240
INITIATION OF PROTEIN SYNTHESIS
[19] P u r i f i c a t i o n a n d P r o p e r t i e s from Pig Liver
[19]
of eIF-2
B y INGRID HARBITZ and JENS G. HAUGE
Slaughterhouse material has the advantage over laboratory animal tissues as the starting point for protein purification that it is available in large quantities at low cost. We describe here the purification of initiation factor eIF-2 from pig liver and some of the properties of this preparation. The factor forms a complex with Met-tRNA~ ~t and GTP, and it binds MettRNA Metto 40 S ribosomal subunits in the absence of the template. ~The factor preparation does not lead to formation of an 80 S initiation complex. Partial purification of eIF-2 from rabbit, rat, and chicken liver has been reported by Cashion and Stanley, 2 and from calf liver by Stringer et al.3 Reagents
Buffer A-X M KCI: 20 mM Tris. HCI, pH 7.6, 0.2 mM EDTA, l0 mM mercaptoethanol, 10% glycerol (v/v), and KCI as indicated Buffer B: 20 mM K phosphate buffer, pH 7.2, 0.2 mM MgClz, 50 mM NH4CI, 7 mM mercaptoethanol, and 5% glycerol (v/v) Buffer C-X M K phosphate: 10% glycerol (v/v), 10 mM mercaptoethanol and K phosphate buffer, pH 7.5, as indicated L-[3'~S]Methionine, [8-3H]GTP, and [8-:~H]GDP (Amersham) Membrane filters (Gelman, mixed nitrocellulose and cellulose acetate, type GN-6) DEAE-cellulose (Whatman DE-52) Phosphocellulose (Sigma) Hydroxyapatite, spheroidal (BDH) Poly(A,U,G) (Miles) E s c h e r i c h i a coli B (Miles) Rat liver tRNA M~t, 60% pure (Biogenics Research Corporation) Calf liver tRNA, crude (Boehringer Mannheim) ['~S]Met-tRNAM~'t: Rat or calf liver tRNA was aminoacylated with L-[3~S]methionine by incubation with E. coli aminoacyl-tRNA synthetases. The synthetases were prepared according to Stanley. 4 The incubation mixture contained per milliliter 5 mg of liver tRNA, ' I. Harbitz and J. G. Hauge,Arch. Biochem. Biophys. 176, 766 (1976). 2 L. M. Cashion and W. M. Stanley, Jr., Biochim. Biophys. Acta 324, 410 (1973). '~E. A. Stringer, A. Chaudhuri, and U. Maitra, Biochem. Biophys. Res. Commun. 76, 586 (1977). 4 W. M. Stanley, Jr., Anal. Biochem. 48, 202 (1972). METHODS
IN ENZYMOLOGY,
VOL. LX
Copyright ~ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. I S B N 0-12-181960-4
[19]
PIG LIVER INITIATION FACTOR e l F - 2
241
5 mg of synthetases, 2 0 / z M [asS]methionine (specific activity, 15 Ci/mmol), 4 m M ATP, 1 m M CTP, 16 m M MgC12, 14 m M Tris .HCI, pH 7.5, and 18 m M mercaptoethanol. After incubation at 37 ° for 30 min, the reaction mixture was deproteinized with phenol and the tRNA was precipitated with ethanol. The precipitated tRNA was dissolved in 0.1 m M EDTA and passed through a Sephadex G-25 column. The eluted [asS]Met-tRNA~let was precipitated, washed with ethanol, and dissolved in 0.1 m M EDTA to a concentration of 2 nmol of [:~sS]Met-tRNArMet/ml, frozen in liquid nitrogen, and stored at - 8 0 ° Ribosomal subunits were prepared from rat liver according to Falvey and Staehelin, a with the modifications introduced by Schreier and Staehelin. 