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[13] Initiation factors for protein synthesis from wheat germ
[13]
SYNTHESIS INITIATION FACTORS FROM WHEAT GERM
181
easily by polyacrylamide gel electrophoresis and can be used directly or inserted into published fractionation procedures if additional purification is necessary.
[13] I n i t i a t i o n F a c t o r s f o r P r o t e i n from Wheat Germ
By
Synthesis
BENJAMIN V . TREADWELL, LJUBICA MAUSER,
and W1LLIAM G. ROBINSON Factors involved in the formation of initiation complexes for protein synthesis have been isolated from a number of eukaryotic tissues.~-l° In virtually every procedure crude ribosomes are prepared and extracted with buffered solutions of 0.5-0.7 M potassium chloride. In wheat germ, on the other hand, all the factors that we have attempted to detect have been present in the high,speed supernatant. These include eIF-2,1~ a presumptive elF-3, and elF-5: dissociation factor activity is also present in the supernatant. From the standpoint of preparative procedures, therefore, wheat germ offers a number of advantages. It is inexpensive, readily available in large quantity, and does not require prior isolation of ribosomes. In addition, the supernatant also contains eIF-MI, which catalyzes the GTP-independent binding of Met-tRNA or AcPhe-tRNA to 40 S ribosomes in the presence of AUG or poly(U), respectively. The physiological function of this factor is not known, but since it behaves similarly to eIF-2 on ion-exchange chromatography, the purification procedure for eIF-2 can be used to prepare both eIF-2 and eIF-M I. Wheat germ eIF-M 1, eIF-2, and dissociation factor are functionally interchangeable with factors from other tissues; eIF-3 and eIF-5 have not yet been tested in this respect. t M. H. Schreier, B. Erni, and T. Staehelin, J. Mol, Biol. 116, 727 (1977). C. L. Woodley, Y. C. Chen, and N. K. Gupta, this series, Vol. 30, p. 141. :3 D. Picciano and W. F. Anderson, this series, Vol. 30, p. 171. 4 G. Kramer, A. B. Henderson, P. Pinphanichakarn, M. H. Wallis, and B. Hardesty, Proc. Natl. Acad. Sci. U.S.A. 74, 1445 (1977). 5 R. S. Ranu and I. G. Wool, J. Biol. Chem. 251, 1926 (1976). G. E. Blair, H. H. M. Dahl, E. Truelsen, and J. C. Lelong, Nature (London) 265, 651 (1977). 7 j. F. Hejtmancik and J. P. Comstock, Biochemistry 15, 3804 (1976). L. L. Spremulli, B. J. Walthall, S. R. Lax, and J. M. Ravel, Arch. Biochem. Biophys. 178, 565 (1977). M. Giesen, R. Roman, S. N. Seal, and A. Marcus, J. Biol. Chem. 251, 6057 (1976). ~o H. A. Thompson, I. Sadnik, J. Scheinbuks, and K. Moldave,Biochemistry 16,2221 (1977). N The nomenclature for the eukaryotic initiation factors conforms to the system proposed at an International Symposium on Protein Synthesis in October 1976 at the National Institutes of Health in Bethesda, Maryland.
M E T H O D S 1N E N Z Y M O L O G Y , VOL. LX
Copyright ~) 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181960-4
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INITIATION OF PROTEIN SYNTHESIS
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The function of elF-2 is to form a complex with GTP and Met-tRNA~ that is subsequently bound to 40 S ribosomes. An additional factor that is required for the transfer of Met-tRNAj to the 40 S subunit has been reported 4,12to occur in reticulocytes, but its presence in wheat germ has not been demonstrated. Met-tRNAi is transferred to 40 S ribosomes in the presence of GTP, highly purified wheat germ elF-2, and magnesium ions. Measurement of the amount of either the elF-2 •GTP. Met-tRNAi complex or the 40 S complex in reaction mixtures can serve as the basis of an elF-2 assay. The ribosomal complex assay, however, can be used to advantage only with elF-2 preparations that are free of elF-M1 since, under the conditions of the assay, elF-MI catalyzes the destruction of the 40 S complex formed with elF-2. A third but more cumbersome assay is the restoration by elF-2 of protein synthesis in a wheat germ system inhibited by antibody to elF-2. Wheat germ supernatant also contains a protein that seems to be analogous to the cofactor for ternary complex formation in reticuiocytes reported by Gupta. 13At the protein concentration ordinarily used in the elF-2 assay, addition of the cofactor (Co-elF-2) has no effect on activity. When amounts of elF-2 are used that give barely detectable amounts of ternary complex, addition of Co-elF-2 stimulates complex formation about 3-fold. The Co-elF-2 preparation alone has no elF-2 activity. Eukaryotic ribosomal subunits are often separated by centrifugation in sucrose gradients containing high concentrations of both potassium chloride (up to I M) and magnesium acetate (up to 12 mM). Wheat germ subunits prepared by these procedures have very little activity in binding assays and are inactive in translating globin mRNA in a reconstituted protein synthesizing system. The small subunit is damaged more severely by the high salt than the 60 S subunit. Highly active wheat germ subunits can be prepared, however, with sucrose gradients at low concentrations of potassium chloride and magnesium acetate as described by Weeks. 14Subunits prepared in this way recombine spontaneously, translate natural mRNAs, and are fully active in binding assays. Assays
eIF-M/ Assay The activity of eIF-M1 preparations is determined by measuring the amount of [14C]aminoacyl-tRNA retained on Millipore filters under appro12 A. Majumdar, R. Roy, A. Das, A. Dasgupta, and N. K. Gupta, Biochem. Biophys. Res. Commun. 78, 161 (1977). 13 S. H. Reynolds, A. Dasgupta, S. Palmieri, A. Majumdar, and N. K. Gupta, Arch. Biochem. Biophys. 184, 328 (1977). 14 D. P. Weeks, D. P. S. Verma, S. N. Seal, and A. Marcus, Nature (London) 236, 167 (1972).
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SYNTHESIS INITIATION FACTORS FROM WHEAT GERM
183
priate conditions. 15 Two aminoacyl-tRNAs, fMet-tRNA and AcPhetRNA (both prepared with E. coli tRNA; but eukaryotic aminoacyltRNAs are also active) can be used; since the assay conditions are different for each of them, they are described separately. The complex consisting of fMet-tRNA, AUG, and 40 S subunit forms readily at 0°, but decomposes rapidly as the temperature is raised. In order to obtain consistent results, therefore, all reagents and transfer pipettes are kept cold while the complex is being collected and washed on the filter. Reaction mixtures contain 90 mM Tris .HC1, pH 7.5, 150 mM KCI, 4 mM Mg(Ac).,, 1.5 mM dithiothreitol, 0.3 A260 unit of 40 S ribosomal subunits from wheat germ, 0.05 A 2~0unit of AUG, 15 pmol of f[14C]Met-tRN A (400 cpm/pmol), and eIF-Ml. The final volume is 60/zl. [14C]Met-tRNA can be substituted for f[14C]Met-tRNA. After the samples have been maintained at 0° for 20 min, the reaction is terminated by adding 2 ml of ice-cold buffer containing 60 mM KC1, 50 mM Tris-HCl, pH 7.8, and 10 mM Mg(Ac)2. The dilute mixture is immediately filtered through a Millipore filter, which is subsequently washed with three 2-ml portions of diluting buffer. Filters are placed in scintillation vials, dried under a heat lamp, suspended in a solution of Omnifluor in toluene, and counted in a Beckman scintillation counter. When AcPhe-tRNA is used as substrate, the resulting complex is more stable, and for this reason is more convenient as a routine assay. The optimum magnesium ion concentration for this assay is 7 raM. Sixtymicroliter reaction mixtures contain 90 mM Tris, pH 7.5, 120 mM KC1, 7 mM Mg(Ac).,, 2 mM dithiothreitol, 0.3 A.,60unit of 40 S subunits from wheat germ, 1.0 A260 u n i t of poly(U), 15 pmol of Ac[14C]Phe-tRNA (800cpm/pmol), and eIF-M 1. Incubation is for 20 rain at 25°, after which the samples are diluted, filtered, washed, dried, and counted as described for the fMet-tRNA binding assay. A unit of eIF-M 1 is defined as the amount that catalyzes the binding of 1 pmol substrate in 20 rain in the standard assay. Specific activity is defined as units per milligram of protein. elF-2 Assay The assay for eIF-2 is based upon the retention of a ternary complex containing Met-tRNA~, eIF-2, and GTP on Millipore filters. A reaction mixture containing 20 mM HEPES, pH 7.56, 0.2 mM GTP, 100 mM KCI, 2 mM 2-mercaptoethanol, 3 pmol of wheat germ [35S]Met-tRNA, and eIF-2 in a volume of 50/xl is incubated at 25° for l0 rain. GTP is omitted from controls. The reaction is terminated by adding 2 ml of ice-cold buffer containing 20 mM HEPES, pH 7.56, 100 mM KCI, and 2 mM ~ M. Zasloffand S. Ochoa, this series. Vol. 30, p. 197.
