- Email: [email protected]
[17] Aminoacyl-tRNA synthetases from cultured CHO cells
[17]
AMINOACYL-tRNA SYNTHETASES FROM C H O CELLS
229
this method was 77. These chromatographic patterns serve as a useful reference for determining various tRNA functions in this model mammalian cell culture system. Because of the interspecies similarities between mammalian tRNAs, a model system such as CHO is very useful in studies directed toward understanding nonclassical roles for tRNA. The emergence of CHO as an outstanding somatic cell genetic system adds greatly to its usefulness along these lines. A catalog of tRNA isoacceptors in a defined mammalian system represents an essential first step in the elucidation of possible tRNA involvement in mammalian metabolism and cell regulation.
[17] A m i n o a c y l - t R N A
Synthetases Cells
from Cultured CHO
B y ARNOLD E . H A M P E L , M . D U A N E E N G E R , a n d PRESTON O . RITTER
The role of aminoacyl-tRNA synthetases in protein biosynthesis has been generally recognized for many years. More recently, evidence has accumulated that these enzymes may also play a role in the regulation of cellular metabolism 1and in amino acid transport. 2-4 At the same time the existence of particulate synthetases in eukaryotic systems has become generally accepted.4 Chinese hamster ovary (CHO) cells provide a good model system for more detailed studies on the structure and function of mammalian aminoacyl-tRNA synthetases. This cell line is stable and amenable to controlled physiological and biochemical perturbations. Conditionally lethal synthetase mutants are also available. 5'6 The detailed study of any enzyme is greatly enhanced by the availability of a quick, easy, and reproducible assay. This chapter describes such assays as they have been developed for the analysis of CHO aminoacyltRNA synthetases, then describes a procedure for the isolation of the various forms of these enzymes. This information will be of value to anyone working with eukaryotic aminoacyl-tRNA synthetases. i F. C. Neidhardt, J. Parker, and W. C. McKeever, Annu. Rev. Microbiol. 29, 215 (1975). P. A. Moore, D. W. Jayme; and D. L. Oxender, J. Biol.Chem. 252, 7427 (1977). a S. C. Quay, E. L. Kline, and D. L. Oxender, Proc. Natl. Acad. Sci. U.S.A. 72, 3921 (1975). L. L. Kisselev, and O. O. Favorova, Adv. Enzymol. 40, 141 (1974). L. H. Thompson, J. L. Harkins, and C. P. Stanners, Proc. Natl. Acad. Sci U.S.A. 70, 3094 (1973). 6 L. H. Thompson, D. J. Lofgren, and G. M. Adair, Cell 11, 157 (1977). METHODS IN ENZYMOLOGY,VOL. LIX
Copyright© 1979by AcademicPress, Inc. All rightsof reproductionin any formreserved. 1SBN0-12-181959-0
230
TRANSFER R N A AND A M I N O A C Y L - t R N A SYNTHETASES
[17]
Large-Scale Preparation of Rat Liver tRNA for Synthetase Assays Sixty rats are killed by cervical dislocation. Livers are removed immediately, washed with 0.25 M sucrose at 0 °, then passed through a tissue press into 5 volumes of 0.25 M sucrose at 0 °. The resulting slurry is homogenized with 4 passages through a 2000 rpm motor-driven, PotterElvehjem homogenizer containing a Teflon pestle with a clearance of 0.006-0.009 inches. The homogenate is centrifuged at 8000 g for 10 rain at 4 °. The supernatant is adjusted to l0 mM NaCI and 0.2 mM EDTA pH 7.0 with 50 mM NaCI-1 mM EDTA, to 0.5% sodium dodecyl sulfate (SDS) with 20% SDS, and then one-half volume of 88% redistilled phenol is added. The mixture is stirred for 48 hr at room temperature prior to splitting of the phases with centrifugation at 4000 g for 10 rain. The aqueous phase is retreated with phenol, as just described, two more