6 After separation on a sucrose gradient, the 40 S and 60 S subunits were suspended in buffer ~ to a final A.,,~0 of 100, quickly frozen in liquid nitrogen, and stored at - 8 0 ° Assay The GTP-dependent formation of a complex between elF-2 and MettRNA~ et is assayed in 50-/zl reaction mixtures containing 1 pmol of [:~aS[ Met-tRNAfMet. The ionic conditions are 100 mM KCI, 0.2 mM MgCI.,, 25 mM Tris- HCI, pH 7.6, and 0.5 mM GTP. After a 10-rain incubation at 37 °, 3 ml of ice-cold buffer (100 mM KCI, 0.2 mM MgCI.,, 25 mM Tris. HCI, pH 7.6) is added, and the samples are filtered through Gelman filters and washed with 3 ml of cold buffer. The filters are dried and counted in toluene-PPO-POPOP scintillation liquid. Serum albumin (0.2 mg/ml) should be added to the reaction mixture when the specific activities of different eIF-2 preparations are determined. Purification P r o c e d u r e Initiation factors are found in the cell sap as well as on ribosomes, and various workers have used either isolated ribosomes, microsomes, or the particle-flee supernatant as their source. In order to include all these possible sources of elF-2, the postnuclear fraction is subjected to 0.55 M KCI, and the high speed supernatant from this used as the starting point for the fractionation. The liver, obtained from a slaughterhouse, is rinsed in cold water, cut in pieces, and quickly frozen in Dry Ice-ethanol immediately after being removed from the pig. Frozen liver could be stored at --80 ° for some A. K. Falvey and T. Staehelin, J. Mol. Biol. 53, I (1970). " M. H. Schreier and T. Staehelin, J. Mol. Biol. 73, 329 (1973).
242
I N I T I A T I O N OF PROTEIN S Y N T H E S I S
[19]
months without observable loss of initiation factor activity. All steps in the purification procedure should be performed at 0-4 ° . Step 1. Preparation of the Extract. Frozen liver (250 g) is crushed into smaller pieces with a hammer. Cold homogenization buffer (0.2 M KCI, 5 mM Mg acetate, 20 mM Tris-HCI, pH 7.6, and 10 mM mercaptoethanol) is poured over the liver (2 ml of buffer per gram of liver), and the mixture is homogenized for 3 min in a blender while the liver is still partially frozen. The homogenate is centrifuged in a Sorvall SS 34 rotor at 2500 rpm for 10 min. KCI, 4M, is added to the postnuclear supernatant to a final concentration of 0.55 M KCI, and the mixture is stirred for 20 min and centrifuged at 12,000 rpm for 15 min. The postmitochondrial supernatant is centrifuged in a Beckman 60 Ti rotor at 60,000 rpm for 130 min. The supernatant (200 ml) is carefully removed and used for further purification of elF-2 activity. Step 2. Ammonium Sulfate Fractionation. Solid (NH4).,SO4 is added to the postmicrosomal supernatant to 40% saturation (22.6 g,/100 ml). After stirring for 10 min, the precipitate is spun down and discarded. To the clear supernatant, solid (NH4)2SO4 is added to 50% saturation (5.8 g/100 ml). The precipitated protein (about 1200 mg from 200 ml of supernatant) is collected by centrifugation, dissolved in and dialyzed against buffer A-0.9 M KCI overnight. At this point the preparation is frozen and stored at - 8 0 ° while another portion of liver is processed to the same stage. Step 3. Chromatography on DEAE-Cellulose. Step 2 preparations from 500 g of liver are combined (30 ml) and applied to a 2.5 X 40-cm DEAEcellulose column equilibrated with buffer A-90 mM KCI. The column is washed with the same buffer and eluted with a gradient in buffer A from 0.1 to 0.35 M KC1. A flow rate of 20 ml/hr is suitable, and 5-ml fractions are collected and tested for elF-2 activity, elF-2 activity is eluted at 0.16-0.18 M KCI. The active fractions (25 ml) are combined, frozen in liquid nitrogen, and stored at - 8 0 ° until the phosphocellulose chromatography step. Step 4. Chromatography on Phosphocellulose. Phosphocellulose is washed in acid