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Macromolecular complexes of aminoacyl-tRNA synthetases in Drosophila
Insect Biochem., 1976, Vol. 6, pp. 405 to 411. Pergamon Press. Printed in Great Britain.
MACROMOLECULAR COMPLEXES OF AMINOACYL-tRNA SYNTHETASES IN DROSOPHILA STEPHEN J. SHAFER*, STEPHANIEOLEXA, and RALPH HILLMAN Department of Biology, Temple University, Philadelphia, PA 19122, U.S.A. (Received 3 February 1976)
Al~traet--Aminoacyl-tRNA synthetases in Drosophila are present in large macromolecular aggregates having tool. wt in excess of 106 Daltons. After cellular fractionation these enzymes are found in the post-microsomal hard pellet, the post-microsomal soft pellet, and the post-microsomal supernatant fractions. The distribution of specific enzyme activities within these fractions varies and is dependent upon the specific tRNA synthetase being studied.
INTRODUCTION S T t ~ I ~ on the in vitro isolation and characterization of aminoacyl-tRNA synthetases [aminoacyl:tRNA ligases (AMP)] from a variety of organisms have shown that these enzymes are present in a number of enzymatic,ally active 'forms' (reviews in LOr'rFmLD, 1972; KISS~LEV and FAVOROUA, 1974; SOLE and S C U L L , 1974). In organisms at the lower end of the phylogenetic scale (i.e., bacteria, yeast, and slime molds), aminoacyl-tRNA synthetase activity has been isolated specifically from post-microsomal supernatant fractions in the forms of free enzyme and enzyme--substrate complexes. These enzyme-substrate complexes may take the form of specific tRNA's bound to their cognate synthetases, or aminoacyladenylate enzyme complexes. Aminoacyl:tRNA synthetase studies of mammalian and avian systems indicate a second 'form' of the enzyme, one in which synthetase activity is observed in large particles sedimenting with the microsomal pellet with high speed centrifugation. Free enzymes and enzyme-substrate complexes are also found in the post-microsomal supernatant fraction isolated from these systems. There is relatively little known about the molecular characteristics of aminoacyl-tRNA synthetases in Drosophila. Fox et al. (1965) reported that synthetase activity in adults was exclusively associated with the microsomal pellet. On the other hand, CHRISTOPHER et al. (1971) isolated and characterized at least one free enzyme form from the post-microsomal supernatant fraction of Drosophila larvae. A mutant genotype which was an effect on the enzymatic reactions responsible for tRNA aminoacylation in the post-microsomal supernatant fraction of adult flies has also been reported (Ros~ and HILLMAN, 1969). In preparation for a study of this mutant genotype, we have * Present Address: Department of Biology, Dowling College, Oakdale, NY 11769, U.S.A. 405
investigated several aminoacyl:tRNA synthetases from a wild-type (Oregon-R) strain. Extracts from these flies contain free enzyme and enzyme complexes as well as high tool. wt aggregates, possessing ligase activity, associated with the microsomal pellet. The characteristics of these enzyme aggregates vary according to the synthetase being studied. MATERIALS AND METHODS Reagents Tris-(hydroxymethyl) aminoethane was purchased from Sigma Chemical Co.; radioactive amino acids, either 14C or 3H labelled, from ICN and New England Nuclear; ribonuclease-free sucrose from Mann Research Laboratories; Beef liver catalase from Worthington Biochemical Corp.; and, purified E. coli strain B transfer RNA from General Bioehemicals. Reagents for polyacrylamide disc gel electrophoresis were obtained from Canal Industrial Corp. All chemicals were of reagent grade, and glass double distilled water was used in preparing all solutions. Preparation of Drosophila subeellular fractions Gram quantities of adults 0 to 2 days after eclosion (Ore. #on-R strain of Drosophila raelanogaster) were collected according to the procedure of TRAVAGLINIand TARTOF (1972). Whole flies were stored for periods no longer than one month in a liquid nitrogen container before being used. Post-mitochondrial and post-microsomal supernatant and microsomal fractions were prepared according to the method of Rose and H1LLMAN(1969). However, in the present experiments the flies were homogenized with a rotating teflon pestle in a glass tissue grinder vessel. Standard procedure utilized ten up and down strokes per gram of flies homogenized. In experiments comparing the effects of gentle and harsh homogenization, five and twenty strokes per gram of flies were used respectively. In specific experiments, as noted, the microsomal pellet was further subdivided by adding 1-rnl/g-flies-homogenized of homogenizing medium and gently resuspending the loosely packed gray material found above the darker more tightly packed microsomal pellet. The surface of the tightly
406
STEPHEN J. SHAFER, STEPHANIEOLEXA, AND RALPH HILLMAN
packed rnicrosomal pellet was washed several times before being suspended in 1-ml/g-flies-homogenized of homogenization medium. Partially purified synthetases from ammonium sulfate fractions of the post-mitochondrial and post-microsomal supernatant fractions were prepared according to the method of CHRISTOPHER et al. (1971) except as noted. All fractions (except where noted) were dialyzed overnight and tested for aminoacylation activity on the following day. Sonication of microsomal fractions were carried out using a Branson Sonifier Cell Disrupter (Model W185D) with a microtip attachment; a current of 55 W for 30 sec was employed to disrupt the membrane fraction.
Aminoacylation of tRNA In vitro charging was carried out in accordance with the method of ROSE and HILLMAN (1969). The labelled amino acids were neutralized before use by the addition of 0.01 N KOH to the incubation mixture. The tRNA used in the charging reactions was prepared from E. coli, strain B, and used at a concentration of 10 mg/ml.
Sucrose density gradient centrifugation Linear sucrose gradients (5 to 20~) were prepared with ribonuclease-free sucrose dissolved in homogenization buffer and run according to the procedure of VEDEL and D'AouST (1970). A Beckman 65 fixed angle rotor was spun at 35,000 rev/min for 4 hr in a Spinco model L2-65 ultracentrifuge at 4°C. Round bottom, 10 ml, high speed polycarbonate centrifuge tubes with screw caps were utilized. The relative short running time gave good separation and allowed us to quickly characterize nascent ligase activity in crude extracts. To unload the tubes, a 5 lambda micropipet was lowered to the bottom of each tube and the contents removed by means of a peristaltic pump. Fractions were immediately assayed for synthetase activity. Refractive indices of each fraction were measured on a Zeiss Abbe Refractometer-Model A. Molecular weight determinations were made according to the procedure of MARTIN and A~ms (1960) using beef liver catalase (hydrogen peroxide:hydrogen peroxide oxidoreductase, E.C. 1.11.1.6.) as a reference marker. Catalase activity was assayed according to the method of BEERS and SIZER (1952).