times. The final aqueous phase is treated with 2 volumes of ethanol overnight at -20 °. The precipitated RNA is collected with centrifugation and redissolved in 10 mM Tris.HCl (pH 7.5 at 25°)-10 mM NaC1. The ethanol precipitation step is repeated 2 times, then the final pellet is dissolved in a minimum volume (about 20 mg of RNA per milliliter) of 10 mM Tris.HCl (pH 7.5 at 25o)- l0 mM NaC1. Ribosomal RNA is precipitated by adding an equal volume of 2 M NaC1 then leaving the mixture at 4 ° for 5 hr. After centrifugation the pellet is redissolved in a minimum volume of the Tris-NaC1 buffer and rRNA is once again precipitated as just described. The supernatant fractions from the two rRNA precipitations are combined, and the RNA, now essentially free of rRNA, is subjected to three consecutive ethanol precipitations to remove the excess NaC1. The final pellet is dissolved in a minimum volume of 200 mM NaC1-10 mM Tris.HC1 (pH 7.5 at 25 °) and collected on a BD-cellulose (Regis Chemical Company) column that had been titrated to pH 7.0 and equilibrated with the same buffer. It is important that the load not exceed 5 mg of RNA per milliliter of column. The loaded column is washed with 2 column volumes of 200 mM NaCI-10 mM Tris.HC1 (pH 7.5) prior to the elution of the tRNA with 1.8 M NaCI in 10% ethanol. The eluted tRNA is subjected to three consecutive ethanol precipitations as previously described. The tRNA is redissolved in 10 mM KC1-10 mM Tris-HCl (pH 7.5)-1 mM MgC12-0.1 mM dithiothreitol between precipitations. The final pellet is lyophilized to dryness, dissolved in the same buffer, and stored at -20 °. This final t R N A preparation serves as an excellent substrate for CHO aminoacyl-tRNA synthetase assays that involve the acylation of tRNA with labeled amino acids. The tRNA will readily accept all 20 protein
[17]
AMINOACYL-tRNA SYNTHETASESFROMCHO CELLS
231
amino acids and is reactive with highly purified, as well as crude, CHO synthetase preparations. Assay of CHO Aminoacyl-tRNA Synthetases Aminoacyl-tRNA synthetase activities are measured by esterifying radioactive amino acids to their cognate tRNAs. Such reactions are known to be sensitive to pH, ATP concentration, Mg 2+ concentration, salt concentration, etc. and different synthetases usually have different optimal reaction conditions. A final reaction mixture containing 50 mM Tris (pH 7.4 at 25°)-15 mM MgC12-0.5 mM EDTA (disodium salt, adjusted to pH 7.5 before being added to reaction mixture)-5 mM ATP (adjusted to pH 7 with NaOH before being added to reaction mixture)0.35 mM CTP (adjusted to pH 7.0 with NaOH before being added to reaction mixture)-0.35 mg/ml rat liver tRNA-18-20 p~M [14C]amino acid gives satisfactory reactions for CHO synthetases corresponding to the amino acids Ala, Arg, Asn, Asp, Gln, Glu, Gly, Ile, Lys, Met, Pro, Set, Thr, Tyr, and Val. A similar mixture containing 2 /zM [14C]Trp is used to assay for Trp-tRNA synthetase. Cys-, His-, Leu-, and Phe-tRNA synthetases are assayed under the originally described conditions except that Tris is at pH 8.6 and MgCI2 at 8 mM. Mechanically, the reactions are carried out by placing small drops (less that 50 p.l) of reaction mixture, minus synthetase, on a Saran Wrap (check to make certain that a drop of H20 forms a tight bead) covered slide warmer at 37 ° for 1 min prior to the addition of synthetase. The volume and concentration of the reaction mixture, minus synthetase, are adjusted so that the addition of synthetase brings the total volume of the reaction drop to 50/zl and the final reactant concentrations to the desired values. Limiting amounts of