of a solution containing 2 % Ampholine of pH 7 to 11 and 10% (v/v) glycerol. Electrophoresis was initially carried out with a constant current of 1 mA per tube (0.5x6.5 cm) until the voltage increased to 200 volts and then with a constant voltage of 200 volts. Achievement of the near equilibrium with respect to both the pH gradient and the migration of the protein samples was confirmed by using proteins with known p7 values, such as hemoglobin and cytochrome c. After the electrofocusing was completed, the gels were stained with bromophenol blue as described by Awdeh (22). Vol. 82, No. 6, 1977
1635
Analytical Gel Filtration—The molecular weight of EF-1 a was also estimated by gel filtration on Sephadex G-150 (23). The column (1.2 x 53 cm) was equilibrated with Buffer B containing 0.3 M NH 4 CI, to which was applied the sample (0.3 ml) previously dialyzed against the same solution. The column was developed with the same solution at a flow-rate of 4.5 ml/h and 1-ml fractions were collected. The following proteins were used as references for the molecular weight determination; beef liver catalase [EC 1.11.1.6], Boehringer; crystalline bovine serum albumin, Armour; cytochrome c from horse heart, Type II, Sigma; purified pig liver EF-2 (75, 77); and purified EF-Tu from E. coli (18). The elution position of catalase as well as that of cytochrome c was determined spectrophotometrically by the absorbance at 405 nm, and that of albumin at 280 nm. The elution positions of E F - l a , EF-2, and EF-Tu were determined by measuring their enzymatic activities. Amino Acid Analysis—For determination of the amino acid composition of E F - l a , 200/ig of purified E F - l a was dialyzed against glass-distilled water and hydrolyzed under vacuum in 1.0 ml of constant boiling 6 N HCI at 110°C for 24 h and 72 h by the method of Moore and Stein (24). The amino acid analysis was performed on an amino acid analyzer, Japan Electron Optics Laboratory (model JLC-6AH). The values for serine, threonine and tyrosine were extrapolated to zero time to correct for any decomposition during hydrolysis, and those for leucine, isoleucine, and valine were derived from the sample hydrolyzed for 72 h. The half-cystine content was determined from the amount of carboxymethylcysteine formed after reductive carboxymethylation of E F - l a according to Waxdal et al. (25). The tryptophan content was estimated from the ratio of tyrosine to tryptophan as determined spectrophotometrically in 0.1 N NaOH (26). Dissociation Constant of the Binary Complex Containing EF-la and Guanine Nucleotide—The dissociation constant of the EF-la-guanine nucleotide complex was determined by measuring the amounts of the binary complex containing E F - l a and [sH]guanine nucleotide retained on a nitrocellulose membrane, which was formed after incubating a constant amount of E F - l a with various amounts of either ['H]GTP or [ 3 H]GDP. The reaction mixture contained, in a final volume of
1636 0.5 ml, 20 mM Tris-HCl (pH7.5), 5 mM Mg(CH,COO),, 5mM 2-mercaptoethanol, 10% (v/v) glycerol, 100//g of bovine serum albumin, 10 pmol of EF-lo, various concentrations of NH 4 CI, and various amounts of the [JH]guanine nucleotide (500 Ci/mol) as indicated. The reaction was started by the addition of EF-la. After allowing the reaction mixture, at 0°C for 5 min, to reach the equilibrium, it was transferred onto a nitrocellulose membrane filter and filtered. The filter was carefully washed twice with 0.5 ml of a solution containing 20 mM Tris-HCl (pH 7.5), 5 mM Mg(CH 3 COO),, 5 mM 2-mercaptoethanol, and 10% (v/v) glycerol, dried and counted in a liquid scintillation spectrometer.
S. NAGATA, K. IWASAKI, and Y. KAZIRO Warburg and Christian (28). Miscellaneous—[uC]Phe-tRNA (0.7 nmol/mg tRNA) was prepared by aminoacylation of E. coli B tRNA (Schwartz BioResearch) with partially purified phenylalanyl-tRNA synthetase from E. coli Q13. [14C]Phenylalanine (382 Ci/mol) was purchased from Daiichi Pure Chemicals, Tokyo. [3H]GDP and [3H]GTP were obtained from the Radiochemical Centre, Amersham, England. RESULTS
Purification of EF-la from Pig Liver—All procedures were carried out at 0-5°C. Crude extract: Fresh pig liver (1.3 kg) obKinetic Study of Dissociation of Guanine tained from slaughterhouse was cut into small Nucleotide from the EF-la-Guanine Nucleotide pieces and homogenized with 2 liters of Buffer A for 3 min in a Waring Blendor at the top-speed. Complex—The binary complex containing EF-la and [3H]guanine nucleotide was prepared by The homogenate was centrifuged at 15,000 xg for 15 min and the precipitate was discarded. The suincubating the reaction mixture at 37°C for 5 min, which contained 40 mM Tris-HCl (pH 7.5), 10 mM pernatant obtained (the post-mitochondrial superMg(CH 3 COO),, 0.2 M NH4C1, 25% (v/v) glycerol, natant, 2,350 ml) contained 72 mg of protein per ml. 120 fi% of bovine serum albumin, 10 //g of EF-la, Aqueous two-phase separation: To 2 liters of and either 4 /JM [3H]GTP (500 Ci/mol) or 4 /JM the supernatant from the previous step were added [3H]GDP (500 Ci/mol) in a final volume of 0.06 ml. 562.5 ml of 30% (w/w) polyethylene glycol #6000 The rate of dissociation of the EF-la-PHJguanine (Nakarai Chemicals, Kyoto) and 187.5 ml of 20% nucleotide complex was determined by incubating (w/w) dextran T500 (Pharmacia Fine Chemicals, the binary complex prepared as above in the presUppsala). The mixture was stirred for about ence of a large excess (more than 300-fold) of 10 min in a Waring Blendor and the phases were unlabeled GTP. The reaction mixture contained, separated by centrifugation for 15 min at 15,000 in a final volume of 0.55 ml, 20 mM Tris-HCl xgr. The lower dextran phase was saved because (pH 7.5), 5 mM Mg(CHjCOO),, 5 mM 2-mercap- it contained EF-la, EF-1/9^, and EF-2 exclusively. toethanol, 10% (v/v) glycerol, 70 mM NH4C1, and To the lower phase were added 412.5 g NH4CI 0.15 mM GTP, to which was added 0.05 ml of the and the polyethylene glycol phase that had been EF-la-PHJguanine nucleotide complex prepared prepared in a similar manner as above except that as above. The solution was rapidly mixed and the supernatant was replaced by 2 liters of Buffer kept at 0°C. At the specified time, a 0.1-ml A. After dissolving the NH C1 thoroughly, the 4 portion was diluted with 0.5 ml of the dilution mixture was stirred for 10 min, and then the two buffer containing 20 mM Tris-HCl (pH 7.5), 10 mM phases were again separated by centrifugation. Mg(CH,COO),, 0.1 M NH4C1, 50/*g of bovine Since all elongation factors were transferred into serum albumin, and 25% (v/v) glycerol. The the polyethylene glycol phase by this procedure, diluted sample was filtered through a nitrocellulose the upper polyethylene glycol phase was collected. membrane filter. The filter was washed twice with Polyethylene glycol in this phase was removed by the dilution buffer without glycerol and bovine the use of an aqueous two-phase separation system serum ablumin, dried and the remaining EF-la• consisting of polyethylene glycol and ammonium [*H]guanine nucleotide complex was measured. sulfate. To 2,500 ml of the upper phase obtained Protein Concentration—Protein concentrations as above was added 440 g of solid (NH^iSCV were determined by either the colorimetric method After dissolving the (NH4),SO4, the mixture was of Lowry et al. (27) with bovine serum albumin as stirred for 10 min and centrifuged for 15 min at a standard, or the spectrophotometric method of 8,000xg using a horizontal rotor to separate the J. Biochem.