Polyacrylamide disc gel electrophoresis Proteins from designated fractions were separated on 4.5% polyacrylamide gels according to the method of
DAVIS (1964) for the pH 9.5 system. After each run the gels were cut with a lateral gel slicer into slices 1.5 mm thick. Each slice was placed in a test tube containing 0.15 ml homogenizing medium and allowed to stand for 90 min. All procedures were carried out in the cold. A 0.10 ml aliquot from each tube was then tested for aminoacylation activity.
Column chromatography Samples prepared for Sephadex column chromatography were homogenized gently in the buffer reported by CHRISTOPHER et al. (1971), but were not subjected to ammonium sulfate fractionation. The only change was the replacement of mercaptoethanol by 2 mM dithiothreitol (DTT). The postmicrosomal supernatant and the loosely packed microsomal pellet were isolated and dialyzed overnight against a buffer containing 20 mM Tris (pH 7.6), 2 mM DTT and 10% glycerol. 5-ml samples from both the supernatant and loosely packed microsomal pellet were placed on 2.3 cm x 50cm G200 columns which had been equilibrated with the dialysis buffer, and 1 ml fractions were eluted from these columns using the dialysis buffer with a flow rate of 8 ml/hr. Void volumes were determined with Blue Dextran and protein determinations were by the method of WARBURG and CHRISTIAN (1941). RESULTS AND CONCLUSIONS
Ligase activity in soluble and insoluble fractions Preliminary evidence for the presence, in Drosoof a m i n o a c y l : t R N A synthetases in large, quickly sedimenting 'aggregates' comes from observations of the distribution of various synthetases within the supernatant and pellet of the post-mitochondrial supernatant fraction after centrifugation at 125,000 g for 90 min (Table 1). The distribution of charging activities for any specific synthetase varies b o t h in accordance with the fraction assayed and with the time of storage of the synthetase preparation. The distributions within the fractions also vary dependent upon the specific synthetase tested. In general, those cnzymes (i.e.: Lysyl and Alanyl-tRNA synthetase) which initially have a high relative activity in the s u p e r n a t a n t fraction lose supernatant activity after
phila,
Table 1. Distribution of total activity/mg protein of specific synthetases in fractions isolated after centrifugation of the post-mitochondrial supernatant fraction from adults 0 to 2 days after emergence. Assays were immediately after dialysis and after storage for 24 hr at 4°C Fraction Synthetase Lysine Alanine Leucine Valine
Time of assay (hr)
Supernatant
Soft pellet (membrane)
Pellet (microsome)
0 24 0 24 0 24 0 24
0.76 0.70 0.715 0.56 0.10 0.20 0.15 0.29
0.14 0.21 0.280 0.43 0.81 0.58 0.80 0.64
0.10 0.09 0.005 0.0l 0.09 0.22 0.05 0.07
Synthetases in Drosophila Table 2. Per cent change in activity of specific synthetases in fractions isolated from the post-mitochondrial supernatant after 'harsh' as opposed to 'gentle' homogenization of flies 0 to 2 day after emergence
407 R Phenylolonyl - t RNA Synt he~Jle
30" 403020-
Per cent activity after 'harsh' homogenization*
1040-
Synthetase Alanine Valine Phenylalanine Leucine Lysine
Supernatant
Soft pellet
I04~ 114Yo 108% 129% 149~o
457o 80~ 75~ 81% 125~o
Pellet 50~ 34% 75% 35~o 28Yo
Activity after 'gentle' homogenization set to 100%.
Ionyl -tRNA Synthgtose
30-
? o_ x
§
20|0 30
! :T e/e~
Volyl- tRNA Syllthetose
2O I0 30
I
2O
storage. Those enzymes which have an initial low supernatant and high soft pellet activity (i.e. Leucyland valyl-tRNA synthetase) show, after storage, an increased relative activity of supernatan.~ enzyme and a decreased relative activity of soft pellet enzyme. These data may be interpreted as a breakdown of enzyme from more dense to less dense aggregates during storage or as a differential loss of enzymatic activity which is dependent upon the density of the molecular aggregations. The data do not distinguish between the two hypotheses. The effects of physical disruption on the distribution of synthetase activities within each of the various fractions has been studied after gentle and harsh homogenization procedures. Differential homogenization has a significant effect on the distribution of synthetase activity (Table 2). Each synthetase shows a definite increase in activity in the supernatant fraction after harsh homogenization. Concomitantly, there is a decrease in activity, except in the case of lysyl-tRNA synthetase, in the pelleted fractions. The percent increase in supernatant activity after harsh homogenization varies for each ligase, with alanyl:tRNA ligase activity showing the lowest increase (4Y/o) and lysyltRNA synthetase activity showing the greatest increase (49~o). This is consistent with the hypothesis that there is differential stability of these enzymes within the 'aggregate(s)'.
Fig. 1. Sucrose density gradient centrifugation. Post-microsomal supernatant fractions were layered on 5 to 20~o isokinetic sucrose gradients and spun at 35,000 rev/min for 4 hr. After spinning, 0.25-ml fractions were collected (bottom unloading) and each fraction assayed for specific synthetase activity (as described in methods). Beef liver catalase was used as a reference marker (0----(3).
Table 3. Per cent of original synthetase activity remaining in the microsomal pellet after three resuspensions of the pellet in homogenizing buffer. Activity given in dis/rain t-RNA-14C charged × 10-3
Sucrose density gradient centrifugation
Activity Synthetase Alanine Valine Phenylalanine Leucine Lysine
Supernatant 341 347 732 354 982
7o
Hard pellet Remaining 10,6 47.4 575 2008 5153
3,0 I 1.9 44.0 85.0 84.0
I0 5O 4O 30 20
I0
,;s 5o FRACTION
Repeated washing of the microsomal pellet obtained after normal homogenization releases ligase activity into the soluble fraction but does not completely remove activity from the hard pellet. The amount released is variable and is dependent upon the particular synthetase being studied (Table 3). These results may be interpreted to mean either that the extent of binding of each ligase to the microsomal aggregate material differs, or that the solubility of each synthetase is different due to a different structural configuration of each synthetase within the complex.
