ANALYTICAL
BIOCHEMISTRY
82,
29-37 (1977)
The Determination of Aminoacyl Adenylate Thin-Layer Chromatography’ HIERONIM
by
Z.JAKUBOWSKI,~ ANDRZEJ PASTUZYN, AND ROBERT B. LOFTFIELD
Department of Biochemistry, University of New Mexico, Albuquerque, New Mexico 87131 Received November 23. 1976; accepted April 25, 1977 A rapid and convenient method is presented by which one may determine the extent of formation of Enz.(AA - AMP) in the presence of isotopic amino acid, isotopic nucleotides, tRNA, enzyme, etc. Separation of the components of the reaction mixture is achieved by chromatography on tic cellulose. Aminoacyl adenylate separates from other solutes and is characterized by its chemical and enzymatic reactions. The method may be used to determine the equilibrium constant for the synthesis of Enz.(AA - AMP) which is very sensitive to salt concentration.
As early as 195.5, aminoacyl adenylates were proposed as intermediates in the activation of amino acids for protein biosynthesis (1,2). Aminoacyl adenylates have been synthesized chemically and enzymatically and have the properties that would be expected of intermediates in the formation of aminoacyl-tRNAs (3-6). Several assay methods have been developed for estimation of the amount of enzyme-bound aminoacyl adenylate in a reaction mixture (8- 11). We describe an assay that permits the estimation of total aminoacyl adenylate in the presence of amino acids, aminoacyl-tRNA, ATP. etc. MATERIALS
AND METHODS
Amino acid:tRNA ligases specific for isoleucine, valine, leucine, and phenylalanine were prepared from Escherichia co/i (12). The enzymes were estimated to be 40-50% pure by Sephadex isolation of Enz. [14C]-Ile - AMP under saturating conditions (85% pure as estimated by gel electrophoresis). The tRNA”” was about 90% pure (1700 pmoYOD,,,J. 14C-labeled amino acids and [14C]ATP were purchased from New England Nuclear Corporation and AmershamKearle and were used at maximal specific ’ Supported by Grant Np 103 from the American Cancer Society and by Grant CA 08000 from the United States Public Health Service. * Permanent address: Institute of Biochemistry, Agricultural University, Wolynska 35. Poznan 60-637, Poland. 29 Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN 0003-2697
30
JAKUBOWSKI,
PASTUSZYN,
AND LOFTFIELD
activities (280-556 mCi/mmol). Precoated tic plates on transparent plastic supports were purchased from Brinkman (cellulose, 0.1 mm; silica gel, 0.2 mm) or from Eastman (cellulose, 0.16 mm). All other chemicals and solvents were commercial products of reagent quality. Enzyme-bound aminoacyl adenylate was formed by reacting 20-30 PM 14C-labeled amino acid, 1 mM ATP, 10 mM MgCl,, and ca. l-2 PM ligase for 5 min at 15°C in 100 ~1 of 0.1 M Tris, pH 7.4, containing 1 mM mercaptoethanol and 0.2% bovine serum albumin. The mixture was applied to the top of a 0.5 x 16-cm column of Sephadex G-100 that had been equilibrated at 4°C with 10 mM Tris, pH 7.4, containng 10 mM mercaptoethanol. Elution with the Tris buffer yielded all of the enzyme-bound aminoacyl adenylate in less than 200 ~1, well separated from the unreacted free amino acid (11). For tic chromatography, aliquots of 3 or 4 ~1 are applied to the origin of 20-cm plates, occasionally using warm air to accelerate drying. Development is always with 4 butanol: 1 acetic acid: 1 water (13). The solvent rises through 8 cm of cellulose in 60 min at 21°C or in 90 min at 4°C. Development of the silica gel plates is slower: 2 hr at 21” C and 3 hr at 4°C. Some Eastman cellulose plates were satisfactory, but more recent purchases containing an improved binder develop so slowly with this solvent that they cannot be used. The tic plates are dried, cut into l- or 0.5-cm strips, and assayed for radioactivity by immersion in scintillation cocktail (0.05% POPOP:O.5% PPO:toluene) followed by counting in a scintillation spectrophotometer at, room temperature. The efficiency of counting 14C-labeled compounds on the tic plates was 57-60% and the backgrounds were between 30 and 55 cpm. Cellulose plates have an advantage over silica gel plates in that the 14C-labeled substances are eluted from the silica gel into the toluene. This introduces some variability in the counting and precludes the use of the scintillation cocktail for more than a single assay. RESULTS
Figure 1 shows the behavior of enzyme-bound [14C]isoleucyl adenylate chromatographed on tic cellulose at 4°C with butanol/acetic acid/water. Essentially, no radioactivity remains at the origin, while 90-95% of the isotope is found in a symmetrical peak about 2 cm from the origin. As shown in Fig. 1, this peak is well separated from free isoleucine as well as from other components of the reaction mixture. The results for the phenylalanine, leutine, and valine systems are similar except for slight changes in the migration, as shown in Table 1. If silica gel tic plates are used, there tends to be somewhat greater decomposition of aminoacyl adenylate to amino acid. Similarly, if cellulose plates are developed at 21°C phenylalanyl adenylate and leucyl adenylate partially decompose. In other respects, silica gel plates and development at 4 or 21°C give comparable results.