synthetase are added. After incubation for 5 rain individual reactions are terminated by placing a 1.5 cm in diameter Whatman 3 MM filter paper disk [previously soaked with 50/A of 10% trichloroacetic acid (TCA), dried, and labeled with a pencil] on each incubation drop. There is a limit to the time that the drops can be incubated on the slide warmer because evaporation becomes a problem after about 10 min. Two minutes after a TCA-soaked disk is placed on a reaction drop the disk is removed and placed in ice-cold 10% TCA until the completion of a set of assays. The disks can remain in this cold TCA for at least 2 hr without affecting the final results. After the completion of a set of assays, all reaction disks are washed together 3 times in 10% TCA (5 ml/disk) and 3 times in 5% TCA (5 ml/disk) allowing 4 rain for each wash. The wash removes unesterified [14C]amino acid. The disks
232
TRANSFER R N A AND AMINOACYL-tRNA SYNTHETASES
[17]
are dried under a heat lamp after two 30-sec ether rinses. The TCAinsoluble radioactivity on each disk, corresponding to [~4C]aminoacyltRNA, is determined by placing the disks in separate 5-ml portions of scintillation fluid (5 g of PPO and 0.3 g of dimethyl POPOP per liter of toluene) and counting. Counts per minute per disk are proportional to synthetase concentration at limiting enzyme concentrations. Growth of CHO Cells and Preparation of Subcellular Fractions A hypodiploid line of CHO cells 7 is grown in Ham's F-10 medium with L-glutamine, without NaHCO3, supplemented with 15% calf sera, 100 units penicillin and 100/~g streptomycin per milliliter. Cells are grown to a density of 4.5 to 5.0 × 105 cells/ml (still in exponential growth with a doubling time of 16-17 hr) and collected with centrifugation in batches of 109 cells (about 2 g of packed cells) from 2 liters of media. The cells are washed once with 0.25 M sucrose, resuspended in 18 ml of 100 mM KCI-10 mM Tris.HC1 (pH 7.5 at 25°)-1.0 mM MgC12-0.1 mM dithiothreitol (buffer A), quick-frozen in a Dry Ice-ethanol bath, and stored at -90 °" About 109 stored cells are thawed, then taken to 20 ml with the addition of 2 ml of 10% NP40 in buffer A. Rupture of~he cells is brought about by mixing for 5 sec followed by occasional short-term mixing over 30 min with intermittent cooling in an ice bath. The disrupted cells are centrifuged at 1000 g for 30 min at 4 °. The pellet, containing intact nuclei (with rim of cytoplasm) and cell debris, is resuspended in 10 ml of 10 mM KCI- 10 mM Tris.HCl (pH 7.5)- 1 mM MgCIz-0.1 mM dithiothreitol40% glycerol (buffer C). This suspension is labeled fraction I. The supernatant fraction is transferred to a centrifuge tube for an IEC A211 rotor, underlaid with 1.5 mi of 42% (w/v) nuclease-free sucrose in buffer A, then centrifuged at 45,000 rpm for 2 hr at 3 °. The pellet, resuspended in 4 ml of buffer C to yield fraction II, represents a microsomal fraction containing polysomes and other material larger than 40 S ribosomal subunits. The supernatant fraction (buffer plus sucrose layer) is mixed, then transferred to another centrifuge tube for the IEC A211 rotor. The contents of this tube are underlaid with 2 ml of 30% (w/v) sucrose in 10 mM KCI-10 mM Tris (pH 7.4 at 250)-1 mM MgClz-0.1 mM dithiothreitol (buffer B), and centrifuged at 45,000 rpm (140,000 gay) for 16 hr at 3 °. The supernatnat fraction is dialyzed for 6 hr against 2 liters of buffer C at 4 ° (fraction IV). The pellet, a particulate, postribosomal fraction, is rinsed with buffer B, then suspended in 1.5 ml of buffer B with the aid 7 j. Tjio and T. T. Puck, J. Exp. Med. 108, 259 (1958).