Approximate mol. wt values have been determined for specific synthetases by centrifugation of crude post-mitochondrial extracts, or partially purified ammonium sulfate cuts of these extracts, on 5 to 20~ linear sucrose gradients. Results with crude extracts are seen in Fig. 1. Phenylalanyl:tRNA synthetase has a single peak in the soluble portion of the gradient. A mol. wt of 185,000 has been obtained for the phenylalanine enzyme using beef liver catalase (11.4S, average tool. wt 147,000 (SAMEJIMAand YANG, 1963; KIS~LEV et al., 1967)) as a marker. Leueyl-tRNA synthe-
408
STEPHEN J. SHAFER,STEPHANIEOLEXA,AND RALPH HILLMAN
Table 4. Molecular weight values of aminoacyl:tRNA synthetases determined from sucrose density gradient centrifugation of post-mitochondrial supernatant fluids Peak I Synthetase Alanine Valine Phenylalanine Leucine Lysine
Sedimentation coefficient 5.0 S 6.6 S 9.4 S 6.1 S 7.6 S
Peak II Mol. wt
Sedimentation coefficient
71,000 109,000 185,000 90,000 134,000
7.7 S 9.6 S . . . 10.8 S
tase, the only other enzyme studied which exhibits single peak activity from a crude extract, has a mol. wt of 90,000. Three synthetases (alanyl, valyl, lysyl) exhibit multiple peak activities from crude homogenates. Molecular weight values for all the synthetases studied are summarized in Table 4. Peak I mol. wt values for each of these synthetases agree with values obtained for corresponding aminoacylating enzymes in prokaryotic and other eukaryotic organisms (review by LOFTFIELD,1971). Peak II values for alanyl-, valyl- and lysyl-tRNA synthetases are approximately 70,000 to 80,000 mol. wt higher than values for their corresponding peaks I's. The possibility that Peak II represents 'complexes' of enzyme and tRNA in a molecule ratio of 1:3 or 1"4 can not be ruled out. Ratios of enzyme to tRNA with these values have been observed in complexes isolated from other eukaryotic organisms (LANKS et al., 1971). After further purification by ammonium sulfate precipitation, phenylalanyl and leucyl-tRNA synthetases show no change in sedimentation coefficients in the sucrose density gradients. Peaks I and II for lysyl and valyl-tRNA synthetase remain constant after ammonium sulfate precipitation, but peak III for each of these synthetases is lost.
Disruption of the microsomal pellet The distribution of synthetase activity after disruption of the microsomal pellet by sonification has been examined (Table 5). Total enzymatic activity of the
Peak III Mol. wt
136,000 190,000 . . . . 220,000
Sedimentation coefficient
Mol. wt
-12.3 S
-277,000
. 14.0 S
320,000
. .
.
phenylalanyl and valyl-tRNA synthetases from this pellet are unaffected by the sonication; leucyl and lysyl-tRNA synthetase activities are markedly reduced. In all four cases studied, however, sonification results in a release of synthetase activity from the pellet fraction and a redistribution of this activity into the post 125,000 g supernatant fraction. Sucrose density gradient analyses of sonicated microsomal pellet fractions have been analyzed and the results may be seen in Fig. 2. A major portion of the activity released from the membrane fraction sediments in correspondence with that found in the peak I's of the unsonicated fraction. In the cases of valyl, leucyl, and lysyl-tRNA synthetases additional peaks of activity representing 'aggregates' of high mol. wt are observed. Treatment of the microsomal pellet with sodium deoxycholate (1~ final concentration at 0 to 4°C for 15 min) completely abolishes all phenylalanyl and leucyl-tRNA synthetase activity in the pellet and supernatant fractions. These results agree with those of NORTON et al. (1965) who observed complete inactivation of synthetases associated with chick microsomes when treated in a similar fashion.
Polyacrylamide disc gel electrophoresis The valyl and leucyl-tRNA synthetases of the postmitochondrial and post-microsomal supernatant fractions have been further characterized by polyacrylamide disc gel electrophoresis. Two bands of enzyme
Table 5. Synthetase activity found in the post-microsomal supernatant and microsomal pellet fractions after sonication and centrifugation of the microsomal pellet Activity counts/min 14C/Fraction x 10-3 Synthetase Phenylalanine Valine Leucine Lysine
Before sonication pellet fraction 30.0 28.6 51.6 158
After sonication Supernatant Pellet 10.0 15.3 12.0 22.0
18.0 10.2 10.0 88.1
Total 28.0 25.5 22.0 110.1
S~thetases in Droso ~hila
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Fig. 4. Distribution of aminoacylating enzyme activity for three amino acids in the post-microsomal supernatant fraction after passage .through a Sephadex G200 column, (O----O) total protein. (~-----@) distribution of enzyme activity. (I I) exclusion volume.
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Fig. 3. Polyacrylamide disc gel electrophoresis. Either post-mitochondrial (O. . . . O) or post-mierosomal (~-----@) supernatant fractions were applied to 4.5% polyacrylamide gels and run at a current of 2.5 mA/tube. The gels were cut with a lateral gel slicer into 1.5 mm thick slices, each slice was placed in a tube containing 0.15 ml homogenizing buffer, and was incubated for 90 rain at 0 to 4°C. An aliquot from each tube was then withdrawn and assayed for synthetase activity (as described in methods).
activity have been observed for both synthetases in the post-mitochondrial supernatant fractions (Fig. 3). Band 1 represents a very large 'aggregate' which barely enters the gel, and band 2 is a faster moving fraction which shows high activity. The large aggregate found at the spacer gel-running gel interface is not present when the distribution of activity is determined for the post-microsomal supernatant fraction. These results parallel those obtained from electrophoretic studies of synthetases by VENI,~,OOR and BLOE~DAL (1972). These workers attribute the activity at the interface band to non-specific protein clumping. In our studies, the interface band is correlated with the presence of a large, quickly sedimenting aggregate from the post-mitochondrial supernatant
410
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that the three enzymes represented in Figs. 4 and 5 show differential size distributions in comparisons of the supernatant and pellet fractions. The reason for this is unclear, although it may represent differential stability of these enzymes within the complex.