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INITIATION OF PROTEIN SYNTHESIS
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2-mercaptoethanol. The diluted reaction mixture is filtered on a Millipore filter, which is washed twice (2 ml each time) with the same buffer used to terminate the reaction. The Millipore filters are then dried and counted in a liquid scintillation counter as described in the procedure for the eIF-MI assay. In all eIF-2 assays, the methionyl derivative of wheat germ tRNA was used. A unit of eIF-2 is the amount that binds 1 pmol of Met-tRNA~ in 10 rain. Specific activity is units per milligram of protein. Co-eIF-2 Assay When the amount of Met-tRNAi bound in the standard eIF-2 assay is plotted against increasing concentrations of eIF-2, a lag in Met-tRNAi binding at low eIF-2 concentration is observed. '" After the lag, Met-tRNA~ binding is linear with respect to increasing eIF-2 concentrations. For the Co-eIF-2 assay, an eIF-2 concentration at the lowest part of the linear portion of the binding curve must be chosen; Met-tRNAi binding is not stimulated by Co-eIF-2 when higher eIF-2 concentrations are used. Except for the addition of Co-eIF-2, the components of this assay are identical to those of the standard eIF-2 assay. Three tubes are used for each assay. The first is a complete standard eIF-2 assay mixture containing eIF-2, but no Co-eIF-2. The second tube contains Co-elF-2, but no eIF-2. The third contains both eIF-2 and Co-eIF-2. Incubation, termination of the reaction, filtration on Millipore filters, and counting are the same as for the standard eIF-2 assay. The sum of the amount of Met-tRNAi bound in the first and second tubes subtracted from the amount bound in the third is the stimulation of binding due to Co-eIF-2. The stimulation is usually 2- to 3-fold. A unit of Co-eIF-2 is the amount which stimulates the binding of 1 pmol of Met-tRNA~ in the standard assay. Specific activity is defined as units per milligram of protein. elF-2-Dependent Binding o f Met-tRNA~ to 40 S Ribosomes The addition of magnesium salts to the standard eIF-2 assay causes a marked decrease in the amount of ternary complex captured on Millipore filters. At 5 m M Mg(Ac)2 very little complex is retained, but when 40 S subunits and A U G are also present in the incubation mixture, the amount of Met-tRNAl retained by filters is equal to the amount bound by eIF-2 alone in the absence of magnesium. In this sense there is a stoichiometric binding o f ternary complex to ribosomes. The composition of the 50-p~l reaction mixture is 20 mM H E P E S , pH 7.56, 100 mM KCI, 2 mM ~ B. V. Treadwell and W. G. Robinson, Biochem. Biophys. Res. Commun. 65, 176 (1975).
[13]
SYNTHESIS INITIATION FACTORS FROM W H E A T GERM
185
2-mercaptoethanol, 5 mM Mg(Ac)2, 5 I~M GTP, 0.05 A2~0unit of AUG, 0.3 A2~0 unit of wheat germ 40 S subunits, 3 pmol of wheat germ ['%S]MettRNAi and eIF-2. The eIF-2 must be free of eIF-M1. The ribosomal subunits are omitted from the blank. Samples are incubated for 10 min at 25° and the reaction is terminated by adding 2 ml of cold buffer containing 20 mM HEPES, pH 7.56, 100 mM KC1, 2 mM 2-mercaptoethanol, and 5 mM Mg(Ac)2. Filtration, washing, and counting are the same as described for the standard eIF-2 assay. Preparation of Wheat Germ Ribosomal Subunits Materials Raw wheat germ: fully active ribosomal subunits can be prepared from samples of raw wheat germ that do not yield an active $30 protein-synthesizing system. The failure to synthesize protein is usually due to a defect in the supernatant. Brands of wheat germ used to prepare active translation systems are high in lipid, which complicates somewhat the preparation of ribosomes. Wheat germ suitable for ribosome preparation can be purchased from Dixie Portland Co., Arkansas City, Kansas. Buffer A: 90 mM KCI, 4 mM Mg(Ac)2, 2 mM CaCI.,, 6 mM KHCO:~ Buffer B: 40 mM KCI, 0.33 mM Mg(Ac)2, I mM 2-mercaptoethanol, 10 mM Tris-HCl, pH 7.8 Buffer C: 40 mM KCI, 80 ~ Mg(Ac)2, 3 mM 2-mercaptoethanol, 2 mM Tris .HCI, pH 7.8 Buffer D: 40 mM KC1, 8 mM Mg(Ac)~, 3 mM 2-mercaptoethanol, 2 mM Tris.HC1, pH 7.8 Buffer E: 80 mM KCI, 8 mM Mg(Ac)2, 8 mM 2-mercaptoethanol, 4 mM Tris .HCI, pH 7.8 Mg(Ac)2, 1 M Tris.HCI, I M, pH 7.8 Washed sea sand Procedure. Fifteen grams of wheat germ are ground for 10 rain in a mortar with an equal volume of sand. The grinding and all subsequent steps are done at 0o-4 °. The dry mixture, after grinding, is suspended in 150 ml of buffer A, and about 20 ml of the same buffer are used for rinsing the mortar when its contents are transferred to a beaker. Sand and other insoluble material are removed by centrifuging the combined suspension and rinses at 15,000 rpm in a Sorvall SS-34 rotor for 12 rain. After this and all subsequent centrifugations, a yellow lipid layer is scooped from the top of the solutions, which are then poured though cheese cloth. The volume of the supernatant is measured (about 140 ml) and 0.1 volume of 0.1 M Mg(Ac)._, (about 14 ml) and 0.1 volume of 1 M Tris .HCI, pH 7.8 (about 14
186
INITIATION OF PROTEIN SYNTHESIS
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ml) are added. A precipitate that forms is removed by centrifuging the extract at 15,000 rpm in a Sorvall SS-34 rotor for 15 rain. The upper seven-eighths of the supernatant is retained and transferred to tubes in which it is centrifuged in a Beckman 60 Ti rotor at 38,000 rpm for 2 hr. The supernatant is discarded, and the centrifuge tubes containing the ribosomal pellets are allowed to stand in ice for an hour. During this time a small amount of debris is released from the surface of the clear pellets of 80 S ribosomes. A minimal volume of buffer B is used to gently rinse the pellets before they are suspended in a total volume of 3 ml of buffer B. The concentration of 80 S ribosomes should be 600-650 A.,60 units/ml. To prepare 40 S and 60 S ribosomal subunits, 0.5 ml of the 80 S ribosome preparation is immediately layered onto each of three 50-ml 15-30% (w/v) sucrose gradients in buffer C and centrifuged for 14 hr at 21,000 rpm in a Beckman SW 25.2 rotor. Fractions (0.7-0.8 ml) from each gradient are collected, and their absorbancy at 260 nm is determined and plotted against tube number. By extrapolating the leading arm of the 40 S peak and the trailing arm of the 60 S peak, 40 S and 60 S fractions are chosen that give minimal cross-contamination. Fractions containing 40 S subunits from the three gradients are combined, and the magnesium ion concentration is raised to 8 mM by adding the calculated amount of 1 M Mg(Ac)~. The preparation is diluted to 25 ml with buffer D. An identical procedure is followed with the fractions containing 60 S subunits. The two 25-ml samples are centrifuged overnight at 35,000 rpm in a Beckman 60 Ti rotor in order to concentrate the subunits. The clear, colorless pellets are gently rinsed with buffer E and carefully suspended in a volume of about 0.2 ml of the same buffer. The concentrated suspension is centrifuged at 4000 rpm for 20 rain in the Sorvall SS-34 rotor, and the precipitate is discarded. The supernatant is diluted with buffer E so that the 40 S subunits have a concentration of 200 A26o units/ml, and the 60 S 400 A26o units/ml. Each sample is diluted with 1 volume of glycerol and stored at - 2 0 °. The subunits remain fully active for several months, but upon very prolonged storage activity in Met-tRNA~ binding experiments begins to decline. As determined in a poly(U) translational assay, the 40 S subunits are free of 60 S contamination, but the 60 S subunits have about 10% contamination with the smaller subunit. Purification of eIF-2 and EIF-M1 Materials Wheat germ. The wheat germ used for isolation of ribosomal subunits is satisfactory also for this preparation. Acetone DEAE-Cellulose (Whatman DE-23)
[13]
187
SYNTHESIS I N I T I A T I O N FACTORS FROM W H E A T GERM
DEAE-Sephadex A-50 (Pharmacia) CM-Sephadex C-50 (Pharmacia) Hydroxyapatite (Clarkson Hypatite C) Potassium phosphate buffer, 1 M, pH 7.0 Buffer A: 10 mM Tris .HCI, pH 7.5, 10 mM 2-mercaptoethanol, 0.1 mM EDTA, and varying concentrations of KC1 indicated by a number in parentheses, thus A (100) is buffer A containing 100 mM KC1. Buffer B: 20 mM H E P E S - K O H , pH 7.56, 20 mM 2-mercaptoethanol, 0.1 mM EDTA, and varying concentrations of KC1 indicated by a number in parentheses, thus B (100) is buffer B containing 100 mM KC1. Buffer C: 50 mM Tris .HCI, pH 7.5, 10 mM 2-mercaptoethanol, 0.1 mM EDTA, 100 mM KCI, 50% glycerol (v/v) Procedure. A summary of the purification procedure is given in Table I. All operations are carried out at 4° unless otherwise stated. In chromatographic procedures the presence of UV-absorbing material in eluates is continuously monitored with an LKB Uvicord apparatus. Dialysis of samples against buffer C (contains 50% glycerol) before storage in steps 3-5 results in a three-fourths reduction in volume. This can be a useful technique for concentrating protein solutions. Step I. Preparation of Acetone Powder. One-hundred-gram portions of commercial wheat germ are homogenized in a Waring blender for 45 sec with 500 ml of acetone that has been chilled to - 1 5 °. The homogenate is quickly filtered by suction on a large Biichner funnel, and the filtrate is washed in rapid succession with 100 ml of acetone and 200 ml of ether at 15°. The light gray powder is spread in a thin layer on paper and allowed -
TABLE I PURIFICATION OF EIF-2 AND EIF-M1
Purification step DEAE-cellulose, a m m o n i u m sulfate CM-Sephadex C-50 DEAE-Sephadex A-50 Hydroxyapatite Fractions 4 to 10 Fractions 22 to 30
Volume (ml)
250 15 2 2 2
Protein (rag)
20,000 84 18 4.1 2.0
elF-MI" (pmol/mg/20 min)
elF-2 h (pmol/mg/10 rain
-9,000 19,200
-70 148
-37,000
300 --
The standard AcPhe-tRNA binding assay was used to determine elF-MI activity. b The ternary complex formation with GTP and ['~'~S]Met-tRNAi was used to assay elF-2.