PURIFICATION AND PROPERTIES OF EF-la FROM PIG LIVER three phases: the top (polyethylene glycol), the middle (precipitate) and the bottom (ammonium sulfate) phases. The bottom phase containing all of the elongation factors was carefully saved by aspiration and the proteins were precipitated by the addition of 416 g (20g/100ml) of (NH 4 ) 8 SO 4 . After stirring overnight, the precipitate was collected by centrifugation for 30 min at 15,000 X g. Ammonium sulfate fraction: The precipitated proteins from the previous step were extracted three times with 200 ml, 150 ml, and 150 ml of 50% saturated ammonium sulfate solution in Buffer A (29.1 g of (NH 4 ),SO 4 per 100 ml of Buffer A). The undissolved materials after the extraction were dissolved in Buffer B, which was designated as the 30-50% saturated ammonium sulfate fraction. The proteins extracted with 50% saturated ammonium sulfate solution were precipitated by adding (NH 4 ),SO 4 (19g/100ml) to give 80% saturation, and the precipitate was collected by centrifugation for 30 min at 15,000 x g. The pellet was dissolved in Buffer B and designated as the 50-80% saturated ammonium sulfate fraction. It was found that the 50-80% saturated ammonium sulfate fraction contained the low molecular weight form of EF-1 (EF-la), together with EF-2, while the 30-50% saturated ammonium sulfate fraction contained the high molecular weight form of EF-1. Therefore, the former was used as the crude fraction of EF-1 a for further purification. The whole procedure described above was repeated and the two 50-80% saturated ammonium sulfate fractions were combined. Thus, 150 ml of the fraction containing 100 mg protein per ml were obtained from 2.2 kg of pig liver. First CM-Sephadex column chromatography: The 50-80% saturated ammonium sulfate fraction was extensively dialyzed against Buffer B containing 50 mM NH4C1 and applied to a column (3 x47 cm) of CM-Sephadex C-50 previously equilibrated with Buffer B containing 40 mM NHjCl. The column was then developed successively with 1 liter of Buffer B containing 58 mM, 165 mM, and 350 mM NH4C1 at a flow-rate of 20 ml/h. The E F - l a activity was found in the fractions eluted with Buffer B containing 350 mM NH 4 C1. The active fractions were combined to give 210 ml of a solution, which contained 2.4 mg protein per ml. Since the fractions eluted with Buffer B containing 165 mM NH 4 CI contained most of the EF-2 Vol. 82, No. 6, 1977
1637
activity, they were combined and used as starting material for the purification of EF-2 (15). DEAE-Sephadex column chromatography: The combined fractions containing E F - l a described above were dialyzed overnight against 5 liters of Buffer D containing 80% saturated (NH 4 ),SO 4 (65 g (NH 4 ),SO 4 per 100 ml). The precipitate formed after dialysis was collected by centrifugation and dissolved in a small amount of Buffer C containing 5 mM NH4C1. The solution was then dialyzed overnight against the same buffer. The dialyzed material was applied to a column of DEAE-Sephadex A-25 (1.2x16 cm) previously equilibrated with Buffer C, and the column was washed with Buffer C containing 5 mM NH4C1 at a flow-rate of 10 ml/h. The flow-through fractions containing the E F - l a activity were pooled, and dialyzed overnight against Buffer B containing 180 mM NHiCI. After dialysis, 27 ml of the fraction were obtained, which contained 12.3 mg protein per ml. Second CM-Sephadex column chromatography: The dialyzed solution from the previous step was applied to a column of CM-Sephadex C-50 (1.6 x 75 cm) equilibrated with Buffer B containing 170 mM NH 4 CI. After washing the column with Buffer B containing 180 mM NH 4 C1, elution was carried out with a linear gradient of NH4CI from 185 to 315 mM in Buffer B in a total volume of 1.5 liters. The flow-rate was 5 ml/h and 2.5-ml fractions were collected. As shown in Fig. 1, E F - l a was eluted as four peaks at 225, 245, 270, and 290 mM NH 4 C1, which were designated as Peaks I, II, III, and IV, respectively. Each peak was separately combined, and the pooled fractions were dialyzed against 5 liters of Buffer D containing 80% saturated (NH 4 ) t SO 4 prepared as above. The precipitate appearing was collected by centrifugation and dissolved in a small volume of Buffer B, and the solution was dialyzed overnight, against Buffer B containing 0.3 M NH 4 C1. After removing the insoluble materials by centrifugation at 15,000 x g for 20 min, the clarified supernatant was used as the purified E F - l a . The purified E F - l a could be stored at — 80°C without any appreciable loss of the activity for at least one year, provided that the protein concentration was higher than 1 mg/ml. A summary of typical purification of E F - l a is given in Table I, which indicates that the overall purification of E F - l a was about 340-fold starting
1638
S. NAGATA, K. IWASAKI, and Y. KAZIRO
from the post-mitochondrial supernatant with a yield of 24%. Purity—The purified EF-la was tested for homogeneity by Na-DodSO4 polyacrylamide gel electrophoresis and isoelectric focusing in polyacrylamide gel. Figure 2A shows the results obtained by Na-DodSO4 gel electrophoresis of the four peaks obtained on CM-Sephadex column chromatography. Peaks II, III, and IV migrated
as a single band, but Peak I also showed a faint minor band, although not visible in the figure. The mixture of the four peaks migrated as a single band, which indicates that there was no detectable difference in the electrophoretic mobility of the proteins in these four peaks. The purity of each peak was also analyzed by isoelectric focusing in polyacrylamide gel. As shown in Fig. 2B, Peaks II, III, and IV were again homogeneous B pH7.0
Peak I II in
Fig. 1. Second CM-Sephadex C-50 column chromatography of EF-la. The active fractions obtained by chromatography on a DEAE-Sephadex A-25 column were dialyzed and applied to a column of CM-Sephadex C-50 (1.6x75 cm). Elution was carried out with a linear gradient of NHjCl from 185 to 315mM as described in the text. The total volume of the buffer used was 1,500 ml and 2.5-ml fractions were collected at a flow-rate of 5 ml/h. O, EF-lor activity; X, concentration of NH4C1; and , absorbance at 280 nm.