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DISCUSSION
.10
In Drosophila, as in other eukaryotic organisms, aminoacyl-tRNA synthetases are associated with large 'aggregates' which sediment with the microsomal fraction after high speed centrifugation. The distribution of specific synthetase activities in sub-cellular fractions varies for each amino acid studied. There are several possible explanations for this variability. Either the number of enzyme molecules in each aggregate differs (for example, more leucyl-tRNA synthetases than alanyl-tRNA synthetases are attached to the complex), or the original aggregate size is the same for all synthetases, and the strength of molecular association differs. The results we have observed are consistent with those of investigators who have isolated and characterized synthetases in other eukaryotic organisms. Studies utilizing Drosophila as a test organism however, have distinct advantages over those utilizing other systems. The relative ease of obtaining large numbers of isogenic organisms, the advantages of a well-studied genetic system, and the presence of a wide range of mutants--especially those affecting translational processes of protein synthesis (e.g. Abnormal abdomen)--confer upon Drosophila an advantage not shared by other eukaryotes.
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Acknowledgements--This research was supported by Grant GM 18080 from the U.S. Public Health Service.
FRACTION NUMBER
REFERENCES Fig. 5. Distribution of aminoacylating enzyme activity for three amino acids in the post-microsomal soft pellet fraction after passage through a Sephadex G200 column. (O----O) total protein. ( 0 - - - 0 ) distribution of enzyme activity. (I I) exclusion volume. fraction. Both the band and the aggregate are absent in the post-microsomal supernatant fraction. Column chromatography
The distributions of aminoacylating enzyme activities in elution fractions from a G200 column of both supernatant (Fig. 4) and pelleted (Fig. 5) post-microsomal centrifugates clearly show the multimolecular characteristics of the aminoacylating enzymes. Since the exclusion of the G200 column is approximately 106 Daltons, and since the mol. wt of active enzymes have been shown to be considerably less than 106; those enzymes present in the void volume of both the supernatant and soft pellet fraction must be present as multimolecular complexes. It is of some interest
BEERS R. F. JR. and SIZER I. W. (1952) A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J. bioL Chem. 195, 133-140. CHRISTOPHER C. W., JONES M. E., and STAFFORDD. W. (1971) Phenylalanine tRNA synthetase from Drosophila melanogaster. Biochim. Biophys. Acla 228, 682~87. DAVIS B. J. (1964) Disc Electrophoresis, II; Method and application to human serum proteins. Ann. N.Y. Acad. Sci. 121, 404-412. FOX A, S., KAN J., KANG S. H., and WALLIS R. (1965) Protein synthesis in celt-free preparations from Drosophila melanogaster. J. biol. Chem. 240, 2059-2065. K[SSELEV L. L. and FAVOROVAO. O. (1974) AminoacyltRNA Synthetases: Some Recent Results and Achievements. Adv Enzymology 40, 141-238. KISELEV N. A., SHPITZBERGC. L,, and VAINSHTEINB. K. (1967) Crystallization of catalase in the form of tubes with monomolecular walls. J. molec. Biol. 25, 433-441. LANKS K. W., SCISCENT]J., WEINSTEIN I. B., and CANTOR C. R. (1971) Studies on rat liver phenylalanyl transfer ribonucleic acid synthetase. J. biol. Chem. 246, 3494-3499.
Synthetases in Drosophila LOFTFIELD R. B. (1971) The aminoacylation of transfer ribonucleic acid. In Protein Synthesis (Ed. by McCONKEY E. H.), 1, 1-88. Marcel Dekker, New York. LOFTFIELDR. B. (1972) The mechanism of aminoacylation of transfer RNA. In Progress in Nucleic Acid Research and Molecular Biology (Ed. by DAVlDSON J. N. and COnN W. E.), 12, 87-128. Academic Press, New York. M~,~aa~q R. G. and AM~SB. N. (1960) A method for determining the sedimentation behavior of enzymes: Application to protein mixtures. J. biol. Chem. 236, 1372-1379. NORTON S. J., KEY M. D., and SCHOLeSS. W. (1965) Some studies of the association of amino acid-activating enzymes with isolated microsomes of chick embryo. Arch. Biochem. Biophys. 109, 7-12. Rose R. and HILLMANR. (1969) In vitro studies of protein synthesis in Drosophila. Biochem. Biophys. Res. Comm. 35, 197-204.
411
SAMmlUthT. and YANG J. T. (1963) Reeonstitution of aciddenatured catalase. J. biol. Chem. 238, 3256-3261. SOLED. and SCmMMELP. R. (1974) Aminoacyl-tRNA Synthetases. In The Enzymes (Ed. by BOXES P. D.), 3rd ed. pp. 489-538. Academic Press, New York. TRAVAGLINIE. C. and TARTOF D. (1972) 'Instant' Drosophila: A method for mass culturing large numbers of Drosophila. Drosophila Inf. Serv. 48, 157. VEDEL F. and D'AousT M. J. (1970) Rapid separation of ribosomal RNA by sucrose density gradient centrifugation with a fixed-angle rotor. Anal. Biochem. 35, 54-59. VEN~-~GOORC. and BLOEMEr~OALH. (1972) Occurrence and particle character of aminoacyl-tRNA synthetases in the postmicrosomal fraction from rat liver. European J. Bigchem. 26, 462-473. WAgBtrgG D. and CHgXSTIANW. (1941) Isolierung und Kristallisation des G/irungsferments Enolase. Biochem. Z. 310, 384.