188
I N I T I A T I O N OF P R O T E I N S Y N T H E S I S
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to dry at room temperature until the odors of acetone and ether can no longer be detected. Each 100 g of wheat germ yields approximately 80 g of acetone powder. No loss of activity is observed after prolonged storage of the acetone powder at - 15°. Step 2. Extraction, DEAE-Cellulose Treatment, and Ammonium Sulfate Fractionation. Three hundred and twenty grams of wheat germ acetone powder are suspended in 1600 mi of buffer A (100), and the mixture is stirred for 2.5 hr. The suspension is centrifuged at 15,000g for 20 min, and the supernatant is poured into a beaker containing 700 ml of settled DEAE-cellulose (DE-23), which has been equilibrated with buffer A (100). The DEAE-cellulose suspension is stirred and filtered with suction through Whatman No. 1 filter paper on a Bfichner funnel. The packed DEAEcellulose is suspended in about 400 ml of buffer A (100) and again filtered. The filter cake can be used as starting material for the preparation of Co-elF-2. The combined filtrates are brought to 40% saturation with ammonium sulfate and centrifuged at 15,000 g for 20 min. The resulting precipitate is discarded, and the supernatant is adjusted to 65% saturation with solid ammonium sulfate. The protein pellet obtained after centrifugation at 15,000 g for 20 min is dissolved in 135 ml of buffer A (100), and the protein solution is dialyzed against 4 liters of the same buffer overnight. Step 3. Chromatography on CM-Sephadex C-50. The success of chromatography on CM-Sephadex C-50 is somewhat dependent upon the procedure by which the column is prepared. CM-Sephadex C-50 (33 g) is allowed to swell in an excess of 0.5 M KCI for at least 3 days at 4°. The supernatant is decanted and the remaining volume of settled ion exchanger (about 800 ml) is diluted 5-fold with cold distilled water. The suspension is briefly stirred and allowed to settle; the supernatant is decanted. The settled Sephadex is resuspended in buffer A (100) and allowed to settle; the supernatant is decanted, and the thick Sephadex suspension is used to fill a 5 X 60 cm column. The column is immediately attached to a reservoir containing buffer A (100) at a pressure head of about 1 meter. After 350-400 ml of buffer has flowed through the column it is sufficiently packed for application of the sample. The dialyzed preparation from the preceding step (25 g of protein in 300 ml) is applied to the column, and washing with buffer A (200) is begun immediately. Washing is continued until the absorbance of the effluent reaches a low steady level; this is usually after about 1800 ml of buffer have passed through the column. The factors elF-2 and elF-M 1 are eluted with buffer A (300). Both these factors are in a single UV-absorbing peak after about 500 ml of buffer has passed through the column. The active fractions are pooled and concentrated to a volume of 15-20 ml with an Amicon ultrafiltration apparatus fitted with a PM-10 membrane. The concentrated
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S Y N T H E S I S I N I T I A T I O N FACTORS FRO M W H E A T GERM
189
solution is dialyzed against buffer C before storage at - 15°, and before it is used in the next step it is dialyzed for 2-3 hr against buffer B (50). Step 4. Chromatography on DEAE-Sephadex A-50. The protein solution from the preceding step is applied to a DEAE-Sephadex A-50 column (2.5 × 40 cm), which has been prepared similarly to the preparation of cation exchanger in the preceding section, except that it is equilibrated with buffer B (50). The column is washed with buffer B (100) after the sample has been applied until no protein can be detected in the eluate. The protein that elutes with buffer B (200) is collected and concentrated by ultrafiltration, as in the preceding step, to 4 ml. The preparation is dialyzed against buffer C for storage at - 15° and against buffer A (100) for 2-3 hr before use in the next step. Step 5. Hydroxyapatite Chromatography. The sample from the preceding step is applied to a hydroxyapatite column (1.5 X 11 cm) equilibrated with buffer A (100). The column is then washed with 100 mM potassium phosphate, pH 7.0, containing 10 mM 2-mercaptoethanol until no UVabsorbing material can be detected eluting from the column. The column is then developed with a linear gradient (100 × 100 ml) from 100 mM potassium phosphate, pH 7.0, to 500 mM potassium phosphate, pH 7.0 (both buffer solutions contain 10 mM 2-mercaptoethanol), and 4-ml fractions are collected. The peak of eIF-2 activity elutes from the column at 280 mM potassium phosphate and the peak of elF-M 1 activity at 400 mM potassium phosphate. Fractions containing elF-MI activity but no elF-2 activity are pooled, and fractions with elF-2 activity but no elF-MI are also pooled. Both these fractions are concentrated to 2 ml by Amicon ultrafiltration with a PM-10 membrane and dialyzed against buffer C before use or storage.
Comments elF-2. The eIF-2 fractions chosen from the hydroxyapatite chromatography are free of elF-M1, but they represent a little less than half of the total eIF-2 in the eluate. The remaining eIF-2 is present in intermediate fractions contaminated with eI F-M 1. Rechromatography of these fractions has not been of practical value, but the fractions can still be used in experiments in which the presence of eIF-MI is not an important factor. Upon storage at --15 ° eIF-2 gradually loses activity, but when frozen in small aliquots in buffer C at - 8 0 °, full activity is retained for at least 6 months. The elution profiles of eIF-2 and eIF-M1 on Sephadex G-200 are identical; they both have estimated molecular weights by this technique of 145,000. Upon electrophoresis in sodium dodecyl sulfate on polyacrylamide gels, three major bands are obtained from eIF-2 with molecular
190
INITIATION OF PROTEIN SYNTHESIS
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weights of 37,000, 41,000, and 57,000. Wheat germ elF-2, therefore, is assumed to be like elF-2 from reticulocytes in consisting of three different polypeptide chains. Ternary complex formation with wheat germ elF-2 is inhibited by N-ethylmaleimide and p-chloromercuribenzoate. It is also inhibited by EDTA. The probable reason for this is that complex formation requires a low concentration of magnesium, ~7which is normally supplied as an impurity in other components of the standard reaction mixture. There is no difference in activity at 25 ° and 37 ° in the usual 10-min assay. The concentration of GTP (0.2 mM) routinely used in the ternary complex assay is a large excess over the amount actually needed. With the amount ofelF-2 and Met-tRNA~ employed in the standard assay, maximal complex formation occurs at a GTP concentration of about 0.5/xM. GTP concentration becomes an important consideration in regard to the binding of Met-tRNA~ to 40 S ribosomes because some inhibition of binding appears to occur at the high concentration used for ternary complex formation. For this reason a lower GTP concentration (5/zM) is used for formation of the 40 S complex. Under the conditions of the standard assays, Met-tRNAi bound in both the ternary and 40 S complexes does not exchange with free Met-tRNAt. Experimental conditions for Met-tRNAi.AUG.40 S complex formation with elF-2 are similar to those for binding Met-tRNA~ to the small subunits with elF-M 1. A major difference in the two systems, however, is the temperature at which the assay is performed. The complex produced in the presence of elF-M1 rapidly decomposes at temperatures above 0°. The decomposition is catalyzed by elF-M 1 itself. On the other hand, the elF-2 complex is stable at 25°, but is formed very slowly at 0°. Addition ofelF-M 1 at 25° to a Met-tRNAi .AUG .40 S complex formed with elF-2 catalyzes the destruction of the complex. For this reason Met-tRNAi. AUG .40 S complex formation with elF-2 can be detected only if the elF-2 preparation is free of elF-M1. In the systems described here elF-2 acts stoichiometrically; it does not turn over. In the absence of AUG, a Met-tRNAi.40 S complex is formed with elF-2. However, up to 5 times more complex is formed when tile trinucleotide is present than when it is absent. The reason for this observation is that AUG stabilizes the ribosomal complex. Both types of complex, with and without AUG, are sufficiently stable to survive analytical sucrose density gradients. The complex formed in the absence of AUG is not destroyed by elF-M1. elF-M1. The physical and biological properties of what germ elF-MI ~rL. M. Cashion, G. L. Dettman, and W. M. Stanley,Jr., this series, Vol. 30, p. 153.