DETERMINATION
OF AMINOACYL
31
ADENYLATE
AMP
CPM. 5oo-
ATP
-
ISOLEUCINE
-RNA 400
-
PROTEIN -
L0
300-
ZOO-
loo-
, .I 0
i
,\.
II -\r
I
2
,
,._,_
5
6
_,
,,c=--1. 3 4 CENTIMETERS
7
8
FIG. 1. tic chromatography of enzyme. isoleucyl adenylate complex. An aliquot of 4 ~1 of Sephadex G-50 (fine)-purified isoleucine:tRNA ligase.isoleucyl adenylate complex was applied to the origin of a cellulose tic plate and dried immediately. The chromatogram was developed with 4 butanol: 1 acetic acid: 1 water at 0°C. Strips of the tic plate (0.5 cm) were cut and radioassayed by immersion in scintillation counting liquid. The location of protein was determined with Coomassie blue stain (20), isoleucine was determined with ninhydrin, nucleotides were determined by quenching, and [i4C]Ile-tRNA was determined by radioassay.
Figure 2 illustrates the experimental data that establish that the major peak in Fig. 1 is isoleucyl adenylate. A mixture of [14C]isoleucine, ATP, MgC&, and isoleucine;tRNA ligase was incubated for 5 min at 15°C and pH 7.4, after which 20 ~1 was pipeted as a strip at the origin of a cellulose tic plate. Trace A in Fig. 2 is the result. About two-thirds of the [14C]isoleutine moves as free isoleucine, and one-third moved only 2.0 cm. The presumed isoleucyl adenylate was eluted from the cellulose by scraping the powder into 0.5 ml of 0.1 M sodium cacodylate, pH 5.4. TABLE
1
tic CHROMATOGRAPHY AND THEIR
OF ISOLEIJCINE, VALINE, LEUCINE, AND PHENYLALANINE ADENYLATES ON CELLULOSE USING 4 BUTANOL: 1 WATER: 1 ACETIC ACID AT 4”Ca R, (AA
Isoleucine Valine Leucine Phenylalanine
- AMP) 0.25 0.19 0.25 0.22
R, (AA) 0.69 0.53 0.70 0.56
n Aliquots of 4 ~1 of a solution of Enz. [14C]AA - AMP were applied to the origin line of a cellulose tic plate, dried, and developed at 4°C as described under Materials and Methods. W-labeled amino acid was applied and developed separately.
32
JAKUBOWSKI,
PASTUSZYN,
AND LOFTFIELD
C.p.M.lI
CP.M -20000
-0
- 1000
-0
CENTIMETERS
FIG. 2. Chemical characteristics of isoleucyl adenylate. (A) An ahquot (20 ~1) of a reaction mixture containing 22 PM [‘*C]isoleucine, 1 mM ATP, 1 mM mercaptoethanol, 0.1% bovine serum albumin, 10 mM MgCl,, and 6 PM iso1eucine:tRNA ligase was applied to the origin of tic cellulose, dried, and developed. The cellulose carrying isoleucyl adenylate (1 .O- 1.5 cm) was scraped from the tic plate and extracted at 0°C with 500 ~1 of pH 5.4,O. 1 M sodium cacodylate buffer. (B) After standing for 4 hr at room temperature, 40 ~1 of the extract of A was rechromatographed. (C) Thirty microliters of the extract of A was combined with 10 ~1 of 1.0 M KOH and chromatographed after 10 min. (D) One hundred fifty microliters of the extract of A was combined with 1 PM tRNA lie, 10 mM MgCl,, 1 mM mercaptoethanol, and 0.5 pM isoleucine enzyme. Aliquots of 30 ~1 were removed at 2, 5, 10, and 20 min and assayed for [14C]iso1eucyl. tRNA formation by trichloroacetic acid precipitation. Reaction was almost complete in 10 min at 0°C. A final aliquot of 30 ~1 at 20 min was chromatographed and yielded chromatogram trace D. A control omitting enzyme yielded no [i4C]Ile-tRNA and gave a chromatogram similar to B.