rv•DMV MI
pHllD Peak
1
n ra
Fig. 2. A. Na-DodSO4 gel electrophoresis of purified EF-la. The four peaks of EF-la obtained by chromatography on a CM-Sephadex column were subjected to Na-DodSO4 gel electrophoresis. From left to right: Peak I, Peak II, Peak III, Peak IV (3 pig of protein to each gel), and a mixture of the four peaks (3 fig, of each peak). B. Isoelectric focusing of EF-la. Isoelectric focusing in %% acrylamide gel containing 2% carrier ampholite was carried out as described in " MATERIALS AND METHODS." From left to right: Peak I, Peak II, Peak III, Peak IV (10 fig of protein to each gel), and a mixture of the four peaks (10 fig of each peak).
TABLE I. Summary of the purification of EF-la from pig liver. Step Post-mitochondrial supernatant1 Aqueous twe-phase separation system and ammonium sulfate fractionation First CM-Sephadex column chromatography DEAE-Sephadex column chromatography Second CM-Sephadex column chromatography Peak I Peak U Peak III Peak IV
Protein (mg)
Activity (units x 10"*)
Specific activity (units/mg)
288,000
16.5
0.057
15,000
6.94
504
6.29 4.37
12.5 13.2
0.53 1.52 1.44 0.43
16.5 18.3 17.9 18.0
333
32.0 83.0 80.5 24.0
0.364
» Obtained from 2.2 kg of pig liver. / . Biochem.
PURIFICATION AND PROPERTIES OF EF-lo FROM PIG LIVER
1639
but Peak I also contained a minor component. It is noteworthy that the proteins in each peak were focused at slightly different positions, indicating that they had slightly different isoelectric points. This was further confirmed by electrofocusing of a mixture of the four peaks, in which four major bands were detectable. From the positions of these bands, the isoelectric points of proteins in Peaks I, II, III, and IV were calculated to be 9.3, 9.5, 9.7, and 9.9, respectively. The purified E F - l a did not contain lipid or carbohydrate in any measurable amount when 10 mg of the protein was analyzed by thin layer chromatography and mass spectrometry. Molecular Weight — Since the molecular weights of E F - l a in the four peaks were almost identical as shown in Fig. 2A, its precise value was determined using Peak III. Peak III together with several reference proteins was subjected to Na-DodS0 4 polyacrylamide gel electrophoresis, and the relative mobilities of the reference proteins were plotted against the logarithms of their molecular weights as shown in Fig. 3A. From the figure, the molecular weight of EF-lar was calculated to be 53,000. The molecular weight of Peak III was further determined by gel filtration using a Sephadex G-150 column as described in " MATERIALS AND METHODS." Figure 3B shows a plot of the logarithms of the molecular weights of several reference proteins versus their elution volumes. From the figure, the molecular weight of E F - l a was estimated to be 61,000, which is close to that obtained by Na-DodSO 4 polyacrylamide gel electrophoresis. This indicates that EF-lar is a single polypeptide chain having a molecular weight of 53,000 and no subunit structure. Similar observations were made with Peaks I, II, and IV. Amino Acid Composition—Since Peaks II, III, and IV were homogeneous (Fig. 2), the amino acid compositions of these peaks were determined. As shown in Table II, the proteins in each peak had the same amino acid compositions within experimental error when expressed as mole percent. This indicates that the numbers of amitio acid residues in these proteins were very close to each other, because their molecular weights were the same as described above. The total amount of the basic amino acids (histidine, lysine, and arginine) is 14.5% and that of the acidic amino acids (asparVol. 82, No. 6, 1977
02
QA Qfi MOBILITY
30
40 50 60 ELUTION VOLUME (ml)
Fig. 3. A. Molecular weight determination of EF-lar by Na-DodSOj polyacrylamide gel electrophoresis. Na-DodSO4 gel electrophoresis of EF-la and the reference proteins was carried out as described in " MATERIALS AND METHODS." The relative mobilities of the reference proteins are plotted against the logarithms of their molecular weights. The molecular weight of EF-la was determined from its relative mobility with the aid of this plot. The abbreviations and molecular weights (in parentheses) of the reference proteins or their subunits are: EF-2, purified EF-2 from pig liver (96,500); EF-G, purified EF-G from E. coli (83,000); BSA, bovine serum albumin (68,000); CAT, catalase from beef liver (60,000); EF-Tu, purified EF-Tu from E. coli (47,000); LDH, lactate dehydrogenase from rabbit muscle (36,000); and CYT, cytochrome c from horse heart (13,000). B. Analytical gel filtration of EF-la on a Sephadex G-150 column. EF-la and the reference proteins were chromatographed as described in " MATERIALS AND METHODS." The elution volumes of the reference proteins were plotted against the logarithms of their molecular weights. The abbreviations and molecular weights (in parentheses) of the reference proteins are: CAT, catalase from beef liver (230,000); EF-2, purified EF-2 from pig liver (110,000); BSA, bovine serum albumin (68,000); EF-Tu, purified EF-Tu from E. coli (48,000); and CYT, cytochrome c from horse heart (13,000).