[17]
AM1NOACYL-tRNA
SYNTHETASES
FROM
CHO
233
CELLS
of a small magnetic stir bar (resultant suspension termed fraction III). Stirring is done in an ice bath, care being taken not to stir rapidly enough to cause foaming. Solution is usually effected after 3 hr. Some synthetase studies have involved further separation of the particulate postribosomal particles in fraction III. In these experiments approximately 2 ml of fraction III, plus a small rinse volume, were layered on a 10 to 30% exponential sucrose (in buffer B) gradient in a 60-ml centrifuge tube for a Spinco SW-25.2 rotor. After centrifugation for 22 hr at 25,000 rpm at 3 °, the gradient was fractionated by pumping concentrated sucrose into the bottom of the tube to force the gradient through a spectrophotometer flow cell. Distribution of Aminoacyl-tRNA Synthetases in the Subcellular Fractions of CHO Cells Q
The distribution of CHO aminoacyl-tRNA synthetase activities in the four major subcellular fractions is summarized in Table I. All the synTABLE
I
RELATIVE SUBCELLULAR DISTRIBUTION OF AMINOACYL-tRNA SYNTHETASES IN C H O CELLS
Amino acid
Fraction IV (supernatant)
Fraction III (postribosomal)
Fraction II (microsomal)
Fraction I (nuclear)
Ala
+ + +
+
-
-
Arg
+ +
+ +
-
-
Asp
+
+ +
+
+
Asn
++
++
-
-
Cys
+ +
+ +
-
-
Glu
-
+ + +
-
-
Glu
-
+ + +
-
-
+
-
Gly
+++
His lle
+ +
Leu
+
+ +
Lys
+
++
Met
-
+++
-
+ + +++
-
-
-
-
-
-
++
+
-
-
Phe
++
++
++
-
Pro
+ +
+ +
+
-
Ser
+ + +
+
-
-
Thr
+ +
+
-
-
++
-
-
Tyr
Trp
+++ + +
+ +
-
-
Val
++
++
-
-
234
TRANSFER R N A AND A M I N O A C Y L - t R N A SYNTHETASES
[18]
thetases appear to have predominant activity in fraction III or fraction IV, although some of the enzymes are distributed throughout all four fractions. The particulate postribosomal aminoacyl-tRNA synthetases (fraction III) can themselves be divided into three classes on the basis of their sedimentation patterns in the 10 to 30% (w/v) exponential sucrose gradients run in buffer B; class I synthetases (those for Ala, Asn, Ser, Trp, and Tyr) with a single activity peak at 6-10 S, class II synthetases (those for Cys, His, Phe, and Val) with a 8-10 S form plus a 15-21 S form, and class III synthetases (those for Asp, Arg, Gln, Glu, Ile, Leu, Lys, Met, Pro, and Thr) with an approximately 30 S form plus, in some cases, one or more slower sedimenting forms.S The sucrose gradients have little or no glycyl-tRNA synthetase activity. Summary
..
Methods have been described that should allow others to readily initiate a study of aminoacyl-tRNA synthetases in CHO cells. These cells represent an excellent model system for the study of the diverse structures and functions of eukaryotic synthetases. Many of the procedures should also prove applicable to other eukaryotic systems. This research was supported by NIH Grant 19506. 8 p. Ritter, M. D. Enger, and A. H a m p e l , in " O n c o - D e v e l o p m e n t a l Gene E x p r e s s i o n " (W. H. F i s h m a n and S. Sell, eds.), p. 47. A c a d e m i c Press, N e w York, 1976.
[18] T r y p t o p h a n y l - t R N A
Synthetase
from Beef Pancreas
By LEV L. KlSSELEV, OL'GA O. FAVOROVA, and GALINA K. KOVALEVA
Isolation of highly purified preparations of various aminoacyl-tRNA synthetases (EC 6.1.1), mainly from unicellular organisms, has been described in a number of publications (see reviews~'Z). As a rule, the purification procedure consists of 6-9 steps and yields 10-30 mg of the enzyme per 1 kg of packed cells. However, various physical and chemical investigations require isolation of considerable amounts of highly purified enzyme preparations by using a convenient and rather simple procedure. 1 L. L. Kisselev and O. O. F a v o r o v a , Adv. Enzymol. 40, 141 (1974). 2 D. $611 and P. R. Schimmel, in - T h e E n z y m e s " (P. Boyer, ed.), 3rd ed., Vol. 10, p. 489. Academic Press, N e w York, 1974.