(i) After 10 min at 0°C) the suspension was filtered and 40 ~1 was rerun after 4 hr at 4°C on tic cellulose; approximately half the material behaved liked isoleucyl adenylate and half now ran with isoleucine (curve B, Fig. 2). (ii) Thirty microliters was combined with 10 ~1 of 1 .O N KOH and, after 10 min at room temperature, 30 ~1 was applied to tic cellulose; all of the radioactivity moves with isoleucine (curve C, Fig. 2). (iii) Two 150~~1 aliquots were incubated at 0°C with 10 mM MgC&, 1 mM mercaptoethanol, 1.0 PM tRNAie, and 0.5 or 0.0 PM Enzlie. At intervals of 2 to 20 min ahquots were removed, precipitated with trichloroacetic acid, and assayed for [14C]Ile-tRNA1Le. Transfer was almost complete in 10 min and was complete in 20 min with a total yield of 80%. An aliquot taken at 20 min and chromatographed on cellulose gave curve D in Fig. 2 showing about 90% of the total radioactivity associated with the origin as Ile-tRNA. The
DETERMINATION
OF AMINOACYL
ADENYLATE
33
yM ENZYME
FIG. 3. Relationship of yield of isoleucyl adenylate to enzyme concentration; 22.5 PM [14C]isoleucine, 10 mM MgC12, 1 mM ATP, 1 mM mercaptoethanol, and the indicated concentration of isoleucine:tRNA ligase were combined in 0.1 M Tris, pH 7.4, and permitted to react for 5 min at 16°C. Aliquots of 4 ~1 were transferred to tic cellulose and analyzed as described. Above 2 f&M enzyme, the yield of Ile - AMP is 80% and independent of enzyme concentration, white the yield falls to abodt 20% at 0.2 PM enzyme.
parallel run in the absence of Em+ transferred no isoleucine to tRNA as determined by either the trichloroacetic acid assay or the tic assay (which resembled curve B). This confirms our impression that the isoleucyl adenylate peak contains no undenatured enzyme. (iv) When the original incubation mixture contained [14C]ATP as well as P4C]isoleucine, hydrolysis of the presumed isoleucyl adenylate yielded equimolar amounts of AMP and isoleucine. The amount of radioactivity appearing in the isoleucyl adenylate peak is exactly proportional to the size of the aliquot applied to the origin. However, in a series of reactions all of which would be expected to have enzyme 80% saturated with isoleucyl adenylate, the yield of isoleucyl adenylate activity is not proportional to the amount of enzyme below 0.5 PM (Fig. 3). We cannot explain this defect, which may be a consequence of denaturation at low enzyme concentrations. Alternatively, it is possible that the equilibrium is displaced as very small amounts of enzyme bind to relatively large amounts of cellulose. Regardless of the explanation, caution is in order and a modification of our technique may be necessary when low concentrations of enzyme are used. In general, however, it seems that no substantial changes in the reaction mixture occur after application of the aliquot to the tic plate. For instance, in an effort to determine the equilibrium constant for the reaction, at 0°C and pH 5.4, Enz. [14C]Ile-AMP
+ PPi s Enz + ATP + [14C]Ile,
34
JAKUBOWSKI,
PASTUSZYN,
AND
LOFTFIELD
we varied initial concentrations of isoleucine, pyrophosphate, and salt over wide ranges. In every case, the formation of Enz. Be - AMP had reached equilibrium at 1 min and did not change for at least 8 min. The constancy of K,, (Table 2) over large changes in concentrations of substrates and products suggests that the equilibrium was not shifted significantly during application of the sample or during development. The presence of the neutral, “indifferent” salt, KCl, changes the K,, dramatically. We previously reported that KC1 has an almost insignificant effect on the rate of ATP:PP, exchange (14). The interacting charges on ATP. Mg and Ile are probably - 1 and -0 and those on Ile - AMP and PPi* Mg are about 0 and -2, respectively, at pH 5.0. From this, one would anticipate almost no simple Debye-Hiickel dependence on ionic strength. Presumably addtional charges are involved if one considers the enzyme, but it seems unlikely that 0.3 M KC1 could account for a 20-fold change in the K,,. If one postulates the formation of an inert Enz*(KCl), complex, Enz + Be + ATP % Enz. Ile - AMP + PPi, + Kw nKC1 11 K, Enz*(KCl), one derives the relationship log(2--