MACROMOLECULAR COMPLEXES OF AMINOACYL-tRNA SYNTHETASES IN DROSOPHILA STEPHEN J. SHAFER*, STEPHANIEOLEXA, and RALPH HILLMAN Department of Biology, Temple University, Philadelphia, PA 19122, U.S.A. (Received 3 February 1976)
Al~traet--Aminoacyl-tRNA synthetases in Drosophila are present in large macromolecular aggregates having tool. wt in excess of 106 Daltons. After cellular fractionation these enzymes are found in the post-microsomal hard pellet, the post-microsomal soft pellet, and the post-microsomal supernatant fractions. The distribution of specific enzyme activities within these fractions varies and is dependent upon the specific tRNA synthetase being studied.
INTRODUCTION S T t ~ I ~ on the in vitro isolation and characterization of aminoacyl-tRNA synthetases [aminoacyl:tRNA ligases (AMP)] from a variety of organisms have shown that these enzymes are present in a number of enzymatic,ally active 'forms' (reviews in LOr'rFmLD, 1972; KISS~LEV and FAVOROUA, 1974; SOLE and S C U L L , 1974). In organisms at the lower end of the phylogenetic scale (i.e., bacteria, yeast, and slime molds), aminoacyl-tRNA synthetase activity has been isolated specifically from post-microsomal supernatant fractions in the forms of free enzyme and enzyme--substrate complexes. These enzyme-substrate complexes may take the form of specific tRNA's bound to their cognate synthetases, or aminoacyladenylate enzyme complexes. Aminoacyl:tRNA synthetase studies of mammalian and avian systems indicate a second 'form' of the enzyme, one in which synthetase activity is observed in large particles sedimenting with the microsomal pellet with high speed centrifugation. Free enzymes and enzyme-substrate complexes are also found in the post-microsomal supernatant fraction isolated from these systems. There is relatively little known about the molecular characteristics of aminoacyl-tRNA synthetases in Drosophila. Fox et al. (1965) reported that synthetase activity in adults was exclusively associated with the microsomal pellet. On the other hand, CHRISTOPHER et al. (1971) isolated and characterized at least one free enzyme form from the post-microsomal supernatant fraction of Drosophila larvae. A mutant genotype which was an effect on the enzymatic reactions responsible for tRNA aminoacylation in the post-microsomal supernatant fraction of adult flies has also been reported (Ros~ and HILLMAN, 1969). In preparation for a study of this mutant genotype, we have * Present Address: Department of Biology, Dowling College, Oakdale, NY 11769, U.S.A. 405
investigated several aminoacyl:tRNA synthetases from a wild-type (Oregon-R) strain. Extracts from these flies contain free enzyme and enzyme complexes as well as high tool. wt aggregates, possessing ligase activity, associated with the microsomal pellet. The characteristics of these enzyme aggregates vary according to the synthetase being studied. MATERIALS AND METHODS Reagents Tris-(hydroxymethyl) aminoethane was purchased from Sigma Chemical Co.; radioactive amino acids, either 14C or 3H labelled, from ICN and New England Nuclear; ribonuclease-free sucrose from Mann Research Laboratories; Beef liver catalase from Worthington Biochemical Corp.; and, purified E. coli strain B transfer RNA from General Bioehemicals. Reagents for polyacrylamide disc gel electrophoresis were obtained from Canal Industrial Corp. All chemicals were of reagent grade, and glass double distilled water was used in preparing all solutions. Preparation of Drosophila subeellular fractions Gram quantities of adults 0 to 2 days after eclosion (Ore. #on-R strain of Drosophila raelanogaster) were collected according to the procedure of TRAVAGLINIand TARTOF (1972). Whole flies were stored for periods no longer than one month in a liquid nitrogen container before being used. Post-mitochondrial and post-microsomal supernatant and microsomal fractions were prepared according to the method of Rose and H1LLMAN(1969). However, in the present experiments the flies were homogenized with a rotating teflon pestle in a glass tissue grinder vessel. Standard procedure utilized ten up and down strokes per gram of flies homogenized. In experiments comparing the effects of gentle and harsh homogenization, five and twenty strokes per gram of flies were used respectively. In specific experiments, as noted, the microsomal pellet was further subdivided by adding 1-rnl/g-flies-homogenized of homogenizing medium and gently resuspending the loosely packed gray material found above the darker more tightly packed microsomal pellet. The surface of the tightly
406
STEPHEN J. SHAFER, STEPHANIEOLEXA, AND RALPH HILLMAN
packed rnicrosomal pellet was washed several times before being suspended in 1-ml/g-flies-homogenized of homogenization medium. Partially purified synthetases from ammonium sulfate fractions of the post-mitochondrial and post-microsomal supernatant fractions were prepared according to the method of CHRISTOPHER et al. (1971) except as noted. All fractions (except where noted) were dialyzed overnight and tested for aminoacylation activity on the following day. Sonication of microsomal fractions were carried out using a Branson Sonifier Cell Disrupter (Model W185D) with a microtip attachment; a current of 55 W for 30 sec was employed to disrupt the membrane fraction.
Aminoacylation of tRNA In vitro charging was carried out in accordance with the method of ROSE and HILLMAN (1969). The labelled amino acids were neutralized before use by the addition of 0.01 N KOH to the incubation mixture. The tRNA used in the charging reactions was prepared from E. coli, strain B, and used at a concentration of 10 mg/ml.
Sucrose density gradient centrifugation Linear sucrose gradients (5 to 20~) were prepared with ribonuclease-free sucrose dissolved in homogenization buffer and run according to the procedure of VEDEL and D'AouST (1970). A Beckman 65 fixed angle rotor was spun at 35,000 rev/min for 4 hr in a Spinco model L2-65 ultracentrifuge at 4°C. Round bottom, 10 ml, high speed polycarbonate centrifuge tubes with screw caps were utilized. The relative short running time gave good separation and allowed us to quickly characterize nascent ligase activity in crude extracts. To unload the tubes, a 5 lambda micropipet was lowered to the bottom of each tube and the contents removed by means of a peristaltic pump. Fractions were immediately assayed for synthetase activity. Refractive indices of each fraction were measured on a Zeiss Abbe Refractometer-Model A. Molecular weight determinations were made according to the procedure of MARTIN and A~ms (1960) using beef liver catalase (hydrogen peroxide:hydrogen peroxide oxidoreductase, E.C. 1.11.1.6.) as a reference marker. Catalase activity was assayed according to the method of BEERS and SIZER (1952).