METHODS IN ENZYMOLOGY, VOL. LIX
Copyright ~) 1979by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-18195%0
AMINOACYL-tRNA SYNTHETASES FROM C H O CELLS
229
this method was 77. These chromatographic patterns serve as a useful reference for determining various tRNA functions in this model mammalian cell culture system. Because of the interspecies similarities between mammalian tRNAs, a model system such as CHO is very useful in studies directed toward understanding nonclassical roles for tRNA. The emergence of CHO as an outstanding somatic cell genetic system adds greatly to its usefulness along these lines. A catalog of tRNA isoacceptors in a defined mammalian system represents an essential first step in the elucidation of possible tRNA involvement in mammalian metabolism and cell regulation.
[17] A m i n o a c y l - t R N A
Synthetases Cells
from Cultured CHO
B y ARNOLD E . H A M P E L , M . D U A N E E N G E R , a n d PRESTON O . RITTER
The role of aminoacyl-tRNA synthetases in protein biosynthesis has been generally recognized for many years. More recently, evidence has accumulated that these enzymes may also play a role in the regulation of cellular metabolism 1and in amino acid transport. 2-4 At the same time the existence of particulate synthetases in eukaryotic systems has become generally accepted.4 Chinese hamster ovary (CHO) cells provide a good model system for more detailed studies on the structure and function of mammalian aminoacyl-tRNA synthetases. This cell line is stable and amenable to controlled physiological and biochemical perturbations. Conditionally lethal synthetase mutants are also available. 5'6 The detailed study of any enzyme is greatly enhanced by the availability of a quick, easy, and reproducible assay. This chapter describes such assays as they have been developed for the analysis of CHO aminoacyltRNA synthetases, then describes a procedure for the isolation of the various forms of these enzymes. This information will be of value to anyone working with eukaryotic aminoacyl-tRNA synthetases. i F. C. Neidhardt, J. Parker, and W. C. McKeever, Annu. Rev. Microbiol. 29, 215 (1975). P. A. Moore, D. W. Jayme; and D. L. Oxender, J. Biol.Chem. 252, 7427 (1977). a S. C. Quay, E. L. Kline, and D. L. Oxender, Proc. Natl. Acad. Sci. U.S.A. 72, 3921 (1975). L. L. Kisselev, and O. O. Favorova, Adv. Enzymol. 40, 141 (1974). L. H. Thompson, J. L. Harkins, and C. P. Stanners, Proc. Natl. Acad. Sci U.S.A. 70, 3094 (1973). 6 L. H. Thompson, D. J. Lofgren, and G. M. Adair, Cell 11, 157 (1977). METHODS IN ENZYMOLOGY,VOL. LIX
Copyright© 1979by AcademicPress, Inc. All rightsof reproductionin any formreserved. 1SBN0-12-181959-0
230
TRANSFER R N A AND A M I N O A C Y L - t R N A SYNTHETASES
[17]
Large-Scale Preparation of Rat Liver tRNA for Synthetase Assays Sixty rats are killed by cervical dislocation. Livers are removed immediately, washed with 0.25 M sucrose at 0 °, then passed through a tissue press into 5 volumes of 0.25 M sucrose at 0 °. The resulting slurry is homogenized with 4 passages through a 2000 rpm motor-driven, PotterElvehjem homogenizer containing a Teflon pestle with a clearance of 0.006-0.009 inches. The homogenate is centrifuged at 8000 g for 10 rain at 4 °. The supernatant is adjusted to l0 mM NaCI and 0.2 mM EDTA pH 7.0 with 50 mM NaCI-1 mM EDTA, to 0.5% sodium dodecyl sulfate (SDS) with 20% SDS, and then one-half volume of 88% redistilled phenol is added. The mixture is stirred for 48 hr at room temperature prior to splitting of the phases with centrifugation at 4000 g for 10 rain. The aqueous phase is retreated with phenol, as just