lj=nlog[KCl]-nlog(K,),
where K’,, is the apparent K,, measured in the presence of KC1 and K, is the dissociation constant for Enz*(KCl),. Figure 4 shows that there is a linear relationship between [(K’,,IK,J - l] and log [KC11 over a IO-fold change in K’eq. If this interpretation is correct, n is 2.9 and K, is 0.11 M. DISCUSSION Yarus and Berg (15) reported that isoleucine:tRNA ligase was adsorbed tightly to nitrocellulose filters. They used the technique to determine binding constant, specificity, kinetic constants, etc., for the specific complex Enz. tRNA1le (16). Thiebe and Hirsch (10) have improved the binding and washing procedures and extended the assay to the determination of phenylalanine and phenylalanyl adenylate bound to phenylalanine:tRNA ligase (yeast). Interestingly, Thiebe (17) found that, under some conditions, one molecule of ligase binds one molecule of phenylalanine and one molecule of the Phe - AMP, both of which are transferable to tRNA. Bartmann et al. (9) developed a similar assay based on the affinity of phenylalanine:tRNA ligase (E. coli) for DEAE-cellulose. They established a stoichiometry of binding of phenylalanine to enzyme of 2:1, exactly
DETERMINATION
OF AMINOACYL TABLE
DETERMINATION
2
K,, ATDIFFERENTINITIALCONCENTRATIONSOF
OF
ISOLEUCINE,
35
ADENYLATE
AND POTASSIUM
PYROPHOSPHATE,
CHLORIDE"
[Enzl [Enz’Ile
- AMP]
(FM)
(PM)
WI (PM)
3.55 2.70 0.75 0.09
3.55 13 101 loo0
19 19.8 21.8 22.4
0.65 1.50 3.45 4.1
9.79 8.46 9.92 10.22
-
[ppilo (M) 0 10-5 10-4 10-Z
Wil
PM
x 10VM
Welo(PM) 7.5 22.5 75
2.7 3.5 4.0
2.7 3.5 4.0
4.8 19 71
1.5 0.7 0.2
9.87 10.85 8.87
3.5 3.2 2.4 1.6
3.5 3.2 2.4 1.6
19 19.3 20.1 20.9
0.7 1.0 1.8 2.6
10.85 18.85 62.81 212.26
Wllo (M) 0 0.1 0.2 0.3
(2Unless otherwise indicated, the reaction mixture included, in 40 ~1 of 0.1 M sodium cacodylate buffer, pH 5.4: 1 mM ATP, 10 mM MgCl,, 22.5 FM [**C]isoleucine, 4.2 PM Enz’ie, 1mM mercaptoethanol, and 0.02% bovine serum albumin. After mixing and incubation at WC, 4pl aliquots were removed at intervals of from 1 to 10 min and chromatographed. There was no time-dependent change in the amount of [r4C]isoleucyI adenylate, from which we infer that enzymatic or nonenzymatic hydrolysis of adenylate is not significant in these determinations. [PPJ, [Enz], and [Ile] were estimated by adding the [Enz’lle - AMP] to initial [PP,],, or subtracting it from initial [Enz], or [isoleucine],, respectively.
equivalent to Thiebe’s observation. Bartmann did not use labeled ATP so it is not possible to confirm in E. coli Thiebe’s conclusion in yeast that the activated complex contains twice as much amino acid as adenylate. Both of these techniques appear to be at least 90% quantitative and are certainly useful for the phenylalanine system. Each depends on finding conditions where the ligase is bound quantitatively to the filter and, as such, may not be generally applicable. The vast majority of amino acid:tRNA ligases form stable complexes with aminoacyl adenylates which can be isolated by Sephadex chromatography (11). This has been used to quantitate the amount of enzyme, the major problem being that synthesis of the adenylate may be reversed as amino acid and ATP concentrations are reduced by the Sephadex filtration. Our technique resembles the Sephadex isolation in simplicity of principle and generality of application, but without the complication of reversal of synthesis. Furthermore, it becomes a simple matter to determine K,, and
36
JAKUBOWSKI,
-1.0
PASTUSZYN,
AND LOFTFIELD
-.8 Log [KC11
-.6
-.4
FIG. 4. Variation of apparent equilibrium constant with potassium chloride concentration. The data of Table 2 are replotted to determine the order of reaction of KC1 with enzyme and its binding constant on the assumption that KC1 competitively inhibits formation of Enz.Ile - AMP. K,, is the average of eight values ofK ,gderived from a total of 40 determinations of Be - AMP under different conditions. Each estimate of K’,, is based on four or five determinations of IIt - AMP is the presence of various KCI concentrations after incubations of 0.5 to 8 min.