and alkali. Step 3 preparations from 1 kg of liver are combined (50 ml) and applied to a 1.5 X 18 cm phosphocellulose column equilibrated with buffer A-0.18 M KC1. The column is washed with buffer A-0.3 M KC1 and eluted with buffer A-0.6 M KC1. The flow rate is 15 ml/hr, and 3-ml fractions are collected and tested for elF-2 activity. The active fractions (9 ml, 3 mg of protein) are combined and concentrated to 2 ml by ultrafiltration (Amicon UM-10). The concentrated elF-2 preparation is dialyzed against buffer C- 10 mM K phosphate, frozen in liquid nitrogen, and stored at - 8 0 ° until the hydroxyapatite chromatography step. Step 5. Chromatography on Hydroxyapatite. Step 4 preparation from 1 kg of liver (2 ml, 3 mg of protein) is applied to a 0.9 X 5 cm hydroxyapatite column equilibrated with buffer C-10 mM K phosphate. The column is
[19]
PIG LIVER INITIATION FACTOR e l F - 2
243
washed with buffer C- 10 mM K phosphate and eluted with buffer C-0.15 M K phosphate. The flow rate is 15 ml/hr, and 2-ml fractions are collected. The active fractions (4-6 ml, 1 mg of protein) are combined and concentrated to less than 1 ml by ultrafiltration. The eIF-2 preparation is quickly frozen in liquid nitrogen and stored at - 8 0 °. As an alternative concentration procedure for the eluate from the hydroxyapatite column, we have dialyzed the eluate against 40% polyethylene glycol in buffer A-0.12 M KC1. When the preparation was sufficiently concentrated, the polyethylene glycol was removed by dialysis against buffer A-0.12 M KC1. Although this concentration procedure resulted in very small final volumes, it did lead to greater losses of activity than the ultrafiltration procedure. Table I summarizes the data for a processing of 1 kg of liver through steps I-4. A further approximately 3-fold purification is achieved in step 5. A protein with molecular weight 40,500 is the main contaminant removed in this step (Fig. 1).
-•
.iv,..
.
.7
(.
.
.
FIG. 1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of a step 4 (left column) and step 5 (right column) elF-2 preparation. The electrophoresis was performed in a 15% separating slab gel as described by U. K. Laemmli [Nature (London) 227, 680 (1970)].
244
INITIATION OF PROTEIN SYNTHESIS
INITIATION OF PROTEIN SYNTHESIS
[19] P u r i f i c a t i o n a n d P r o p e r t i e s from Pig Liver
[19]
of eIF-2
B y INGRID HARBITZ and JENS G. HAUGE
Slaughterhouse material has the advantage over laboratory animal tissues as the starting point for protein purification that it is available in large quantities at low cost. We describe here the purification of initiation factor eIF-2 from pig liver and some of the properties of this preparation. The factor forms a complex with Met-tRNA~ ~t and GTP, and it binds MettRNA Metto 40 S ribosomal subunits in the absence of the template. ~The factor preparation does not lead to formation of an 80 S initiation complex. Partial purification of eIF-2 from rabbit, rat, and chicken liver has been reported by Cashion and Stanley, 2 and from calf liver by Stringer et al.3 Reagents