tic acid and glutamic acid) is 18.8%. The fact that the p / values of these proteins were around 9.7 suggests that these acidic amino acids may exist in their amide forms (asparagine and glutamine), although no direct amide group analysis was performed. The half-cystine residues in E F - l a determined as carboxymethylcysteine after reductive carboxymethylation was about 1.2 mol %, which indicates that one molecule of EF-1 a contains approximately
S. NAGATA, K. IWASAKJ, and Y. KAZIRO ]
1640
TABLE II. Amino acid analysis of EF-la. Amino acid analyses of EF-la in Peaks II, III, and IV were carried out as described in " MATERIALS AND METHODS," and the results are expressed as mol %. The amount of half-cystine was determined as carboxymethylcysteine after reductive carboxymethylation, and that of tryptophan was estimated by the spectrophotometric method. The content of amides was not determined. Residue Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine Tryptophan
Peak II
Peak III
Peak IV
9.79 5.61 4.73 9.22 5.66 9.54 8.46 1.05 9.62 2. 12
9.58 6.18 4.93 8.95 5.45 10.30 8.25 1.24 8.81 2.19 6.81 6.16 2.58 3.18 2.40 8.38 3.58 0.87
9.43 5.81 5.05 9.36 5.84 9.92 8.34 1.15 9.21 1.81 6.41 5.76 2.38 3.12 2.52 8.22 3.58 0.82
6.68 6.15 2.44 3.44 2.48 8.50 3.69 0.81
6 half-cystine residues, assuming its molecular weight to be 53,000. This was further confirmed by titrating the sulfhydryls with radioactive pchloromercuribenzoate (pCMB) according to Robinson and Maxwell (29). When EF-la was titrated with [uC]pCMB in the presence of 6 M guanidine hydrochloride, it was found that EF-la contained 4 mol of sulfhydryl residues per mol, while when titration was carried out in the presence of both 0.2 M Na,SO, and 6 M guanidine hydrochloride, 5 mol of sulfhydryl residues were detected per mol (data not shown). This observation indicated that 1 mol of EF-la contained 4 mol of sulfhydryl residues and 1 mol of disulfide, or 6 mol of half-cystine. Stability—EF-la was very unstable, but the stability could be markedly increased by the ad-
2 5 10 20 50 pCMB CONCENTRATION (
Purification and Properties of Polypeptide Chain Elongation Factor-la from Pig Liver Shigekazu NAGATA, Kentaro IWASAKI, and Yoshito KAZIRO Institute of Medical Science, University of Tokyo, Takanawa, Minato-ku, Tokyo 108 Received for publication, July 18, 1977
Eukaryotic polypeptide chain elongation factor-la (EF-la) has been purified from pig liver by steps including aqueous two-phase separation, ammonium sulfate fractionation, and three successive column chromatographies on CM-Sephadex, DEAE-Sephadex, and CM-Sephadex. On the last column chromatography on CM-Sephadex, EF-1 a activity was eluted as four peaks. These peaks except the first one appear to be homogeneous as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and isoelectric focusing in polyacrylamide gel. There was no difference between these four peaks in the activity to promote the binding of Phe-tRNA to ribosomes in the presence of guanyl-5'-yl methylenediphosphonate. They had similar molecular weights and similar amino acid compositions but different isoelectric points which varied from 9.3 to 9.9. The molecular weight of EF-lar was estimated to be 53,000 and 61,000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and by gel filtration on Sephadex G-150, respectively, indicating that EF-la is composed of a single polypeptide. EF-la appears to contain 6 half-cystine residues per molecule, at least one of which is essential for its activity. The effect of the concentration of NH4CI on the dissociation constant of the binary complex containing EF-la and guanine nucleotides (EF-la-GDP or EF-la-GTP) was extensively investigated, and it was found that the logarithm of the dissociation constant of EF-la-GDP increased proportionally to the square root of the concentration of NH4C1, while that of EF-1 a-GTP decreased. Similar relationships were observed between the rate constant of dissociation of EF-la-GDP or EF-la-GTP and the concentration of NH4CI. When the rate constant of association was calculated from these values, the logarithm of the constant of EF-la-GDP and that of EF-1 aGTP found to decrease proportionally to the square root of the NHjCl concentration.
In the eukaryotic system, the polypeptide chain elongation reaction requires two complementary factors, EF-11 and EF-2, of which the former
catalyzes the GTP-dependent binding of aminoacyltRNA to the aminoactyl site of ribosomes and the latter promotes the GTP-depcndent translocation
1 A uniform nomenclature for elongation factors, EF-1, EF-2, EF-Tu, EF-Ts, and EF-G, is used in this paper (sec Ref. /). Abbreviations: GMP-P(CH,)P, guanyl-5'-yl methylenediphosphonate; GMP-P(NH)P, guanyl-5'-yl imidodiphosphate; Na-DodSO4, sodium dodecyl sulfate; DTT, dithiothreitol; pCMBp-chloromercuribenzoate.
Vol. 82, No. 6, 1977
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1634 of peptidyl-tRNA from the aminoacyl to the peptidyl site on the same ribosomes (2). In spite of many attempts with various tissues, the molecular form of EF-1 has not been well elucidated because of its heterogeneity (5-7). Schneir and Moldave (3) found three forms of EF-1 with different molecular weights ranging from 100,000 to 400,000 in rat liver, while McKeehan and Hardesty (4) reported that EF-1 from rabbit reticulocytes had a molecular weight of 186,000. It has been further suggested that the high molecular weight form of EF-1 (EF-1H) isolated from calf brain and Krebs ascites cells was an aggregated form of the light species (EF-1L) having a molecular weight of 50,000 to 60,000 (6, 7). On the other hand, EF-1 H purified from wheat germ and Artemia salina cysts has been reported to consist of three different polypeptides (8, 9). In the course of the purification of EF-1 from pig liver we found that the factor could be resolved into two complementary factors, EF-1 a and EF-1/3;- (JO). Furthermore, as reported in the previous papers (77, 12), it was found that in a variety of tissues including liver, reticulocytes and Artemia salina cysts, there were two species of EF-1 a differing in their molecular weights, of which the low molecular weight form was found to be the major one, when it was stabilized by the addition of glycerol to the solution (77, 12). Thus, extensive purification of the low molecular weight form of EF-1 a has been attempted to obtain a homogeneous preparation (77). Purified EF-1 a has been found to form not only a binary complex of EF-1 a-GDP or EF-la-GTP, but also a ternary complex consisting of EF-1 a, Phe-tRNA, and GTP (13). These results clearly indicate that the low molecular weight form of EF-1 a is functionally equivalent to EF-Tu in the bacterial system. Recently, we were able to purify EF-1 fiy from pig liver to a homogeneous state and showed that it stimulates three reactions, namely, polyphenylalanine synthesis in the presence of EF-1 a and EF-2, the EF-lar-dependent binding of Phe-tRNA to ribosomes and the exchange of bound GDP to EF-1 a with exogenous GTP (14-16). From these observations we have concluded that EF-10;functions similarly to bacterial EF-Ts. Since the previous report (77) on the purification of the low molecular weight form of EF-lcr from pig liver was preliminary, the detailed pro-