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SYNTHESISINITIATION FACTORS FROM WHEAT GERM
191
are similar to those of elF-MI isolated from other tissues. 15,18,19Full activity of the purified factor is retained upon storage in 50% glycerol (buffer C) at - 1 5 ° for several weeks, but after this time activity begins to decline because of the oxidation of sulfhydryl groups. In the early stages of inactivation, dialysis of the sample against buffer C containing fresh 2-mercaptoethanol will completely restore activity. Samples frozen at --80 ° retain activity for longer times. As estimated by gel filtration on Sephadex G-200, the molecular weight of elF-M1 is 145,000. The value obtained by electrophoresis in sodium dodecyl sulfate on polyacrylamide gels is 41,000, which could be an indication that the native protein is a tetramer. The factor acts catalytically, and, as determined for a molecular weight of 145,000, it has a turnover number estimated roughly to be about 10. Although the isolation procedure described here uses a wheat germ acetone powder extract as starting material, the same procedure can be used to purify eIF-M 1 from a 100,000 g wheat germ supernatant. In addition, wheat germ ribosomes also contain eIF-M 1 that can be extracted with 700 mM KCI and also.purified by this same procedure. The supernatant and ribosomal eIF-Mls are indistinguishable. Highly purified elF-MI binds to 40 S wheat germ ribosomes. One method of demonstrating this is to mix 40 S ribosomes and eIF-M 1, and separate the ribosome-eIF-M 1 complex from free eIF-MI. The 40 S ribosome.eIF-M1 complex is active in binding aminoacyl-tRNA. Binding of eIF-M 1 to ribosomes is the most likely explanation for the fact that 40 S ribosomes, prepared by the low-salt procedure described here, frequently give high blanks in the eIF-M1 assay. As the ribosome preparations age, the blank values decrease. P rep ar atio n of Co-eIF-2 Materials
Wheat germ, ion-exchange chromatography materials, and buffers A and C are the same as described for the preparation of elF-2 and elF-MI. Procedure. The starting material for the preparation of Co-elF-2 is the filtered DEAE-cellulose from step 2 in the purification of elF-2 and elF-M 1. All operations are carried out at 4°. Table II is a summary of the purification procedure. Step 1. Elution o f Co-elF-2 and A m m o n i u m Sulfate Precipitation. The 1~E. Gasior and K. Moldave,J. Mol. Biol. 66, 391 (1972). ~9W. C. Merrick and W. F. Anderson,J. Biol. Chem. 250, 1197(1975).
192
INITIATION OF PROTEIN SYNTHESIS
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TABLE II PURIFICATION OF C o - E I F - 2
Purification step DEAE-cellulose, ammonium sulfate DEAE-cellulose CM-Sephadex C-50
Volume (ml)
Protein (mg)
Co-eIF-2 (pmol/mg/10 rain)
57 2.5 0.7
1900 69 6. I
-9 60
moist DEAE-cellulose filter cake from step 2 of the e l F - 2 - e l F - M 1 preparative procedure is suspended in 55 ml of buffer A (400). The suspension is occasionally stirred over a period of 30 min and filtered with gentle suction through Whatman No. 1 filter paper on a Bfichner funnel. Solid ammonium sulfate is added to the clear yellow filtrate to 80% saturation, and the precipitated protein is removed by centrifugation at 15,000 rpm in the SS-34 Sorvall rotor. The solid pellet, which contains the Co-elF-2, can be stored at - 15° for several months without loss of activity. Before assay or use in the next step, it is dissolved in approximately 25 ml of buffer A (50) and dialyzed overnight against the same buffer. A precipitate which forms at this low salt concentration is removed by centrifugation and discarded. Step 2. Chromatography on DEAE-Cellulose. The protein solution from the preceding step is applied to a DEAE-cellulose column (2.5 X 35 cm) equilibrated in buffer A (50), and the column is washed with the same buffer. The major protein peak that washes from the column is retained and concentrated to approximately 20 ml by ultrafiltration using an Amicon cell with a PM- 10 membrane. If the sample is to be stored at - 15°, it should first be dialyzed against buffer C. Before use in the next step it is dialyzed against buffer A (100). Step 3. Chromatography on CM-Sephadex C-50. The Co-elF-2 preparation is applied to a CM-Sephadex C-50 column (2.5 X 30 cm) equilibrated with buffer A (100). The column is washed successively with buffer A (100) and A (200). Each time the washing is continued until the amount of protein in the column effluent is close to zero. The protein fraction that elutes from the column with buffer A (100) contains some Co-elF-2, but the bulk of the activity is in the A (200) eluate. This sample is concentrated to 4 ml by ultrafiltration as in step 2 and dialyzed against buffer C for storage at - 15°.
Comments An unusual aspect of the purification procedure is that Co-eIF-2 in the crude extract binds to DEAE-cellulose, but in a later step it is not retained
[14]
WHEAT GERM INITIATION FACTORS
193
on the same anion exchanger even at a lower salt concentration. A possible reason for this is that Co-eIF-2 may be complexed with another protein in the crude extract, and it is the complex that binds to DEAE-cellulose. A subsequent manipulation, such as elution from the DEAE-cellulose with 400 mM potassium chloride, precipitation with ammonium sulfate, or dialysis against 50 mM KC1, could disrupt the complex, thereby altering the behavior of the cofactor. A distinct advantage of this preparative procedure is that Co-eIF-2 is readily separated from eIF-2. The buffer A (400) eluate is virtually free of eIF-2. Occasionally some preparations have a high GTPindependent binding of Met-tRNAi which is probably due to nonspecific binding. A 2- to 3-fold stimulation of Met-tRNAi binding to elF-2 by Co-eIF-2 is routinely obtained although a maximum of about 5-fold has been observed in a few cases. Addition of Co-elF-2 to the standard reaction mixture for binding Met-tRNA~ to 40 S ribosomes does not cause an increase in the amount of 40 S complex formed. The mechanism of action of Co-eIF-2 is not known, but it would be attractive to assume that it forms a complex with eIF-2 and that this complex is the species which combines with GTP and Met-tRNAi. If this hypothesis is correct it should be possible to show a complete dependence upon Co-eIF-2 by very highly purified eIF-2. In the reticulocyte system .'° it has been shown that purified, but not crude, eIF-2 is stimulated by the cofactor. ~0 A. Dasgupta, A. Majumdar, A. D. George, and N. K. Gupta, Biochem. Biophys. Res. Cornmun. 71, 1234 (1976).
[14] I s o l a t i o n a n d P u r i f i c a t i o n o f P r o t e i n S y n t h e s i s Initiation Factors from Wheat Germ 1
By
B E N J. W A L T H A L L ,
LINDA L. SPREMULLI,
SANDRA R. LAX, and JOANNE M. RAVEL
Crude extracts of wheat germ are capable of translating mRN As from a variety of sources. '-4 Wheat germ extracts are easy to prepare, are low in nuclease activity, have low endogenous levels of mRNA, and translate exogenously supplied mRNAs with high efficiency and fidelity. Marcus This work was supported in part by U.S. Public Health Service Grant GM-18775. K. Marcu and B. Dudock, Nucl. Acids Res. 1, 1385 (1974). :~ B. E. Roberts and B. M. Patersofi, Proc. Natl. Acad. Sci. U.S.A. 70, 2330 (1973). 4 0 . E. Beck and H. G. G a s s e n , Biochern. Biophys. Res. Commun. 74, 16 (1977), and references cited in this paper.