Buffer A-X M KCI: 20 mM Tris. HCI, pH 7.6, 0.2 mM EDTA, l0 mM mercaptoethanol, 10% glycerol (v/v), and KCI as indicated Buffer B: 20 mM K phosphate buffer, pH 7.2, 0.2 mM MgClz, 50 mM NH4CI, 7 mM mercaptoethanol, and 5% glycerol (v/v) Buffer C-X M K phosphate: 10% glycerol (v/v), 10 mM mercaptoethanol and K phosphate buffer, pH 7.5, as indicated L-[3'~S]Methionine, [8-3H]GTP, and [8-:~H]GDP (Amersham) Membrane filters (Gelman, mixed nitrocellulose and cellulose acetate, type GN-6) DEAE-cellulose (Whatman DE-52) Phosphocellulose (Sigma) Hydroxyapatite, spheroidal (BDH) Poly(A,U,G) (Miles) E s c h e r i c h i a coli B (Miles) Rat liver tRNA M~t, 60% pure (Biogenics Research Corporation) Calf liver tRNA, crude (Boehringer Mannheim) ['~S]Met-tRNAM~'t: Rat or calf liver tRNA was aminoacylated with L-[3~S]methionine by incubation with E. coli aminoacyl-tRNA synthetases. The synthetases were prepared according to Stanley. 4 The incubation mixture contained per milliliter 5 mg of liver tRNA, ' I. Harbitz and J. G. Hauge,Arch. Biochem. Biophys. 176, 766 (1976). 2 L. M. Cashion and W. M. Stanley, Jr., Biochim. Biophys. Acta 324, 410 (1973). '~E. A. Stringer, A. Chaudhuri, and U. Maitra, Biochem. Biophys. Res. Commun. 76, 586 (1977). 4 W. M. Stanley, Jr., Anal. Biochem. 48, 202 (1972). METHODS
IN ENZYMOLOGY,
VOL. LX
Copyright ~ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved. I S B N 0-12-181960-4
[19]
PIG LIVER INITIATION FACTOR e l F - 2
241
5 mg of synthetases, 2 0 / z M [asS]methionine (specific activity, 15 Ci/mmol), 4 m M ATP, 1 m M CTP, 16 m M MgC12, 14 m M Tris .HCI, pH 7.5, and 18 m M mercaptoethanol. After incubation at 37 ° for 30 min, the reaction mixture was deproteinized with phenol and the tRNA was precipitated with ethanol. The precipitated tRNA was dissolved in 0.1 m M EDTA and passed through a Sephadex G-25 column. The eluted [asS]Met-tRNA~let was precipitated, washed with ethanol, and dissolved in 0.1 m M EDTA to a concentration of 2 nmol of [:~sS]Met-tRNArMet/ml, frozen in liquid nitrogen, and stored at - 8 0 ° Ribosomal subunits were prepared from rat liver according to Falvey and Staehelin, a with the modifications introduced by Schreier and Staehelin. 6 After separation on a sucrose gradient, the 40 S and 60 S subunits were suspended in buffer ~ to a final A.,,~0 of 100, quickly frozen in liquid nitrogen, and stored at - 8 0 ° Assay The GTP-dependent formation of a complex between elF-2 and MettRNA~ et is assayed in 50-/zl reaction mixtures containing 1 pmol of [:~aS[ Met-tRNAfMet. The ionic conditions are 100 mM KCI, 0.2 mM MgCI.,, 25 mM Tris- HCI, pH 7.6, and 0.5 mM GTP. After a 10-rain incubation at 37 °, 3 ml of ice-cold buffer (100 mM KCI, 0.2 mM MgCI.,, 25 mM Tris. HCI, pH 7.6) is added, and the samples are filtered through Gelman filters and washed with 3 ml of cold buffer. The filters are dried and counted in toluene-PPO-POPOP scintillation liquid. Serum albumin (0.2 mg/ml) should be added to the reaction mixture when the specific activities of different eIF-2 preparations are determined. Purification P r o c e d u r e Initiation factors are found in the cell sap as well as on ribosomes, and various workers have used either isolated ribosomes, microsomes, or the particle-flee supernatant as their source. In order to include all these possible sources of elF-2, the postnuclear fraction is subjected to 0.55 M KCI, and the high speed supernatant from this used as the starting point for the fractionation. The liver, obtained from a slaughterhouse, is rinsed in cold water, cut in pieces, and quickly frozen in Dry Ice-ethanol immediately after being removed from the pig. Frozen liver could be stored at --80 ° for some A. K. Falvey and T. Staehelin, J. Mol. Biol. 53, I (1970). " M. H. Schreier and T. Staehelin, J. Mol. Biol. 73, 329 (1973).