described, two more times. The final aqueous phase is treated with 2 volumes of ethanol overnight at -20 °. The precipitated RNA is collected with centrifugation and redissolved in 10 mM Tris.HCl (pH 7.5 at 25°)-10 mM NaC1. The ethanol precipitation step is repeated 2 times, then the final pellet is dissolved in a minimum volume (about 20 mg of RNA per milliliter) of 10 mM Tris.HCl (pH 7.5 at 25o)- l0 mM NaC1. Ribosomal RNA is precipitated by adding an equal volume of 2 M NaC1 then leaving the mixture at 4 ° for 5 hr. After centrifugation the pellet is redissolved in a minimum volume of the Tris-NaC1 buffer and rRNA is once again precipitated as just described. The supernatant fractions from the two rRNA precipitations are combined, and the RNA, now essentially free of rRNA, is subjected to three consecutive ethanol precipitations to remove the excess NaC1. The final pellet is dissolved in a minimum volume of 200 mM NaC1-10 mM Tris.HC1 (pH 7.5 at 25 °) and collected on a BD-cellulose (Regis Chemical Company) column that had been titrated to pH 7.0 and equilibrated with the same buffer. It is important that the load not exceed 5 mg of RNA per milliliter of column. The loaded column is washed with 2 column volumes of 200 mM NaCI-10 mM Tris.HC1 (pH 7.5) prior to the elution of the tRNA with 1.8 M NaCI in 10% ethanol. The eluted tRNA is subjected to three consecutive ethanol precipitations as previously described. The tRNA is redissolved in 10 mM KC1-10 mM Tris-HCl (pH 7.5)-1 mM MgC12-0.1 mM dithiothreitol between precipitations. The final pellet is lyophilized to dryness, dissolved in the same buffer, and stored at -20 °. This final t R N A preparation serves as an excellent substrate for CHO aminoacyl-tRNA synthetase assays that involve the acylation of tRNA with labeled amino acids. The tRNA will readily accept all 20 protein
[17]
AMINOACYL-tRNA SYNTHETASESFROMCHO CELLS
231
amino acids and is reactive with highly purified, as well as crude, CHO synthetase preparations. Assay of CHO Aminoacyl-tRNA Synthetases Aminoacyl-tRNA synthetase activities are measured by esterifying radioactive amino acids to their cognate tRNAs. Such reactions are known to be sensitive to pH, ATP concentration, Mg 2+ concentration, salt concentration, etc. and different synthetases usually have different optimal reaction conditions. A final reaction mixture containing 50 mM Tris (pH 7.4 at 25°)-15 mM MgC12-0.5 mM EDTA (disodium salt, adjusted to pH 7.5 before being added to reaction mixture)-5 mM ATP (adjusted to pH 7 with NaOH before being added to reaction mixture)0.35 mM CTP (adjusted to pH 7.0 with NaOH before being added to reaction mixture)-0.35 mg/ml rat liver tRNA-18-20 p~M [14C]amino acid gives satisfactory reactions for CHO synthetases corresponding to the amino acids Ala, Arg, Asn, Asp, Gln, Glu, Gly, Ile, Lys, Met, Pro, Set, Thr, Tyr, and Val. A similar mixture containing 2 /zM [14C]Trp is used to assay for Trp-tRNA synthetase. Cys-, His-, Leu-, and Phe-tRNA synthetases are assayed under the originally described conditions except that Tris is at pH 8.6 and MgCI2 at 8 mM. Mechanically, the reactions are carried out by placing small drops (less that 50 p.l) of reaction mixture, minus synthetase, on a Saran Wrap (check to make certain that a drop of H20 forms a tight bead) covered slide warmer at 37 ° for 1 min prior to the addition of synthetase. The volume and concentration of the reaction mixture, minus synthetase, are adjusted so that the addition of synthetase brings the total volume of the reaction drop to 50/zl and the final reactant concentrations to the desired values. Limiting