Polyacrylamide disc gel electrophoresis Proteins from designated fractions were separated on 4.5% polyacrylamide gels according to the method of
DAVIS (1964) for the pH 9.5 system. After each run the gels were cut with a lateral gel slicer into slices 1.5 mm thick. Each slice was placed in a test tube containing 0.15 ml homogenizing medium and allowed to stand for 90 min. All procedures were carried out in the cold. A 0.10 ml aliquot from each tube was then tested for aminoacylation activity.
Column chromatography Samples prepared for Sephadex column chromatography were homogenized gently in the buffer reported by CHRISTOPHER et al. (1971), but were not subjected to ammonium sulfate fractionation. The only change was the replacement of mercaptoethanol by 2 mM dithiothreitol (DTT). The postmicrosomal supernatant and the loosely packed microsomal pellet were isolated and dialyzed overnight against a buffer containing 20 mM Tris (pH 7.6), 2 mM DTT and 10% glycerol. 5-ml samples from both the supernatant and loosely packed microsomal pellet were placed on 2.3 cm x 50cm G200 columns which had been equilibrated with the dialysis buffer, and 1 ml fractions were eluted from these columns using the dialysis buffer with a flow rate of 8 ml/hr. Void volumes were determined with Blue Dextran and protein determinations were by the method of WARBURG and CHRISTIAN (1941). RESULTS AND CONCLUSIONS
Ligase activity in soluble and insoluble fractions Preliminary evidence for the presence, in Drosoof a m i n o a c y l : t R N A synthetases in large, quickly sedimenting 'aggregates' comes from observations of the distribution of various synthetases within the supernatant and pellet of the post-mitochondrial supernatant fraction after centrifugation at 125,000 g for 90 min (Table 1). The distribution of charging activities for any specific synthetase varies b o t h in accordance with the fraction assayed and with the time of storage of the synthetase preparation. The distributions within the fractions also vary dependent upon the specific synthetase tested. In general, those cnzymes (i.e.: Lysyl and Alanyl-tRNA synthetase) which initially have a high relative activity in the s u p e r n a t a n t fraction lose supernatant activity after
phila,
Table 1. Distribution of total activity/mg protein of specific synthetases in fractions isolated after centrifugation of the post-mitochondrial supernatant fraction from adults 0 to 2 days after emergence. Assays were immediately after dialysis and after storage for 24 hr at 4°C Fraction Synthetase Lysine Alanine Leucine Valine
Time of assay (hr)
Supernatant
Soft pellet (membrane)
Pellet (microsome)
0 24 0 24 0 24 0 24
0.76 0.70 0.715 0.56 0.10 0.20 0.15 0.29
0.14 0.21 0.280 0.43 0.81 0.58 0.80 0.64
0.10 0.09 0.005 0.0l 0.09 0.22 0.05 0.07
Synthetases in Drosophila Table 2. Per cent change in activity of specific synthetases in fractions isolated from the post-mitochondrial supernatant after 'harsh' as opposed to 'gentle' homogenization of flies 0 to 2 day after emergence
407 R Phenylolonyl - t RNA Synt he~Jle
30" 403020-
Per cent activity after 'harsh' homogenization*
1040-
Synthetase Alanine Valine Phenylalanine Leucine Lysine
Supernatant
Soft pellet
I04~ 114Yo 108% 129% 149~o
457o 80~ 75~ 81% 125~o
Pellet 50~ 34% 75% 35~o 28Yo
Activity after 'gentle' homogenization set to 100%.
Ionyl -tRNA Synthgtose
30-
? o_ x
§
20|0 30
! :T e/e~
Volyl- tRNA Syllthetose
2O I0 30
I
2O
storage. Those enzymes which have an initial low supernatant and high soft pellet activity (i.e. Leucyland valyl-tRNA synthetase) show, after storage, an increased relative activity of supernatan.~ enzyme and a decreased relative activity of soft pellet enzyme. These data may be interpreted as a breakdown of enzyme from more dense to less dense aggregates during storage or as a differential loss of enzymatic activity which is dependent upon the density of the molecular aggregations. The data do not distinguish between the two hypotheses. The effects of physical disruption on the distribution of synthetase activities within each of the various fractions has been studied after gentle and harsh homogenization procedures. Differential homogenization has a significant effect on the distribution of synthetase activity (Table 2). Each synthetase shows a definite increase in activity in the supernatant fraction after harsh homogenization. Concomitantly, there is a decrease in activity, except in the case of lysyl-tRNA synthetase, in the pelleted fractions. The percent increase in supernatant activity after harsh homogenization varies for each ligase, with alanyl:tRNA ligase activity showing the lowest increase (4Y/o) and lysyltRNA synthetase activity showing the greatest increase (49~o). This is consistent with the hypothesis that there is differential stability of these enzymes within the 'aggregate(s)'.
Fig. 1. Sucrose density gradient centrifugation. Post-microsomal supernatant fractions were layered on 5 to 20~o isokinetic sucrose gradients and spun at 35,000 rev/min for 4 hr. After spinning, 0.25-ml fractions were collected (bottom unloading) and each fraction assayed for specific synthetase activity (as described in methods). Beef liver catalase was used as a reference marker (0----(3).
Table 3. Per cent of original synthetase activity remaining in the microsomal pellet after three resuspensions of the pellet in homogenizing buffer. Activity given in dis/rain t-RNA-14C charged × 10-3
Sucrose density gradient centrifugation
Activity Synthetase Alanine Valine Phenylalanine Leucine Lysine
Supernatant 341 347 732 354 982
7o
Hard pellet Remaining 10,6 47.4 575 2008 5153
3,0 I 1.9 44.0 85.0 84.0
I0 5O 4O 30 20
I0
,;s 5o FRACTION
Repeated washing of the microsomal pellet obtained after normal homogenization releases ligase activity into the soluble fraction but does not completely remove activity from the hard pellet. The amount released is variable and is dependent upon the particular synthetase being studied (Table 3). These results may be interpreted to mean either that the extent of binding of each ligase to the microsomal aggregate material differs, or that the solubility of each synthetase is different due to a different structural configuration of each synthetase within the complex.