242
I N I T I A T I O N OF PROTEIN S Y N T H E S I S
[19]
months without observable loss of initiation factor activity. All steps in the purification procedure should be performed at 0-4 ° . Step 1. Preparation of the Extract. Frozen liver (250 g) is crushed into smaller pieces with a hammer. Cold homogenization buffer (0.2 M KCI, 5 mM Mg acetate, 20 mM Tris-HCI, pH 7.6, and 10 mM mercaptoethanol) is poured over the liver (2 ml of buffer per gram of liver), and the mixture is homogenized for 3 min in a blender while the liver is still partially frozen. The homogenate is centrifuged in a Sorvall SS 34 rotor at 2500 rpm for 10 min. KCI, 4M, is added to the postnuclear supernatant to a final concentration of 0.55 M KCI, and the mixture is stirred for 20 min and centrifuged at 12,000 rpm for 15 min. The postmitochondrial supernatant is centrifuged in a Beckman 60 Ti rotor at 60,000 rpm for 130 min. The supernatant (200 ml) is carefully removed and used for further purification of elF-2 activity. Step 2. Ammonium Sulfate Fractionation. Solid (NH4).,SO4 is added to the postmicrosomal supernatant to 40% saturation (22.6 g,/100 ml). After stirring for 10 min, the precipitate is spun down and discarded. To the clear supernatant, solid (NH4)2SO4 is added to 50% saturation (5.8 g/100 ml). The precipitated protein (about 1200 mg from 200 ml of supernatant) is collected by centrifugation, dissolved in and dialyzed against buffer A-0.9 M KCI overnight. At this point the preparation is frozen and stored at - 8 0 ° while another portion of liver is processed to the same stage. Step 3. Chromatography on DEAE-Cellulose. Step 2 preparations from 500 g of liver are combined (30 ml) and applied to a 2.5 X 40-cm DEAEcellulose column equilibrated with buffer A-90 mM KCI. The column is washed with the same buffer and eluted with a gradient in buffer A from 0.1 to 0.35 M KC1. A flow rate of 20 ml/hr is suitable, and 5-ml fractions are collected and tested for elF-2 activity, elF-2 activity is eluted at 0.16-0.18 M KCI. The active fractions (25 ml) are combined, frozen in liquid nitrogen, and stored at - 8 0 ° until the phosphocellulose chromatography step. Step 4. Chromatography on Phosphocellulose. Phosphocellulose is washed in acid and alkali. Step 3 preparations from 1 kg of liver are combined (50 ml) and applied to a 1.5 X 18 cm phosphocellulose column equilibrated with buffer A-0.18 M KC1. The column is washed with buffer A-0.3 M KC1 and eluted with buffer A-0.6 M KC1. The flow rate is 15 ml/hr, and 3-ml fractions are collected and tested for elF-2 activity. The active fractions (9 ml, 3 mg of protein) are combined and concentrated to 2 ml by ultrafiltration (Amicon UM-10). The concentrated elF-2 preparation is dialyzed against buffer C- 10 mM K phosphate, frozen in liquid nitrogen, and stored at - 8 0 ° until the hydroxyapatite chromatography step. Step 5. Chromatography on Hydroxyapatite. Step 4 preparation from 1 kg of liver (2 ml, 3 mg of protein) is applied to a 0.9 X 5 cm hydroxyapatite column equilibrated with buffer C-10 mM K phosphate. The column is
[19]
PIG LIVER INITIATION FACTOR e l F - 2
243
washed with buffer C- 10 mM K phosphate and eluted with buffer C-0.15 M K phosphate. The flow rate is 15 ml/hr, and 2-ml fractions are collected. The active fractions (4-6 ml, 1 mg of protein) are combined and concentrated to less than 1 ml by ultrafiltration. The eIF-2 preparation is quickly frozen in liquid nitrogen and stored at - 8 0 °. As an alternative concentration procedure for the eluate from the hydroxyapatite column, we have dialyzed the eluate against 40% polyethylene glycol in buffer A-0.12 M KC1. When the preparation was sufficiently concentrated, the polyethylene glycol was removed by dialysis against buffer A-0.12 M KC1. Although this concentration procedure resulted in very small final volumes, it did lead to greater losses of activity than the ultrafiltration procedure. Table I summarizes the data for a processing of 1 kg of liver through steps I-4. A further approximately 3-fold purification is achieved in step 5. A protein with molecular weight 40,500 is the main contaminant removed in this step (Fig. 1).
-•
.iv,..
.
.7
(.
.
.
FIG. 1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of a step 4 (left column) and step 5 (right column) elF-2 preparation. The electrophoresis was performed in a 15% separating slab gel as described by U. K. Laemmli [Nature (London) 227, 680 (1970)].
244
INITIATION OF PROTEIN SYNTHESIS