S. NAGATA, K. IWASAKI, and Y. KAZIRO cedure of its purification, together with some physicochemical properites of the purified EF-1 or are described in the present communication. MATERIALS AND METHODS Buffers—The following buffers were used. Buffer A, 50mM Tris-HCI (pH8.0),' 4 mM Mg(CHjCOO),, 5 mM 2-mercaptoethanol, 25 mM KCI, and 0.35 M sucrose; Buffer B, 20 mM Tris-HCI (pH 7.5), 0.1 min EDTA, 10 mM 2-mercaptoethanol, and 25% (v/v) glycerol; Buffer C, same as Buffer B except that the pH of the Tris-HCI buffer was 9.0; and Buffer D, 20 mM Tris-HCI (pH 7.5), 0.1 mM EDTA, 10 mM 2-mercaptoethanol, and 10% (v/v) glycerol. Ribosomes—Washed 80S ribosomes from Artemia salina cysts were prepared as described previously (77), which had no detectable EF-1 nor EF-2 activity. Assay—The EF-1 a activity was assayed by measuring the extent of the poly(U)-dependent binding of [ u C]Phe-tRNA to ribosomes in the presence of guanyl-5'-yl methylenediphosphonate (GMP-P(CH.)P) (13). The standard conditions used were as follows: the reaction mixture contained, in a final volume of 0.1 ml, 50 mM TrisHCI (pH7.5), 75 mM KCI, 0.2 mM dithiothreitol (DTT), 0.1 mM GMP-P(CH,)P, 10 /*g of bovine serum albumin, 16 pmol of [uC]Phe-tRNA (382 Ci/ mol), 1.0 Ait0 unit of ribosome-poly(U)-tRNA complex which was prepared as described previously (77), and an appropriate amount of EF-1 a. Incubation was carried out for 5 min at 37°C, and the reaction was stopped by the addition of 3 ml of chilled washing buffer containing 20 mM Tris-HCI (pH7.5), 5mM Mg(CHjCOO),, and 100 mM NH4CI. Then, the diluted sample was poured onto a nitrocellulose membrane filter (0.45 fim pore size, Sartorius-Membranfilter GmbH). The filter was washed twice with 3 ml of the washing buffer, dried and counted in a liquid scintillation spectrometer. Under these conditions, the amount of ["C]Phe-tRNA bound to ribosomes was proportional to that of EF-1 a added to the reaction mixture up to 10 pmol. One unit of EF-1 or is defined as the activity which promoted the binding 1
The pH of the buffer solution was always adjusted at 20°C. / . Biochem.
PURIFICATION AND PROPERTIES OF EF-la FROM PIG LIVER of 1.0 nmol [14C]Phe-tRNA to nbosomcs under the standard conditions. Because GMP-P(CH,)P in the standard assay mixture could be replaced by guanyl-5'-yl imidodiphosphate (GMP-P(NH)P) without affecting the results, 0.1 ITIM GMP-P(NH)P was used in place of GMP-P(CHi)P in some experiments as indicated. The activities of EF-2 and EF-Tu were measured according to Mizumoto et al. (17) and Arai et al. (18), respectively. Polyacrylamide Gel Electrophoresis—Gel electrophoresis in the presence of sodium dodecyl sulfate (Na-DodSO 4 ) was carried out on a slab gel (130x90 mm, 1.2 mm thick) according to Maizel (19). The molecular weight of E F - l a was estimated from its relative electrophoretic mobility to the standard proteins. The proteins used as standards and their sources were as follows: crystalline bovine serum albumin-, Armour; beef liver catalase [EC 1.11.1.6], Boehringer; lactate dehydrogenase [EC 1.1.1.27] from rabbit muscle, Boehringer; cytochrome c from horse heart, Sigma; purified EF-2 prepared from pig liver as described previously (75, 77); EF-Tu purified from E. coli MRE 600 (75); and EF-G purified from E. coli MRE 600 (20). Purified EF-Tu and EF-G were kindly supplied by Drs. K. Arai and N. Arai of this department, respectively. Isoelectric focusing of E F - l a was carried out at 0°C as described by Wrigley (27) with slight modifications. The gel contained 8 % acrylamide, 2% carrier ampholites (a mixture of equal parts of Ampholine of pH 7 to 9 and of pH 9 to 11; LKB Instruments, Inc.), 20% (v/v) glycerol and 0.1 ITIM DTT, and was photopolymerized in the presence of riboflavin. The sample containing 25% (v/v) glycerol was layered on the gel. over which was layered 100^1 of a solution containing 2 % Ampholine of pH 7 to 11 and 10% (v/v) glycerol. Electrophoresis was initially carried out with a constant current of 1 mA per tube (0.5x6.5 cm) until the voltage increased to 200 volts and then with a constant voltage of 200 volts. Achievement of the near equilibrium with respect to both the pH gradient and the migration of the protein samples was confirmed by using proteins with known p7 values, such as hemoglobin and cytochrome c. After the electrofocusing was completed, the gels were stained with bromophenol blue as described by Awdeh (22). Vol. 82, No. 6, 1977
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Analytical Gel Filtration—The molecular weight of EF-1 a was also estimated by gel filtration on Sephadex G-150 (23). The column (1.2 x 53 cm) was equilibrated with Buffer B containing 0.3 M NH 4 CI, to which was applied the sample (0.3 ml) previously dialyzed against the same solution. The column was developed with the same solution at a flow-rate of 4.5 ml/h and 1-ml fractions were collected. The following proteins were used as references for the molecular weight determination; beef liver catalase [EC 1.11.1.6], Boehringer; crystalline bovine serum albumin, Armour; cytochrome c from horse heart, Type II, Sigma; purified pig liver EF-2 (75, 77); and purified EF-Tu from E. coli (18). The elution position of catalase as well as that of cytochrome c was determined spectrophotometrically by the absorbance at 405 nm, and that of albumin at 280 nm. The elution positions of E F - l a , EF-2, and EF-Tu were determined by measuring their enzymatic activities. Amino Acid Analysis—For determination of the amino acid composition of E F - l a , 200/ig of purified E F - l a was dialyzed against glass-distilled water and hydrolyzed under vacuum in 1.0 ml of constant boiling 6 N HCI at 110°C for 24 h and 72 h by the method of Moore and Stein (24). The amino acid analysis was performed on an amino acid analyzer, Japan Electron Optics Laboratory (model JLC-6AH). The values for serine, threonine and tyrosine were extrapolated to zero time to correct for any decomposition during hydrolysis, and those for leucine, isoleucine, and valine were derived from the sample hydrolyzed for 72 h. The half-cystine content was determined from the amount of carboxymethylcysteine formed after reductive carboxymethylation of E F - l a according to Waxdal et al. (25). The tryptophan content was estimated from the ratio of tyrosine to tryptophan as determined spectrophotometrically in 0.1 N NaOH (26). Dissociation Constant of the Binary Complex Containing EF-la and Guanine Nucleotide—The dissociation constant of the EF-la-guanine nucleotide complex was determined by measuring the amounts of the binary complex containing E F - l a and [sH]guanine nucleotide retained on a nitrocellulose membrane, which was formed after incubating a constant amount of E F - l a with various amounts of either ['H]GTP or [ 3 H]GDP. The reaction mixture contained, in a final volume of
1636 0.5 ml, 20 mM Tris-HCl (pH7.5), 5 mM Mg(CH,COO),, 5mM 2-mercaptoethanol, 10% (v/v) glycerol, 100//g of bovine serum albumin, 10 pmol of EF-lo, various concentrations of NH 4 CI, and various amounts of the [JH]guanine nucleotide (500 Ci/mol) as indicated. The reaction was started by the addition of EF-la. After allowing the reaction mixture, at 0°C for 5 min, to reach the equilibrium, it was transferred onto a nitrocellulose membrane filter and filtered. The filter was carefully washed twice with 0.5 ml of a solution containing 20 mM Tris-HCl (pH 7.5), 5 mM Mg(CH 3 COO),, 5 mM 2-mercaptoethanol, and 10% (v/v) glycerol, dried and counted in a liquid scintillation spectrometer.