Approximate mol. wt values have been determined for specific synthetases by centrifugation of crude post-mitochondrial extracts, or partially purified ammonium sulfate cuts of these extracts, on 5 to 20~ linear sucrose gradients. Results with crude extracts are seen in Fig. 1. Phenylalanyl:tRNA synthetase has a single peak in the soluble portion of the gradient. A mol. wt of 185,000 has been obtained for the phenylalanine enzyme using beef liver catalase (11.4S, average tool. wt 147,000 (SAMEJIMAand YANG, 1963; KIS~LEV et al., 1967)) as a marker. Leueyl-tRNA synthe-
408
STEPHEN J. SHAFER,STEPHANIEOLEXA,AND RALPH HILLMAN
Table 4. Molecular weight values of aminoacyl:tRNA synthetases determined from sucrose density gradient centrifugation of post-mitochondrial supernatant fluids Peak I Synthetase Alanine Valine Phenylalanine Leucine Lysine
Sedimentation coefficient 5.0 S 6.6 S 9.4 S 6.1 S 7.6 S
Peak II Mol. wt
Sedimentation coefficient
71,000 109,000 185,000 90,000 134,000
7.7 S 9.6 S . . . 10.8 S
tase, the only other enzyme studied which exhibits single peak activity from a crude extract, has a mol. wt of 90,000. Three synthetases (alanyl, valyl, lysyl) exhibit multiple peak activities from crude homogenates. Molecular weight values for all the synthetases studied are summarized in Table 4. Peak I mol. wt values for each of these synthetases agree with values obtained for corresponding aminoacylating enzymes in prokaryotic and other eukaryotic organisms (review by LOFTFIELD,1971). Peak II values for alanyl-, valyl- and lysyl-tRNA synthetases are approximately 70,000 to 80,000 mol. wt higher than values for their corresponding peaks I's. The possibility that Peak II represents 'complexes' of enzyme and tRNA in a molecule ratio of 1:3 or 1"4 can not be ruled out. Ratios of enzyme to tRNA with these values have been observed in complexes isolated from other eukaryotic organisms (LANKS et al., 1971). After further purification by ammonium sulfate precipitation, phenylalanyl and leucyl-tRNA synthetases show no change in sedimentation coefficients in the sucrose density gradients. Peaks I and II for lysyl and valyl-tRNA synthetase remain constant after ammonium sulfate precipitation, but peak III for each of these synthetases is lost.
Disruption of the microsomal pellet The distribution of synthetase activity after disruption of the microsomal pellet by sonification has been examined (Table 5). Total enzymatic activity of the
Peak III Mol. wt
136,000 190,000 . . . . 220,000
Sedimentation coefficient
Mol. wt
-12.3 S
-277,000
. 14.0 S
320,000
. .
.
phenylalanyl and valyl-tRNA synthetases from this pellet are unaffected by the sonication; leucyl and lysyl-tRNA synthetase activities are markedly reduced. In all four cases studied, however, sonification results in a release of synthetase activity from the pellet fraction and a redistribution of this activity into the post 125,000 g supernatant fraction. Sucrose density gradient analyses of sonicated microsomal pellet fractions have been analyzed and the results may be seen in Fig. 2. A major portion of the activity released from the membrane fraction sediments in correspondence with that found in the peak I's of the unsonicated fraction. In the cases of valyl, leucyl, and lysyl-tRNA synthetases additional peaks of activity representing 'aggregates' of high mol. wt are observed. Treatment of the microsomal pellet with sodium deoxycholate (1~ final concentration at 0 to 4°C for 15 min) completely abolishes all phenylalanyl and leucyl-tRNA synthetase activity in the pellet and supernatant fractions. These results agree with those of NORTON et al. (1965) who observed complete inactivation of synthetases associated with chick microsomes when treated in a similar fashion.
Polyacrylamide disc gel electrophoresis The valyl and leucyl-tRNA synthetases of the postmitochondrial and post-microsomal supernatant fractions have been further characterized by polyacrylamide disc gel electrophoresis. Two bands of enzyme
Table 5. Synthetase activity found in the post-microsomal supernatant and microsomal pellet fractions after sonication and centrifugation of the microsomal pellet Activity counts/min 14C/Fraction x 10-3 Synthetase Phenylalanine Valine Leucine Lysine
Before sonication pellet fraction 30.0 28.6 51.6 158
After sonication Supernatant Pellet 10.0 15.3 12.0 22.0
18.0 10.2 10.0 88.1
Total 28.0 25.5 22.0 110.1
S~thetases in Droso ~hila
30
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Fig. 2. Sucrose density gradient centrifugation. Microsomal pellet fractions were sonicated (as described in methods), layered on 5 to 20% isokinetic sucrose gradients and spun at 35,000 rev/min for 4 hr. After spinning, 0.25 ml fractions were collected (bottom unloading) and resuspended in 0.25 ml of standard homogenizing buffer to which was added enough sucrose to make a 7.5% solution. Each fraction was then assayed for specific synthetase activity (as described in methods). 76"5. i
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Fig. 4. Distribution of aminoacylating enzyme activity for three amino acids in the post-microsomal supernatant fraction after passage .through a Sephadex G200 column, (O----O) total protein. (~-----@) distribution of enzyme activity. (I I) exclusion volume.
0
::I ORIGIN
80
SLICE •
TRACKING DYE
Fig. 3. Polyacrylamide disc gel electrophoresis. Either post-mitochondrial (O. . . . O) or post-mierosomal (~-----@) supernatant fractions were applied to 4.5% polyacrylamide gels and run at a current of 2.5 mA/tube. The gels were cut with a lateral gel slicer into 1.5 mm thick slices, each slice was placed in a tube containing 0.15 ml homogenizing buffer, and was incubated for 90 rain at 0 to 4°C. An aliquot from each tube was then withdrawn and assayed for synthetase activity (as described in methods).
activity have been observed for both synthetases in the post-mitochondrial supernatant fractions (Fig. 3). Band 1 represents a very large 'aggregate' which barely enters the gel, and band 2 is a faster moving fraction which shows high activity. The large aggregate found at the spacer gel-running gel interface is not present when the distribution of activity is determined for the post-microsomal supernatant fraction. These results parallel those obtained from electrophoretic studies of synthetases by VENI,~,OOR and BLOE~DAL (1972). These workers attribute the activity at the interface band to non-specific protein clumping. In our studies, the interface band is correlated with the presence of a large, quickly sedimenting aggregate from the post-mitochondrial supernatant
410
STEPHEN J. SHAFER, STEPHANIE OLEXA, AND RALPH HILLMAN 1
i
1
I
~
0.60.5 0.4 0.3-
0.2 ~
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that the three enzymes represented in Figs. 4 and 5 show differential size distributions in comparisons of the supernatant and pellet fractions. The reason for this is unclear, although it may represent differential stability of these enzymes within the complex.