METHODS 1N ENZYMOLOGY, VOL. LX
Copyright © 1979by AcademicPress. Inc. All rights of reproduction in any form reserved. ISBN 0-12-181960-4
SYNTHESIS INITIATION FACTORS FROM WHEAT GERM
181
easily by polyacrylamide gel electrophoresis and can be used directly or inserted into published fractionation procedures if additional purification is necessary.
[13] I n i t i a t i o n F a c t o r s f o r P r o t e i n from Wheat Germ
By
Synthesis
BENJAMIN V . TREADWELL, LJUBICA MAUSER,
and W1LLIAM G. ROBINSON Factors involved in the formation of initiation complexes for protein synthesis have been isolated from a number of eukaryotic tissues.~-l° In virtually every procedure crude ribosomes are prepared and extracted with buffered solutions of 0.5-0.7 M potassium chloride. In wheat germ, on the other hand, all the factors that we have attempted to detect have been present in the high,speed supernatant. These include eIF-2,1~ a presumptive elF-3, and elF-5: dissociation factor activity is also present in the supernatant. From the standpoint of preparative procedures, therefore, wheat germ offers a number of advantages. It is inexpensive, readily available in large quantity, and does not require prior isolation of ribosomes. In addition, the supernatant also contains eIF-MI, which catalyzes the GTP-independent binding of Met-tRNA or AcPhe-tRNA to 40 S ribosomes in the presence of AUG or poly(U), respectively. The physiological function of this factor is not known, but since it behaves similarly to eIF-2 on ion-exchange chromatography, the purification procedure for eIF-2 can be used to prepare both eIF-2 and eIF-M I. Wheat germ eIF-M 1, eIF-2, and dissociation factor are functionally interchangeable with factors from other tissues; eIF-3 and eIF-5 have not yet been tested in this respect. t M. H. Schreier, B. Erni, and T. Staehelin, J. Mol, Biol. 116, 727 (1977). C. L. Woodley, Y. C. Chen, and N. K. Gupta, this series, Vol. 30, p. 141. :3 D. Picciano and W. F. Anderson, this series, Vol. 30, p. 171. 4 G. Kramer, A. B. Henderson, P. Pinphanichakarn, M. H. Wallis, and B. Hardesty, Proc. Natl. Acad. Sci. U.S.A. 74, 1445 (1977). 5 R. S. Ranu and I. G. Wool, J. Biol. Chem. 251, 1926 (1976). G. E. Blair, H. H. M. Dahl, E. Truelsen, and J. C. Lelong, Nature (London) 265, 651 (1977). 7 j. F. Hejtmancik and J. P. Comstock, Biochemistry 15, 3804 (1976). L. L. Spremulli, B. J. Walthall, S. R. Lax, and J. M. Ravel, Arch. Biochem. Biophys. 178, 565 (1977). M. Giesen, R. Roman, S. N. Seal, and A. Marcus, J. Biol. Chem. 251, 6057 (1976). ~o H. A. Thompson, I. Sadnik, J. Scheinbuks, and K. Moldave,Biochemistry 16,2221 (1977). N The nomenclature for the eukaryotic initiation factors conforms to the system proposed at an International Symposium on Protein Synthesis in October 1976 at the National Institutes of Health in Bethesda, Maryland.
M E T H O D S 1N E N Z Y M O L O G Y , VOL. LX
Copyright ~) 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181960-4
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[13]
The function of elF-2 is to form a complex with GTP and Met-tRNA~ that is subsequently bound to 40 S ribosomes. An additional factor that is required for the transfer of Met-tRNAj to the 40 S subunit has been reported 4,12to occur in reticulocytes, but its presence in wheat germ has not been demonstrated. Met-tRNAi is transferred to 40 S ribosomes in the presence of GTP, highly purified wheat germ elF-2, and magnesium ions. Measurement of the amount of either the elF-2 •GTP. Met-tRNAi complex or the 40 S complex in reaction mixtures can serve as the basis of an elF-2 assay. The ribosomal complex assay, however, can be used to advantage only with elF-2 preparations that are free of elF-M1 since, under the conditions of the assay, elF-MI catalyzes the destruction of the 40 S complex formed with elF-2. A third but more cumbersome assay is the restoration by elF-2 of protein synthesis in a wheat germ system inhibited by antibody to elF-2. Wheat germ supernatant also contains a protein that seems to be analogous to the cofactor for ternary complex formation in reticuiocytes reported by Gupta. 13At the protein concentration ordinarily used in the elF-2 assay, addition of the cofactor (Co-elF-2) has no effect on activity. When amounts of elF-2 are used that give barely detectable amounts of ternary complex, addition of Co-elF-2 stimulates complex formation about 3-fold. The Co-elF-2 preparation alone has no elF-2 activity. Eukaryotic ribosomal subunits are often separated by centrifugation in sucrose gradients containing high concentrations of both potassium chloride (up to I M) and magnesium acetate (up to 12 mM). Wheat germ subunits prepared by these procedures have very little activity in binding assays and are inactive in translating globin mRNA in a reconstituted protein synthesizing system. The small subunit is damaged more severely by the high salt than the 60 S subunit. Highly active wheat germ subunits can be prepared, however, with sucrose gradients at low concentrations of potassium chloride and magnesium acetate as described by Weeks. 14Subunits prepared in this way recombine spontaneously, translate natural mRNAs, and are fully active in binding assays. Assays
eIF-M/ Assay The activity of eIF-M1 preparations is determined by measuring the amount of [14C]aminoacyl-tRNA retained on Millipore filters under appro12 A. Majumdar, R. Roy, A. Das, A. Dasgupta, and N. K. Gupta, Biochem. Biophys. Res. Commun. 78, 161 (1977). 13 S. H. Reynolds, A. Dasgupta, S. Palmieri, A. Majumdar, and N. K. Gupta, Arch. Biochem. Biophys. 184, 328 (1977). 14 D. P. Weeks, D. P. S. Verma, S. N. Seal, and A. Marcus, Nature (London) 236, 167 (1972).
[13]
SYNTHESIS INITIATION FACTORS FROM WHEAT GERM
183
priate conditions. 15 Two aminoacyl-tRNAs, fMet-tRNA and AcPhetRNA (both prepared with E. coli tRNA; but eukaryotic aminoacyltRNAs are also active) can be used; since the assay conditions are different for each of them, they are described separately. The complex consisting of fMet-tRNA, AUG, and 40 S subunit forms readily at 0°, but decomposes rapidly as the temperature is raised. In order to obtain consistent results, therefore, all reagents and transfer pipettes are kept cold while the complex is being collected and washed on the filter. Reaction mixtures contain 90 mM Tris .HC1, pH 7.5, 150 mM KCI, 4 mM Mg(Ac).,, 1.5 mM dithiothreitol, 0.3 A260 unit of 40 S ribosomal subunits from wheat germ, 0.05 A 2~0unit of AUG, 15 pmol of f[14C]Met-tRN A (400 cpm/pmol), and eIF-Ml. The final volume is 60/zl. [14C]Met-tRNA can be substituted for f[14C]Met-tRNA. After the samples have been maintained at 0° for 20 min, the reaction is terminated by adding 2 ml of ice-cold buffer containing 60 mM KC1, 50 mM Tris-HCl, pH 7.8, and 10 mM Mg(Ac)2. The dilute mixture is immediately filtered through a Millipore filter, which is subsequently washed with three 2-ml portions of diluting buffer. Filters are placed in scintillation vials, dried under a heat lamp, suspended in a solution of Omnifluor in toluene, and counted in a Beckman scintillation counter. When AcPhe-tRNA is used as substrate, the resulting complex is more stable, and for this reason is more convenient as a routine assay. The optimum magnesium ion concentration for this assay is 7 raM. Sixtymicroliter reaction mixtures contain 90 mM Tris, pH 7.5, 120 mM KC1, 7 mM Mg(Ac).,, 2 mM dithiothreitol, 0.3 A.,60unit of 40 S subunits from wheat germ, 1.0 A260 u n i t of poly(U), 15 pmol of Ac[14C]Phe-tRNA (800cpm/pmol), and eIF-M 1. Incubation is for 20 rain at 25°, after which the samples are diluted, filtered, washed, dried, and counted as described for the fMet-tRNA binding assay. A unit of eIF-M 1 is defined as the amount that catalyzes the binding of 1 pmol substrate in 20 rain in the standard assay. Specific activity is defined as units per milligram of protein. elF-2 Assay The assay for eIF-2 is based upon the retention of a ternary complex containing Met-tRNA~, eIF-2, and GTP on Millipore filters. A reaction mixture containing 20 mM HEPES, pH 7.56, 0.2 mM GTP, 100 mM KCI, 2 mM 2-mercaptoethanol, 3 pmol of wheat germ [35S]Met-tRNA, and eIF-2 in a volume of 50/xl is incubated at 25° for l0 rain. GTP is omitted from controls. The reaction is terminated by adding 2 ml of ice-cold buffer containing 20 mM HEPES, pH 7.56, 100 mM KCI, and 2 mM ~ M. Zasloffand S. Ochoa, this series. Vol. 30, p. 197.