S. NAGATA, K. IWASAKI, and Y. KAZIRO Warburg and Christian (28). Miscellaneous—[uC]Phe-tRNA (0.7 nmol/mg tRNA) was prepared by aminoacylation of E. coli B tRNA (Schwartz BioResearch) with partially purified phenylalanyl-tRNA synthetase from E. coli Q13. [14C]Phenylalanine (382 Ci/mol) was purchased from Daiichi Pure Chemicals, Tokyo. [3H]GDP and [3H]GTP were obtained from the Radiochemical Centre, Amersham, England. RESULTS
Purification of EF-la from Pig Liver—All procedures were carried out at 0-5°C. Crude extract: Fresh pig liver (1.3 kg) obKinetic Study of Dissociation of Guanine tained from slaughterhouse was cut into small Nucleotide from the EF-la-Guanine Nucleotide pieces and homogenized with 2 liters of Buffer A for 3 min in a Waring Blendor at the top-speed. Complex—The binary complex containing EF-la and [3H]guanine nucleotide was prepared by The homogenate was centrifuged at 15,000 xg for 15 min and the precipitate was discarded. The suincubating the reaction mixture at 37°C for 5 min, which contained 40 mM Tris-HCl (pH 7.5), 10 mM pernatant obtained (the post-mitochondrial superMg(CH 3 COO),, 0.2 M NH4C1, 25% (v/v) glycerol, natant, 2,350 ml) contained 72 mg of protein per ml. 120 fi% of bovine serum albumin, 10 //g of EF-la, Aqueous two-phase separation: To 2 liters of and either 4 /JM [3H]GTP (500 Ci/mol) or 4 /JM the supernatant from the previous step were added [3H]GDP (500 Ci/mol) in a final volume of 0.06 ml. 562.5 ml of 30% (w/w) polyethylene glycol #6000 The rate of dissociation of the EF-la-PHJguanine (Nakarai Chemicals, Kyoto) and 187.5 ml of 20% nucleotide complex was determined by incubating (w/w) dextran T500 (Pharmacia Fine Chemicals, the binary complex prepared as above in the presUppsala). The mixture was stirred for about ence of a large excess (more than 300-fold) of 10 min in a Waring Blendor and the phases were unlabeled GTP. The reaction mixture contained, separated by centrifugation for 15 min at 15,000 in a final volume of 0.55 ml, 20 mM Tris-HCl xgr. The lower dextran phase was saved because (pH 7.5), 5 mM Mg(CHjCOO),, 5 mM 2-mercap- it contained EF-la, EF-1/9^, and EF-2 exclusively. toethanol, 10% (v/v) glycerol, 70 mM NH4C1, and To the lower phase were added 412.5 g NH4CI 0.15 mM GTP, to which was added 0.05 ml of the and the polyethylene glycol phase that had been EF-la-PHJguanine nucleotide complex prepared prepared in a similar manner as above except that as above. The solution was rapidly mixed and the supernatant was replaced by 2 liters of Buffer kept at 0°C. At the specified time, a 0.1-ml A. After dissolving the NH C1 thoroughly, the 4 portion was diluted with 0.5 ml of the dilution mixture was stirred for 10 min, and then the two buffer containing 20 mM Tris-HCl (pH 7.5), 10 mM phases were again separated by centrifugation. Mg(CH,COO),, 0.1 M NH4C1, 50/*g of bovine Since all elongation factors were transferred into serum albumin, and 25% (v/v) glycerol. The the polyethylene glycol phase by this procedure, diluted sample was filtered through a nitrocellulose the upper polyethylene glycol phase was collected. membrane filter. The filter was washed twice with Polyethylene glycol in this phase was removed by the dilution buffer without glycerol and bovine the use of an aqueous two-phase separation system serum ablumin, dried and the remaining EF-la• consisting of polyethylene glycol and ammonium [*H]guanine nucleotide complex was measured. sulfate. To 2,500 ml of the upper phase obtained Protein Concentration—Protein concentrations as above was added 440 g of solid (NH^iSCV were determined by either the colorimetric method After dissolving the (NH4),SO4, the mixture was of Lowry et al. (27) with bovine serum albumin as stirred for 10 min and centrifuged for 15 min at a standard, or the spectrophotometric method of 8,000xg using a horizontal rotor to separate the J. Biochem.