I
-30 HENYLALANINE
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DISCUSSION
.10
In Drosophila, as in other eukaryotic organisms, aminoacyl-tRNA synthetases are associated with large 'aggregates' which sediment with the microsomal fraction after high speed centrifugation. The distribution of specific synthetase activities in sub-cellular fractions varies for each amino acid studied. There are several possible explanations for this variability. Either the number of enzyme molecules in each aggregate differs (for example, more leucyl-tRNA synthetases than alanyl-tRNA synthetases are attached to the complex), or the original aggregate size is the same for all synthetases, and the strength of molecular association differs. The results we have observed are consistent with those of investigators who have isolated and characterized synthetases in other eukaryotic organisms. Studies utilizing Drosophila as a test organism however, have distinct advantages over those utilizing other systems. The relative ease of obtaining large numbers of isogenic organisms, the advantages of a well-studied genetic system, and the presence of a wide range of mutants--especially those affecting translational processes of protein synthesis (e.g. Abnormal abdomen)--confer upon Drosophila an advantage not shared by other eukaryotes.
5
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J 150
Acknowledgements--This research was supported by Grant GM 18080 from the U.S. Public Health Service.
FRACTION NUMBER
REFERENCES Fig. 5. Distribution of aminoacylating enzyme activity for three amino acids in the post-microsomal soft pellet fraction after passage through a Sephadex G200 column. (O----O) total protein. ( 0 - - - 0 ) distribution of enzyme activity. (I I) exclusion volume. fraction. Both the band and the aggregate are absent in the post-microsomal supernatant fraction. Column chromatography
The distributions of aminoacylating enzyme activities in elution fractions from a G200 column of both supernatant (Fig. 4) and pelleted (Fig. 5) post-microsomal centrifugates clearly show the multimolecular characteristics of the aminoacylating enzymes. Since the exclusion of the G200 column is approximately 106 Daltons, and since the mol. wt of active enzymes have been shown to be considerably less than 106; those enzymes present in the void volume of both the supernatant and soft pellet fraction must be present as multimolecular complexes. It is of some interest
BEERS R. F. JR. and SIZER I. W. (1952) A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J. bioL Chem. 195, 133-140. CHRISTOPHER C. W., JONES M. E., and STAFFORDD. W. (1971) Phenylalanine tRNA synthetase from Drosophila melanogaster. Biochim. Biophys. Acla 228, 682~87. DAVIS B. J. (1964) Disc Electrophoresis, II; Method and application to human serum proteins. Ann. N.Y. Acad. Sci. 121, 404-412. FOX A, S., KAN J., KANG S. H., and WALLIS R. (1965) Protein synthesis in celt-free preparations from Drosophila melanogaster. J. biol. Chem. 240, 2059-2065. K[SSELEV L. L. and FAVOROVAO. O. (1974) AminoacyltRNA Synthetases: Some Recent Results and Achievements. Adv Enzymology 40, 141-238. KISELEV N. A., SHPITZBERGC. L,, and VAINSHTEINB. K. (1967) Crystallization of catalase in the form of tubes with monomolecular walls. J. molec. Biol. 25, 433-441. LANKS K. W., SCISCENT]J., WEINSTEIN I. B., and CANTOR C. R. (1971) Studies on rat liver phenylalanyl transfer ribonucleic acid synthetase. J. biol. Chem. 246, 3494-3499.
Synthetases in Drosophila LOFTFIELD R. B. (1971) The aminoacylation of transfer ribonucleic acid. In Protein Synthesis (Ed. by McCONKEY E. H.), 1, 1-88. Marcel Dekker, New York. LOFTFIELDR. B. (1972) The mechanism of aminoacylation of transfer RNA. In Progress in Nucleic Acid Research and Molecular Biology (Ed. by DAVlDSON J. N. and COnN W. E.), 12, 87-128. Academic Press, New York. M~,~aa~q R. G. and AM~SB. N. (1960) A method for determining the sedimentation behavior of enzymes: Application to protein mixtures. J. biol. Chem. 236, 1372-1379. NORTON S. J., KEY M. D., and SCHOLeSS. W. (1965) Some studies of the association of amino acid-activating enzymes with isolated microsomes of chick embryo. Arch. Biochem. Biophys. 109, 7-12. Rose R. and HILLMANR. (1969) In vitro studies of protein synthesis in Drosophila. Biochem. Biophys. Res. Comm. 35, 197-204.
411
SAMmlUthT. and YANG J. T. (1963) Reeonstitution of aciddenatured catalase. J. biol. Chem. 238, 3256-3261. SOLED. and SCmMMELP. R. (1974) Aminoacyl-tRNA Synthetases. In The Enzymes (Ed. by BOXES P. D.), 3rd ed. pp. 489-538. Academic Press, New York. TRAVAGLINIE. C. and TARTOF D. (1972) 'Instant' Drosophila: A method for mass culturing large numbers of Drosophila. Drosophila Inf. Serv. 48, 157. VEDEL F. and D'AousT M. J. (1970) Rapid separation of ribosomal RNA by sucrose density gradient centrifugation with a fixed-angle rotor. Anal. Biochem. 35, 54-59. VEN~-~GOORC. and BLOEMEr~OALH. (1972) Occurrence and particle character of aminoacyl-tRNA synthetases in the postmicrosomal fraction from rat liver. European J. Bigchem. 26, 462-473. WAgBtrgG D. and CHgXSTIANW. (1941) Isolierung und Kristallisation des G/irungsferments Enolase. Biochem. Z. 310, 384.