184
INITIATION OF PROTEIN SYNTHESIS
[13]
2-mercaptoethanol. The diluted reaction mixture is filtered on a Millipore filter, which is washed twice (2 ml each time) with the same buffer used to terminate the reaction. The Millipore filters are then dried and counted in a liquid scintillation counter as described in the procedure for the eIF-MI assay. In all eIF-2 assays, the methionyl derivative of wheat germ tRNA was used. A unit of eIF-2 is the amount that binds 1 pmol of Met-tRNA~ in 10 rain. Specific activity is units per milligram of protein. Co-eIF-2 Assay When the amount of Met-tRNAi bound in the standard eIF-2 assay is plotted against increasing concentrations of eIF-2, a lag in Met-tRNAi binding at low eIF-2 concentration is observed. '" After the lag, Met-tRNA~ binding is linear with respect to increasing eIF-2 concentrations. For the Co-eIF-2 assay, an eIF-2 concentration at the lowest part of the linear portion of the binding curve must be chosen; Met-tRNAi binding is not stimulated by Co-eIF-2 when higher eIF-2 concentrations are used. Except for the addition of Co-eIF-2, the components of this assay are identical to those of the standard eIF-2 assay. Three tubes are used for each assay. The first is a complete standard eIF-2 assay mixture containing eIF-2, but no Co-eIF-2. The second tube contains Co-elF-2, but no eIF-2. The third contains both eIF-2 and Co-eIF-2. Incubation, termination of the reaction, filtration on Millipore filters, and counting are the same as for the standard eIF-2 assay. The sum of the amount of Met-tRNAi bound in the first and second tubes subtracted from the amount bound in the third is the stimulation of binding due to Co-eIF-2. The stimulation is usually 2- to 3-fold. A unit of Co-eIF-2 is the amount which stimulates the binding of 1 pmol of Met-tRNA~ in the standard assay. Specific activity is defined as units per milligram of protein. elF-2-Dependent Binding o f Met-tRNA~ to 40 S Ribosomes The addition of magnesium salts to the standard eIF-2 assay causes a marked decrease in the amount of ternary complex captured on Millipore filters. At 5 m M Mg(Ac)2 very little complex is retained, but when 40 S subunits and A U G are also present in the incubation mixture, the amount of Met-tRNAl retained by filters is equal to the amount bound by eIF-2 alone in the absence of magnesium. In this sense there is a stoichiometric binding o f ternary complex to ribosomes. The composition of the 50-p~l reaction mixture is 20 mM H E P E S , pH 7.56, 100 mM KCI, 2 mM ~ B. V. Treadwell and W. G. Robinson, Biochem. Biophys. Res. Commun. 65, 176 (1975).
[13]
SYNTHESIS INITIATION FACTORS FROM W H E A T GERM
185
2-mercaptoethanol, 5 mM Mg(Ac)2, 5 I~M GTP, 0.05 A2~0unit of AUG, 0.3 A2~0 unit of wheat germ 40 S subunits, 3 pmol of wheat germ ['%S]MettRNAi and eIF-2. The eIF-2 must be free of eIF-M1. The ribosomal subunits are omitted from the blank. Samples are incubated for 10 min at 25° and the reaction is terminated by adding 2 ml of cold buffer containing 20 mM HEPES, pH 7.56, 100 mM KC1, 2 mM 2-mercaptoethanol, and 5 mM Mg(Ac)2. Filtration, washing, and counting are the same as described for the standard eIF-2 assay. Preparation of Wheat Germ Ribosomal Subunits Materials Raw wheat germ: fully active ribosomal subunits can be prepared from samples of raw wheat germ that do not yield an active $30 protein-synthesizing system. The failure to synthesize protein is usually due to a defect in the supernatant. Brands of wheat germ used to prepare active translation systems are high in lipid, which complicates somewhat the preparation of ribosomes. Wheat germ suitable for ribosome preparation can be purchased from Dixie Portland Co., Arkansas City, Kansas. Buffer A: 90 mM KCI, 4 mM Mg(Ac)2, 2 mM CaCI.,, 6 mM KHCO:~ Buffer B: 40 mM KCI, 0.33 mM Mg(Ac)2, I mM 2-mercaptoethanol, 10 mM Tris-HCl, pH 7.8 Buffer C: 40 mM KCI, 80 ~ Mg(Ac)2, 3 mM 2-mercaptoethanol, 2 mM Tris .HCI, pH 7.8 Buffer D: 40 mM KC1, 8 mM Mg(Ac)~, 3 mM 2-mercaptoethanol, 2 mM Tris.HC1, pH 7.8 Buffer E: 80 mM KCI, 8 mM Mg(Ac)2, 8 mM 2-mercaptoethanol, 4 mM Tris .HCI, pH 7.8 Mg(Ac)2, 1 M Tris.HCI, I M, pH 7.8 Washed sea sand Procedure. Fifteen grams of wheat germ are ground for 10 rain in a mortar with an equal volume of sand. The grinding and all subsequent steps are done at 0o-4 °. The dry mixture, after grinding, is suspended in 150 ml of buffer A, and about 20 ml of the same buffer are used for rinsing the mortar when its contents are transferred to a beaker. Sand and other insoluble material are removed by centrifuging the combined suspension and rinses at 15,000 rpm in a Sorvall SS-34 rotor for 12 rain. After this and all subsequent centrifugations, a yellow lipid layer is scooped from the top of the solutions, which are then poured though cheese cloth. The volume of the supernatant is measured (about 140 ml) and 0.1 volume of 0.1 M Mg(Ac)._, (about 14 ml) and 0.1 volume of 1 M Tris .HCI, pH 7.8 (about 14
186
INITIATION OF PROTEIN SYNTHESIS
[13]
ml) are added. A precipitate that forms is removed by centrifuging the extract at 15,000 rpm in a Sorvall SS-34 rotor for 15 rain. The upper seven-eighths of the supernatant is retained and transferred to tubes in which it is centrifuged in a Beckman 60 Ti rotor at 38,000 rpm for 2 hr. The supernatant is discarded, and the centrifuge tubes containing the ribosomal pellets are allowed to stand in ice for an hour. During this time a small amount of debris is released from the surface of the clear pellets of 80 S ribosomes. A minimal volume of buffer B is used to gently rinse the pellets before they are suspended in a total volume of 3 ml of buffer B. The concentration of 80 S ribosomes should be 600-650 A.,60 units/ml. To prepare 40 S and 60 S ribosomal subunits, 0.5 ml of the 80 S ribosome preparation is immediately layered onto each of three 50-ml 15-30% (w/v) sucrose gradients in buffer C and centrifuged for 14 hr at 21,000 rpm in a Beckman SW 25.2 rotor. Fractions (0.7-0.8 ml) from each gradient are collected, and their absorbancy at 260 nm is determined and plotted against tube number. By extrapolating the leading arm of the 40 S peak and the trailing arm of the 60 S peak, 40 S and 60 S fractions are chosen that give minimal cross-contamination. Fractions containing 40 S subunits from the three gradients are combined, and the magnesium ion concentration is raised to 8 mM by adding the calculated amount of 1 M Mg(Ac)~. The preparation is diluted to 25 ml with buffer D. An identical procedure is followed with the fractions containing 60 S subunits. The two 25-ml samples are centrifuged overnight at 35,000 rpm in a Beckman 60 Ti rotor in order to concentrate the subunits. The clear, colorless pellets are gently rinsed with buffer E and carefully suspended in a volume of about 0.2 ml of the same buffer. The concentrated suspension is centrifuged at 4000 rpm for 20 rain in the Sorvall SS-34 rotor, and the precipitate is discarded. The supernatant is diluted with buffer E so that the 40 S subunits have a concentration of 200 A26o units/ml, and the 60 S 400 A26o units/ml. Each sample is diluted with 1 volume of glycerol and stored at - 2 0 °. The subunits remain fully active for several months, but upon very prolonged storage activity in Met-tRNA~ binding experiments begins to decline. As determined in a poly(U) translational assay, the 40 S subunits are free of 60 S contamination, but the 60 S subunits have about 10% contamination with the smaller subunit. Purification of eIF-2 and EIF-M1 Materials Wheat germ. The wheat germ used for isolation of ribosomal subunits is satisfactory also for this preparation. Acetone DEAE-Cellulose (Whatman DE-23)
[13]
187
SYNTHESIS I N I T I A T I O N FACTORS FROM W H E A T GERM
DEAE-Sephadex A-50 (Pharmacia) CM-Sephadex C-50 (Pharmacia) Hydroxyapatite (Clarkson Hypatite C) Potassium phosphate buffer, 1 M, pH 7.0 Buffer A: 10 mM Tris .HCI, pH 7.5, 10 mM 2-mercaptoethanol, 0.1 mM EDTA, and varying concentrations of KC1 indicated by a number in parentheses, thus A (100) is buffer A containing 100 mM KC1. Buffer B: 20 mM H E P E S - K O H , pH 7.56, 20 mM 2-mercaptoethanol, 0.1 mM EDTA, and varying concentrations of KC1 indicated by a number in parentheses, thus B (100) is buffer B containing 100 mM KC1. Buffer C: 50 mM Tris .HCI, pH 7.5, 10 mM 2-mercaptoethanol, 0.1 mM EDTA, 100 mM KCI, 50% glycerol (v/v) Procedure. A summary of the purification procedure is given in Table I. All operations are carried out at 4° unless otherwise stated. In chromatographic procedures the presence of UV-absorbing material in eluates is continuously monitored with an LKB Uvicord apparatus. Dialysis of samples against buffer C (contains 50% glycerol) before storage in steps 3-5 results in a three-fourths reduction in volume. This can be a useful technique for concentrating protein solutions. Step I. Preparation of Acetone Powder. One-hundred-gram portions of commercial wheat germ are homogenized in a Waring blender for 45 sec with 500 ml of acetone that has been chilled to - 1 5 °. The homogenate is quickly filtered by suction on a large Biichner funnel, and the filtrate is washed in rapid succession with 100 ml of acetone and 200 ml of ether at 15°. The light gray powder is spread in a thin layer on paper and allowed -
TABLE I PURIFICATION OF EIF-2 AND EIF-M1
Purification step DEAE-cellulose, a m m o n i u m sulfate CM-Sephadex C-50 DEAE-Sephadex A-50 Hydroxyapatite Fractions 4 to 10 Fractions 22 to 30
Volume (ml)
250 15 2 2 2
Protein (rag)
20,000 84 18 4.1 2.0
elF-MI" (pmol/mg/20 min)
elF-2 h (pmol/mg/10 rain
-9,000 19,200
-70 148
-37,000
300 --
The standard AcPhe-tRNA binding assay was used to determine elF-MI activity. b The ternary complex formation with GTP and ['~'~S]Met-tRNAi was used to assay elF-2.