amounts of synthetase are added. After incubation for 5 rain individual reactions are terminated by placing a 1.5 cm in diameter Whatman 3 MM filter paper disk [previously soaked with 50/A of 10% trichloroacetic acid (TCA), dried, and labeled with a pencil] on each incubation drop. There is a limit to the time that the drops can be incubated on the slide warmer because evaporation becomes a problem after about 10 min. Two minutes after a TCA-soaked disk is placed on a reaction drop the disk is removed and placed in ice-cold 10% TCA until the completion of a set of assays. The disks can remain in this cold TCA for at least 2 hr without affecting the final results. After the completion of a set of assays, all reaction disks are washed together 3 times in 10% TCA (5 ml/disk) and 3 times in 5% TCA (5 ml/disk) allowing 4 rain for each wash. The wash removes unesterified [14C]amino acid. The disks
232
TRANSFER R N A AND AMINOACYL-tRNA SYNTHETASES
[17]
are dried under a heat lamp after two 30-sec ether rinses. The TCAinsoluble radioactivity on each disk, corresponding to [~4C]aminoacyltRNA, is determined by placing the disks in separate 5-ml portions of scintillation fluid (5 g of PPO and 0.3 g of dimethyl POPOP per liter of toluene) and counting. Counts per minute per disk are proportional to synthetase concentration at limiting enzyme concentrations. Growth of CHO Cells and Preparation of Subcellular Fractions A hypodiploid line of CHO cells 7 is grown in Ham's F-10 medium with L-glutamine, without NaHCO3, supplemented with 15% calf sera, 100 units penicillin and 100/~g streptomycin per milliliter. Cells are grown to a density of 4.5 to 5.0 × 105 cells/ml (still in exponential growth with a doubling time of 16-17 hr) and collected with centrifugation in batches of 109 cells (about 2 g of packed cells) from 2 liters of media. The cells are washed once with 0.25 M sucrose, resuspended in 18 ml of 100 mM KCI-10 mM Tris.HC1 (pH 7.5 at 25°)-1.0 mM MgC12-0.1 mM dithiothreitol (buffer A), quick-frozen in a Dry Ice-ethanol bath, and stored at -90 °" About 109 stored cells are thawed, then taken to 20 ml with the addition of 2 ml of 10% NP40 in buffer A. Rupture of~he cells is brought about by mixing for 5 sec followed by occasional short-term mixing over 30 min with intermittent cooling in an ice bath. The disrupted cells are centrifuged at 1000 g for 30 min at 4 °. The pellet, containing intact nuclei (with rim of cytoplasm) and cell debris, is resuspended in 10 ml of 10 mM KCI- 10 mM Tris.HCl (pH 7.5)- 1 mM MgCIz-0.1 mM dithiothreitol40% glycerol (buffer C). This suspension is labeled fraction I. The supernatant fraction is transferred to a centrifuge tube for an IEC A211 rotor, underlaid with 1.5 mi of 42% (w/v) nuclease-free sucrose in buffer A, then centrifuged at 45,000 rpm for 2 hr at 3 °. The pellet, resuspended in 4 ml of buffer C to yield fraction II, represents a microsomal fraction containing polysomes and other material larger than 40 S ribosomal subunits. The supernatant fraction (buffer plus sucrose layer) is mixed, then transferred to another centrifuge tube for the IEC A211 rotor. The contents of this tube are underlaid with 2 ml of 30% (w/v) sucrose in 10 mM KCI-10 mM Tris (pH 7.4 at 250)-1 mM MgClz-0.1 mM dithiothreitol (buffer B), and centrifuged at 45,000 rpm (140,000 gay) for 16 hr at 3 °. The supernatnat fraction is dialyzed for 6 hr against 2 liters of buffer C at 4 ° (fraction IV). The pellet, a particulate, postribosomal fraction, is rinsed with buffer B, then suspended in 1.5 ml of buffer B with the aid 7 j. Tjio and T. T. Puck, J. Exp. Med. 108, 259 (1958).