PURIFICATION AND PROPERTIES OF EF-la FROM PIG LIVER three phases: the top (polyethylene glycol), the middle (precipitate) and the bottom (ammonium sulfate) phases. The bottom phase containing all of the elongation factors was carefully saved by aspiration and the proteins were precipitated by the addition of 416 g (20g/100ml) of (NH 4 ) 8 SO 4 . After stirring overnight, the precipitate was collected by centrifugation for 30 min at 15,000 X g. Ammonium sulfate fraction: The precipitated proteins from the previous step were extracted three times with 200 ml, 150 ml, and 150 ml of 50% saturated ammonium sulfate solution in Buffer A (29.1 g of (NH 4 ),SO 4 per 100 ml of Buffer A). The undissolved materials after the extraction were dissolved in Buffer B, which was designated as the 30-50% saturated ammonium sulfate fraction. The proteins extracted with 50% saturated ammonium sulfate solution were precipitated by adding (NH 4 ),SO 4 (19g/100ml) to give 80% saturation, and the precipitate was collected by centrifugation for 30 min at 15,000 x g. The pellet was dissolved in Buffer B and designated as the 50-80% saturated ammonium sulfate fraction. It was found that the 50-80% saturated ammonium sulfate fraction contained the low molecular weight form of EF-1 (EF-la), together with EF-2, while the 30-50% saturated ammonium sulfate fraction contained the high molecular weight form of EF-1. Therefore, the former was used as the crude fraction of EF-1 a for further purification. The whole procedure described above was repeated and the two 50-80% saturated ammonium sulfate fractions were combined. Thus, 150 ml of the fraction containing 100 mg protein per ml were obtained from 2.2 kg of pig liver. First CM-Sephadex column chromatography: The 50-80% saturated ammonium sulfate fraction was extensively dialyzed against Buffer B containing 50 mM NH4C1 and applied to a column (3 x47 cm) of CM-Sephadex C-50 previously equilibrated with Buffer B containing 40 mM NHjCl. The column was then developed successively with 1 liter of Buffer B containing 58 mM, 165 mM, and 350 mM NH4C1 at a flow-rate of 20 ml/h. The E F - l a activity was found in the fractions eluted with Buffer B containing 350 mM NH 4 C1. The active fractions were combined to give 210 ml of a solution, which contained 2.4 mg protein per ml. Since the fractions eluted with Buffer B containing 165 mM NH 4 CI contained most of the EF-2 Vol. 82, No. 6, 1977
1637
activity, they were combined and used as starting material for the purification of EF-2 (15). DEAE-Sephadex column chromatography: The combined fractions containing E F - l a described above were dialyzed overnight against 5 liters of Buffer D containing 80% saturated (NH 4 ),SO 4 (65 g (NH 4 ),SO 4 per 100 ml). The precipitate formed after dialysis was collected by centrifugation and dissolved in a small amount of Buffer C containing 5 mM NH4C1. The solution was then dialyzed overnight against the same buffer. The dialyzed material was applied to a column of DEAE-Sephadex A-25 (1.2x16 cm) previously equilibrated with Buffer C, and the column was washed with Buffer C containing 5 mM NH4C1 at a flow-rate of 10 ml/h. The flow-through fractions containing the E F - l a activity were pooled, and dialyzed overnight against Buffer B containing 180 mM NHiCI. After dialysis, 27 ml of the fraction were obtained, which contained 12.3 mg protein per ml. Second CM-Sephadex column chromatography: The dialyzed solution from the previous step was applied to a column of CM-Sephadex C-50 (1.6 x 75 cm) equilibrated with Buffer B containing 170 mM NH 4 CI. After washing the column with Buffer B containing 180 mM NH 4 C1, elution was carried out with a linear gradient of NH4CI from 185 to 315 mM in Buffer B in a total volume of 1.5 liters. The flow-rate was 5 ml/h and 2.5-ml fractions were collected. As shown in Fig. 1, E F - l a was eluted as four peaks at 225, 245, 270, and 290 mM NH 4 C1, which were designated as Peaks I, II, III, and IV, respectively. Each peak was separately combined, and the pooled fractions were dialyzed against 5 liters of Buffer D containing 80% saturated (NH 4 ) t SO 4 prepared as above. The precipitate appearing was collected by centrifugation and dissolved in a small volume of Buffer B, and the solution was dialyzed overnight, against Buffer B containing 0.3 M NH 4 C1. After removing the insoluble materials by centrifugation at 15,000 x g for 20 min, the clarified supernatant was used as the purified E F - l a . The purified E F - l a could be stored at — 80°C without any appreciable loss of the activity for at least one year, provided that the protein concentration was higher than 1 mg/ml. A summary of typical purification of E F - l a is given in Table I, which indicates that the overall purification of E F - l a was about 340-fold starting
1638
S. NAGATA, K. IWASAKI, and Y. KAZIRO
from the post-mitochondrial supernatant with a yield of 24%. Purity—The purified EF-la was tested for homogeneity by Na-DodSO4 polyacrylamide gel electrophoresis and isoelectric focusing in polyacrylamide gel. Figure 2A shows the results obtained by Na-DodSO4 gel electrophoresis of the four peaks obtained on CM-Sephadex column chromatography. Peaks II, III, and IV migrated
as a single band, but Peak I also showed a faint minor band, although not visible in the figure. The mixture of the four peaks migrated as a single band, which indicates that there was no detectable difference in the electrophoretic mobility of the proteins in these four peaks. The purity of each peak was also analyzed by isoelectric focusing in polyacrylamide gel. As shown in Fig. 2B, Peaks II, III, and IV were again homogeneous B pH7.0
Peak I II in
Fig. 1. Second CM-Sephadex C-50 column chromatography of EF-la. The active fractions obtained by chromatography on a DEAE-Sephadex A-25 column were dialyzed and applied to a column of CM-Sephadex C-50 (1.6x75 cm). Elution was carried out with a linear gradient of NHjCl from 185 to 315mM as described in the text. The total volume of the buffer used was 1,500 ml and 2.5-ml fractions were collected at a flow-rate of 5 ml/h. O, EF-lor activity; X, concentration of NH4C1; and , absorbance at 280 nm.
rv•DMV MI
pHllD Peak
1
n ra
Fig. 2. A. Na-DodSO4 gel electrophoresis of purified EF-la. The four peaks of EF-la obtained by chromatography on a CM-Sephadex column were subjected to Na-DodSO4 gel electrophoresis. From left to right: Peak I, Peak II, Peak III, Peak IV (3 pig of protein to each gel), and a mixture of the four peaks (3 fig, of each peak). B. Isoelectric focusing of EF-la. Isoelectric focusing in %% acrylamide gel containing 2% carrier ampholite was carried out as described in " MATERIALS AND METHODS." From left to right: Peak I, Peak II, Peak III, Peak IV (10 fig of protein to each gel), and a mixture of the four peaks (10 fig of each peak).