188
I N I T I A T I O N OF P R O T E I N S Y N T H E S I S
[13]
to dry at room temperature until the odors of acetone and ether can no longer be detected. Each 100 g of wheat germ yields approximately 80 g of acetone powder. No loss of activity is observed after prolonged storage of the acetone powder at - 15°. Step 2. Extraction, DEAE-Cellulose Treatment, and Ammonium Sulfate Fractionation. Three hundred and twenty grams of wheat germ acetone powder are suspended in 1600 mi of buffer A (100), and the mixture is stirred for 2.5 hr. The suspension is centrifuged at 15,000g for 20 min, and the supernatant is poured into a beaker containing 700 ml of settled DEAE-cellulose (DE-23), which has been equilibrated with buffer A (100). The DEAE-cellulose suspension is stirred and filtered with suction through Whatman No. 1 filter paper on a Bfichner funnel. The packed DEAEcellulose is suspended in about 400 ml of buffer A (100) and again filtered. The filter cake can be used as starting material for the preparation of Co-elF-2. The combined filtrates are brought to 40% saturation with ammonium sulfate and centrifuged at 15,000 g for 20 min. The resulting precipitate is discarded, and the supernatant is adjusted to 65% saturation with solid ammonium sulfate. The protein pellet obtained after centrifugation at 15,000 g for 20 min is dissolved in 135 ml of buffer A (100), and the protein solution is dialyzed against 4 liters of the same buffer overnight. Step 3. Chromatography on CM-Sephadex C-50. The success of chromatography on CM-Sephadex C-50 is somewhat dependent upon the procedure by which the column is prepared. CM-Sephadex C-50 (33 g) is allowed to swell in an excess of 0.5 M KCI for at least 3 days at 4°. The supernatant is decanted and the remaining volume of settled ion exchanger (about 800 ml) is diluted 5-fold with cold distilled water. The suspension is briefly stirred and allowed to settle; the supernatant is decanted. The settled Sephadex is resuspended in buffer A (100) and allowed to settle; the supernatant is decanted, and the thick Sephadex suspension is used to fill a 5 X 60 cm column. The column is immediately attached to a reservoir containing buffer A (100) at a pressure head of about 1 meter. After 350-400 ml of buffer has flowed through the column it is sufficiently packed for application of the sample. The dialyzed preparation from the preceding step (25 g of protein in 300 ml) is applied to the column, and washing with buffer A (200) is begun immediately. Washing is continued until the absorbance of the effluent reaches a low steady level; this is usually after about 1800 ml of buffer have passed through the column. The factors elF-2 and elF-M 1 are eluted with buffer A (300). Both these factors are in a single UV-absorbing peak after about 500 ml of buffer has passed through the column. The active fractions are pooled and concentrated to a volume of 15-20 ml with an Amicon ultrafiltration apparatus fitted with a PM-10 membrane. The concentrated
[13]
S Y N T H E S I S I N I T I A T I O N FACTORS FRO M W H E A T GERM
189
solution is dialyzed against buffer C before storage at - 15°, and before it is used in the next step it is dialyzed for 2-3 hr against buffer B (50). Step 4. Chromatography on DEAE-Sephadex A-50. The protein solution from the preceding step is applied to a DEAE-Sephadex A-50 column (2.5 × 40 cm), which has been prepared similarly to the preparation of cation exchanger in the preceding section, except that it is equilibrated with buffer B (50). The column is washed with buffer B (100) after the sample has been applied until no protein can be detected in the eluate. The protein that elutes with buffer B (200) is collected and concentrated by ultrafiltration, as in the preceding step, to 4 ml. The preparation is dialyzed against buffer C for storage at - 15° and against buffer A (100) for 2-3 hr before use in the next step. Step 5. Hydroxyapatite Chromatography. The sample from the preceding step is applied to a hydroxyapatite column (1.5 X 11 cm) equilibrated with buffer A (100). The column is then washed with 100 mM potassium phosphate, pH 7.0, containing 10 mM 2-mercaptoethanol until no UVabsorbing material can be detected eluting from the column. The column is then developed with a linear gradient (100 × 100 ml) from 100 mM potassium phosphate, pH 7.0, to 500 mM potassium phosphate, pH 7.0 (both buffer solutions contain 10 mM 2-mercaptoethanol), and 4-ml fractions are collected. The peak of eIF-2 activity elutes from the column at 280 mM potassium phosphate and the peak of elF-M 1 activity at 400 mM potassium phosphate. Fractions containing elF-MI activity but no elF-2 activity are pooled, and fractions with elF-2 activity but no elF-MI are also pooled. Both these fractions are concentrated to 2 ml by Amicon ultrafiltration with a PM-10 membrane and dialyzed against buffer C before use or storage.
Comments elF-2. The eIF-2 fractions chosen from the hydroxyapatite chromatography are free of elF-M1, but they represent a little less than half of the total eIF-2 in the eluate. The remaining eIF-2 is present in intermediate fractions contaminated with eI F-M 1. Rechromatography of these fractions has not been of practical value, but the fractions can still be used in experiments in which the presence of eIF-MI is not an important factor. Upon storage at --15 ° eIF-2 gradually loses activity, but when frozen in small aliquots in buffer C at - 8 0 °, full activity is retained for at least 6 months. The elution profiles of eIF-2 and eIF-M1 on Sephadex G-200 are identical; they both have estimated molecular weights by this technique of 145,000. Upon electrophoresis in sodium dodecyl sulfate on polyacrylamide gels, three major bands are obtained from eIF-2 with molecular
190
INITIATION OF PROTEIN SYNTHESIS
[13]
weights of 37,000, 41,000, and 57,000. Wheat germ elF-2, therefore, is assumed to be like elF-2 from reticulocytes in consisting of three different polypeptide chains. Ternary complex formation with wheat germ elF-2 is inhibited by N-ethylmaleimide and p-chloromercuribenzoate. It is also inhibited by EDTA. The probable reason for this is that complex formation requires a low concentration of magnesium, ~7which is normally supplied as an impurity in other components of the standard reaction mixture. There is no difference in activity at 25 ° and 37 ° in the usual 10-min assay. The concentration of GTP (0.2 mM) routinely used in the ternary complex assay is a large excess over the amount actually needed. With the amount ofelF-2 and Met-tRNA~ employed in the standard assay, maximal complex formation occurs at a GTP concentration of about 0.5/xM. GTP concentration becomes an important consideration in regard to the binding of Met-tRNA~ to 40 S ribosomes because some inhibition of binding appears to occur at the high concentration used for ternary complex formation. For this reason a lower GTP concentration (5/zM) is used for formation of the 40 S complex. Under the conditions of the standard assays, Met-tRNAi bound in both the ternary and 40 S complexes does not exchange with free Met-tRNAt. Experimental conditions for Met-tRNAi.AUG.40 S complex formation with elF-2 are similar to those for binding Met-tRNA~ to the small subunits with elF-M 1. A major difference in the two systems, however, is the temperature at which the assay is performed. The complex produced in the presence of elF-M1 rapidly decomposes at temperatures above 0°. The decomposition is catalyzed by elF-M 1 itself. On the other hand, the elF-2 complex is stable at 25°, but is formed very slowly at 0°. Addition ofelF-M 1 at 25° to a Met-tRNAi .AUG .40 S complex formed with elF-2 catalyzes the destruction of the complex. For this reason Met-tRNAi. AUG .40 S complex formation with elF-2 can be detected only if the elF-2 preparation is free of elF-M1. In the systems described here elF-2 acts stoichiometrically; it does not turn over. In the absence of AUG, a Met-tRNAi.40 S complex is formed with elF-2. However, up to 5 times more complex is formed when tile trinucleotide is present than when it is absent. The reason for this observation is that AUG stabilizes the ribosomal complex. Both types of complex, with and without AUG, are sufficiently stable to survive analytical sucrose density gradients. The complex formed in the absence of AUG is not destroyed by elF-M1. elF-M1. The physical and biological properties of what germ elF-MI ~rL. M. Cashion, G. L. Dettman, and W. M. Stanley,Jr., this series, Vol. 30, p. 153.