[17]
AM1NOACYL-tRNA
SYNTHETASES
FROM
CHO
233
CELLS
of a small magnetic stir bar (resultant suspension termed fraction III). Stirring is done in an ice bath, care being taken not to stir rapidly enough to cause foaming. Solution is usually effected after 3 hr. Some synthetase studies have involved further separation of the particulate postribosomal particles in fraction III. In these experiments approximately 2 ml of fraction III, plus a small rinse volume, were layered on a 10 to 30% exponential sucrose (in buffer B) gradient in a 60-ml centrifuge tube for a Spinco SW-25.2 rotor. After centrifugation for 22 hr at 25,000 rpm at 3 °, the gradient was fractionated by pumping concentrated sucrose into the bottom of the tube to force the gradient through a spectrophotometer flow cell. Distribution of Aminoacyl-tRNA Synthetases in the Subcellular Fractions of CHO Cells Q
The distribution of CHO aminoacyl-tRNA synthetase activities in the four major subcellular fractions is summarized in Table I. All the synTABLE
I
RELATIVE SUBCELLULAR DISTRIBUTION OF AMINOACYL-tRNA SYNTHETASES IN C H O CELLS
Amino acid
Fraction IV (supernatant)
Fraction III (postribosomal)
Fraction II (microsomal)
Fraction I (nuclear)
Ala
+ + +
+
-
-
Arg
+ +
+ +
-
-
Asp
+
+ +
+
+
Asn
++
++
-
-
Cys
+ +
+ +
-
-
Glu
-
+ + +
-
-
Glu
-
+ + +
-
-
+
-
Gly
+++
His lle
+ +
Leu
+
+ +
Lys
+
++
Met
-
+++
-
+ + +++
-
-
-
-
-
-
++
+
-
-
Phe
++
++
++
-
Pro
+ +
+ +
+
-
Ser
+ + +
+
-
-
Thr
+ +
+
-
-
++
-
-
Tyr
Trp
+++ + +
+ +
-
-
Val
++
++
-
-
234
TRANSFER R N A AND A M I N O A C Y L - t R N A SYNTHETASES
[18]
thetases appear to have predominant activity in fraction III or fraction IV, although some of the enzymes are distributed throughout all four fractions. The particulate postribosomal aminoacyl-tRNA synthetases (fraction III) can themselves be divided into three classes on the basis of their sedimentation patterns in the 10 to 30% (w/v) exponential sucrose gradients run in buffer B; class I synthetases (those for Ala, Asn, Ser, Trp, and Tyr) with a single activity peak at 6-10 S, class II synthetases (those for Cys, His, Phe, and Val) with a 8-10 S form plus a 15-21 S form, and class III synthetases (those for Asp, Arg, Gln, Glu, Ile, Leu, Lys, Met, Pro, and Thr) with an approximately 30 S form plus, in some cases, one or more slower sedimenting forms.S The sucrose gradients have little or no glycyl-tRNA synthetase activity. Summary
..
Methods have been described that should allow others to readily initiate a study of aminoacyl-tRNA synthetases in CHO cells. These cells represent an excellent model system for the study of the diverse structures and functions of eukaryotic synthetases. Many of the procedures should also prove applicable to other eukaryotic systems. This research was supported by NIH Grant 19506. 8 p. Ritter, M. D. Enger, and A. H a m p e l , in " O n c o - D e v e l o p m e n t a l Gene E x p r e s s i o n " (W. H. F i s h m a n and S. Sell, eds.), p. 47. A c a d e m i c Press, N e w York, 1976.
[18] T r y p t o p h a n y l - t R N A
Synthetase
from Beef Pancreas
By LEV L. KlSSELEV, OL'GA O. FAVOROVA, and GALINA K. KOVALEVA
Isolation of highly purified preparations of various aminoacyl-tRNA synthetases (EC 6.1.1), mainly from unicellular organisms, has been described in a number of publications (see reviews~'Z). As a rule, the purification procedure consists of 6-9 steps and yields 10-30 mg of the enzyme per 1 kg of packed cells. However, various physical and chemical investigations require isolation of considerable amounts of highly purified enzyme preparations by using a convenient and rather simple procedure. 1 L. L. Kisselev and O. O. F a v o r o v a , Adv. Enzymol. 40, 141 (1974). 2 D. $611 and P. R. Schimmel, in - T h e E n z y m e s " (P. Boyer, ed.), 3rd ed., Vol. 10, p. 489. Academic Press, N e w York, 1974.
METHODS IN ENZYMOLOGY, VOL. LIX
Copyright ~) 1979by AcademicPress, Inc. All rights of reproduction in any form reserved. ISBN 0-12-18195%0