TABLE I. Summary of the purification of EF-la from pig liver. Step Post-mitochondrial supernatant1 Aqueous twe-phase separation system and ammonium sulfate fractionation First CM-Sephadex column chromatography DEAE-Sephadex column chromatography Second CM-Sephadex column chromatography Peak I Peak U Peak III Peak IV
Protein (mg)
Activity (units x 10"*)
Specific activity (units/mg)
288,000
16.5
0.057
15,000
6.94
504
6.29 4.37
12.5 13.2
0.53 1.52 1.44 0.43
16.5 18.3 17.9 18.0
333
32.0 83.0 80.5 24.0
0.364
» Obtained from 2.2 kg of pig liver. / . Biochem.
PURIFICATION AND PROPERTIES OF EF-lo FROM PIG LIVER
1639
but Peak I also contained a minor component. It is noteworthy that the proteins in each peak were focused at slightly different positions, indicating that they had slightly different isoelectric points. This was further confirmed by electrofocusing of a mixture of the four peaks, in which four major bands were detectable. From the positions of these bands, the isoelectric points of proteins in Peaks I, II, III, and IV were calculated to be 9.3, 9.5, 9.7, and 9.9, respectively. The purified E F - l a did not contain lipid or carbohydrate in any measurable amount when 10 mg of the protein was analyzed by thin layer chromatography and mass spectrometry. Molecular Weight — Since the molecular weights of E F - l a in the four peaks were almost identical as shown in Fig. 2A, its precise value was determined using Peak III. Peak III together with several reference proteins was subjected to Na-DodS0 4 polyacrylamide gel electrophoresis, and the relative mobilities of the reference proteins were plotted against the logarithms of their molecular weights as shown in Fig. 3A. From the figure, the molecular weight of EF-lar was calculated to be 53,000. The molecular weight of Peak III was further determined by gel filtration using a Sephadex G-150 column as described in " MATERIALS AND METHODS." Figure 3B shows a plot of the logarithms of the molecular weights of several reference proteins versus their elution volumes. From the figure, the molecular weight of E F - l a was estimated to be 61,000, which is close to that obtained by Na-DodSO 4 polyacrylamide gel electrophoresis. This indicates that EF-lar is a single polypeptide chain having a molecular weight of 53,000 and no subunit structure. Similar observations were made with Peaks I, II, and IV. Amino Acid Composition—Since Peaks II, III, and IV were homogeneous (Fig. 2), the amino acid compositions of these peaks were determined. As shown in Table II, the proteins in each peak had the same amino acid compositions within experimental error when expressed as mole percent. This indicates that the numbers of amitio acid residues in these proteins were very close to each other, because their molecular weights were the same as described above. The total amount of the basic amino acids (histidine, lysine, and arginine) is 14.5% and that of the acidic amino acids (asparVol. 82, No. 6, 1977
02
QA Qfi MOBILITY
30
40 50 60 ELUTION VOLUME (ml)
Fig. 3. A. Molecular weight determination of EF-lar by Na-DodSOj polyacrylamide gel electrophoresis. Na-DodSO4 gel electrophoresis of EF-la and the reference proteins was carried out as described in " MATERIALS AND METHODS." The relative mobilities of the reference proteins are plotted against the logarithms of their molecular weights. The molecular weight of EF-la was determined from its relative mobility with the aid of this plot. The abbreviations and molecular weights (in parentheses) of the reference proteins or their subunits are: EF-2, purified EF-2 from pig liver (96,500); EF-G, purified EF-G from E. coli (83,000); BSA, bovine serum albumin (68,000); CAT, catalase from beef liver (60,000); EF-Tu, purified EF-Tu from E. coli (47,000); LDH, lactate dehydrogenase from rabbit muscle (36,000); and CYT, cytochrome c from horse heart (13,000). B. Analytical gel filtration of EF-la on a Sephadex G-150 column. EF-la and the reference proteins were chromatographed as described in " MATERIALS AND METHODS." The elution volumes of the reference proteins were plotted against the logarithms of their molecular weights. The abbreviations and molecular weights (in parentheses) of the reference proteins are: CAT, catalase from beef liver (230,000); EF-2, purified EF-2 from pig liver (110,000); BSA, bovine serum albumin (68,000); EF-Tu, purified EF-Tu from E. coli (48,000); and CYT, cytochrome c from horse heart (13,000).
tic acid and glutamic acid) is 18.8%. The fact that the p / values of these proteins were around 9.7 suggests that these acidic amino acids may exist in their amide forms (asparagine and glutamine), although no direct amide group analysis was performed. The half-cystine residues in E F - l a determined as carboxymethylcysteine after reductive carboxymethylation was about 1.2 mol %, which indicates that one molecule of EF-1 a contains approximately
S. NAGATA, K. IWASAKJ, and Y. KAZIRO ]
1640
TABLE II. Amino acid analysis of EF-la. Amino acid analyses of EF-la in Peaks II, III, and IV were carried out as described in " MATERIALS AND METHODS," and the results are expressed as mol %. The amount of half-cystine was determined as carboxymethylcysteine after reductive carboxymethylation, and that of tryptophan was estimated by the spectrophotometric method. The content of amides was not determined. Residue Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Histidine Lysine Arginine Tryptophan
Peak II
Peak III
Peak IV
9.79 5.61 4.73 9.22 5.66 9.54 8.46 1.05 9.62 2. 12
9.58 6.18 4.93 8.95 5.45 10.30 8.25 1.24 8.81 2.19 6.81 6.16 2.58 3.18 2.40 8.38 3.58 0.87
9.43 5.81 5.05 9.36 5.84 9.92 8.34 1.15 9.21 1.81 6.41 5.76 2.38 3.12 2.52 8.22 3.58 0.82
6.68 6.15 2.44 3.44 2.48 8.50 3.69 0.81
6 half-cystine residues, assuming its molecular weight to be 53,000. This was further confirmed by titrating the sulfhydryls with radioactive pchloromercuribenzoate (pCMB) according to Robinson and Maxwell (29). When EF-la was titrated with [uC]pCMB in the presence of 6 M guanidine hydrochloride, it was found that EF-la contained 4 mol of sulfhydryl residues per mol, while when titration was carried out in the presence of both 0.2 M Na,SO, and 6 M guanidine hydrochloride, 5 mol of sulfhydryl residues were detected per mol (data not shown). This observation indicated that 1 mol of EF-la contained 4 mol of sulfhydryl residues and 1 mol of disulfide, or 6 mol of half-cystine. Stability—EF-la was very unstable, but the stability could be markedly increased by the ad-
2 5 10 20 50 pCMB CONCENTRATION (