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SYNTHESISINITIATION FACTORS FROM WHEAT GERM
191
are similar to those of elF-MI isolated from other tissues. 15,18,19Full activity of the purified factor is retained upon storage in 50% glycerol (buffer C) at - 1 5 ° for several weeks, but after this time activity begins to decline because of the oxidation of sulfhydryl groups. In the early stages of inactivation, dialysis of the sample against buffer C containing fresh 2-mercaptoethanol will completely restore activity. Samples frozen at --80 ° retain activity for longer times. As estimated by gel filtration on Sephadex G-200, the molecular weight of elF-M1 is 145,000. The value obtained by electrophoresis in sodium dodecyl sulfate on polyacrylamide gels is 41,000, which could be an indication that the native protein is a tetramer. The factor acts catalytically, and, as determined for a molecular weight of 145,000, it has a turnover number estimated roughly to be about 10. Although the isolation procedure described here uses a wheat germ acetone powder extract as starting material, the same procedure can be used to purify eIF-M 1 from a 100,000 g wheat germ supernatant. In addition, wheat germ ribosomes also contain eIF-M 1 that can be extracted with 700 mM KCI and also.purified by this same procedure. The supernatant and ribosomal eIF-Mls are indistinguishable. Highly purified elF-MI binds to 40 S wheat germ ribosomes. One method of demonstrating this is to mix 40 S ribosomes and eIF-M 1, and separate the ribosome-eIF-M 1 complex from free eIF-MI. The 40 S ribosome.eIF-M1 complex is active in binding aminoacyl-tRNA. Binding of eIF-M 1 to ribosomes is the most likely explanation for the fact that 40 S ribosomes, prepared by the low-salt procedure described here, frequently give high blanks in the eIF-M1 assay. As the ribosome preparations age, the blank values decrease. P rep ar atio n of Co-eIF-2 Materials
Wheat germ, ion-exchange chromatography materials, and buffers A and C are the same as described for the preparation of elF-2 and elF-MI. Procedure. The starting material for the preparation of Co-elF-2 is the filtered DEAE-cellulose from step 2 in the purification of elF-2 and elF-M 1. All operations are carried out at 4°. Table II is a summary of the purification procedure. Step 1. Elution o f Co-elF-2 and A m m o n i u m Sulfate Precipitation. The 1~E. Gasior and K. Moldave,J. Mol. Biol. 66, 391 (1972). ~9W. C. Merrick and W. F. Anderson,J. Biol. Chem. 250, 1197(1975).
192
INITIATION OF PROTEIN SYNTHESIS
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TABLE II PURIFICATION OF C o - E I F - 2
Purification step DEAE-cellulose, ammonium sulfate DEAE-cellulose CM-Sephadex C-50
Volume (ml)
Protein (mg)
Co-eIF-2 (pmol/mg/10 rain)
57 2.5 0.7
1900 69 6. I
-9 60
moist DEAE-cellulose filter cake from step 2 of the e l F - 2 - e l F - M 1 preparative procedure is suspended in 55 ml of buffer A (400). The suspension is occasionally stirred over a period of 30 min and filtered with gentle suction through Whatman No. 1 filter paper on a Bfichner funnel. Solid ammonium sulfate is added to the clear yellow filtrate to 80% saturation, and the precipitated protein is removed by centrifugation at 15,000 rpm in the SS-34 Sorvall rotor. The solid pellet, which contains the Co-elF-2, can be stored at - 15° for several months without loss of activity. Before assay or use in the next step, it is dissolved in approximately 25 ml of buffer A (50) and dialyzed overnight against the same buffer. A precipitate which forms at this low salt concentration is removed by centrifugation and discarded. Step 2. Chromatography on DEAE-Cellulose. The protein solution from the preceding step is applied to a DEAE-cellulose column (2.5 X 35 cm) equilibrated in buffer A (50), and the column is washed with the same buffer. The major protein peak that washes from the column is retained and concentrated to approximately 20 ml by ultrafiltration using an Amicon cell with a PM- 10 membrane. If the sample is to be stored at - 15°, it should first be dialyzed against buffer C. Before use in the next step it is dialyzed against buffer A (100). Step 3. Chromatography on CM-Sephadex C-50. The Co-elF-2 preparation is applied to a CM-Sephadex C-50 column (2.5 X 30 cm) equilibrated with buffer A (100). The column is washed successively with buffer A (100) and A (200). Each time the washing is continued until the amount of protein in the column effluent is close to zero. The protein fraction that elutes from the column with buffer A (100) contains some Co-elF-2, but the bulk of the activity is in the A (200) eluate. This sample is concentrated to 4 ml by ultrafiltration as in step 2 and dialyzed against buffer C for storage at - 15°.
Comments An unusual aspect of the purification procedure is that Co-eIF-2 in the crude extract binds to DEAE-cellulose, but in a later step it is not retained
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WHEAT GERM INITIATION FACTORS
193
on the same anion exchanger even at a lower salt concentration. A possible reason for this is that Co-eIF-2 may be complexed with another protein in the crude extract, and it is the complex that binds to DEAE-cellulose. A subsequent manipulation, such as elution from the DEAE-cellulose with 400 mM potassium chloride, precipitation with ammonium sulfate, or dialysis against 50 mM KC1, could disrupt the complex, thereby altering the behavior of the cofactor. A distinct advantage of this preparative procedure is that Co-eIF-2 is readily separated from eIF-2. The buffer A (400) eluate is virtually free of eIF-2. Occasionally some preparations have a high GTPindependent binding of Met-tRNAi which is probably due to nonspecific binding. A 2- to 3-fold stimulation of Met-tRNAi binding to elF-2 by Co-eIF-2 is routinely obtained although a maximum of about 5-fold has been observed in a few cases. Addition of Co-elF-2 to the standard reaction mixture for binding Met-tRNA~ to 40 S ribosomes does not cause an increase in the amount of 40 S complex formed. The mechanism of action of Co-eIF-2 is not known, but it would be attractive to assume that it forms a complex with eIF-2 and that this complex is the species which combines with GTP and Met-tRNAi. If this hypothesis is correct it should be possible to show a complete dependence upon Co-eIF-2 by very highly purified eIF-2. In the reticulocyte system .'° it has been shown that purified, but not crude, eIF-2 is stimulated by the cofactor. ~0 A. Dasgupta, A. Majumdar, A. D. George, and N. K. Gupta, Biochem. Biophys. Res. Cornmun. 71, 1234 (1976).
[14] I s o l a t i o n a n d P u r i f i c a t i o n o f P r o t e i n S y n t h e s i s Initiation Factors from Wheat Germ 1
By
B E N J. W A L T H A L L ,
LINDA L. SPREMULLI,
SANDRA R. LAX, and JOANNE M. RAVEL
Crude extracts of wheat germ are capable of translating mRN As from a variety of sources. '-4 Wheat germ extracts are easy to prepare, are low in nuclease activity, have low endogenous levels of mRNA, and translate exogenously supplied mRNAs with high efficiency and fidelity. Marcus This work was supported in part by U.S. Public Health Service Grant GM-18775. K. Marcu and B. Dudock, Nucl. Acids Res. 1, 1385 (1974). :~ B. E. Roberts and B. M. Patersofi, Proc. Natl. Acad. Sci. U.S.A. 70, 2330 (1973). 4 0 . E. Beck and H. G. G a s s e n , Biochern. Biophys. Res. Commun. 74, 16 (1977), and references cited in this paper.
METHODS 1N ENZYMOLOGY, VOL. LX
Copyright © 1979by AcademicPress. Inc. All rights of reproduction in any form reserved. ISBN 0-12-181960-4