therefore to establish what concentrations of ATP and amino acid will be required to assure an almost quantitative formation of Enz*(AA - AMP). We emphasize that the yield of Enz *(AA - AMP) is not quantitative at low concentrations of enzyme: We find the same deficiency in the assay using Sephadex columns. The Thiebe-Hirsch (10) assay is shown to be linear from about 10 to 150 nM enzyme, while the Bartmann assay was evaluated only at relatively high concentrations of enzyme. The enzymatic activity of amino acid:tRNA ligase preparations has been measured by numerous assay techniques (18). Kinetic assays based on the rate of ATP:PP, exchange or the rate of formation of amino acid hydroxamate measure an activity that may persist when the ability to aminoacylate tRNA has been lost (17,19). Kinetic assays based on aminoacylation of tRNA measure the physiologically important enzymatic activity but are unpredictably sensitive to assay condition and changes in the nature of the buffer, resulting in a 40-fold change in rate. Static assays such as the one described here as well as those of Thiebe and Bartmann have an advantage in being precisely quantitative if reaction conditions assure quantitative formation of Enz . (AA - AMP). Since these assays might measure enzyme that lacks aminoacylation competence, it is probably desirable to use a combination of assays to determine ligase activity. REFERENCES 1. 2.
Hoagland, M. B. (1955) Biochim. Biophys. Acta Berg, P. (1956) J. Biol. Chem. 222, 1025-1034.
16, 288-289.
DETERMINATION
OF AMINOACYL
37
ADENYLATE
3. DeMoss, J. A., Genuth, S. M., and Novelli, G. D. (1956) Proc.
Nut.
Acad.
Sci.
Chem.
Sot.
USA
42,32.5-332. 4.
Castelfranco,
P., Moldave,
K., and Meister,
A. (1958) J. Amer.
80,
2335-2336.
5. Moldave, K. Castelfranco, P., and Meister, A. (1959) J. Biol. Chem. 234, 841-848. 6. Berg, P. (1958)J. Biol. Chem. 233, 608-611. 7. Kingdon, H. S., Webster, L. T., and Davie, E. W. (1958) Proc. Nut. Acad. Sci. USA 44, 757-765.
8. 9. 10. 11. 12.
Norris, A. T., and Berg, P. (1964) Proc. Nat. Acad. Sci. USA 52, 330-337. Bartmann, P., Hanke, T., and Holler, E. (1976) Anal. Biochem. 70, 174- 180. Thiebe, R., and Hirsch, R. (1975) FEBS Left. 60, 338-341. Allende, J. E., and Allende, C. C. (1971) Methods in Enzymol. 20,210-220. Liivgren, T. N. E., Heinonen, J., and Loftfield, R. B. (1975) J. Biol. Chem. 250,38543860.
Randerath, K. (1%6) Thin Layer Chromatography, p. 110, Academic Press, New York. 14. Pastuszyn, A., Liivgren, T. N. E., and Loftfield, R. B. (1974) Fed Proc. 33, 1433. 15. Yarus, M., and Berg, P. (1967) .I. Mol. Biol. 28, 479-490. 16. Yarus, M., and Berg, P. (1969)J. Mol. Biol. 42, 171-189. 17. Thiebe, R. (1975) FEBS L&r. 60, 342-345. 18. Eigner, E. A., and Loftfield, R. B. (1974) Methods in Enzymol. 29, 601-619. 19. Cassio, D. (1968) Eur. J. Biochem. 4. 222-224. 20. Weber, K., and Osbom, M. (1972) Methods in Enzymol. 26C, 3-27. 13.
BIOCHEMISTRY
82,
29-37 (1977)
The Determination of Aminoacyl Adenylate Thin-Layer Chromatography’ HIERONIM
by
Z.JAKUBOWSKI,~ ANDRZEJ PASTUZYN, AND ROBERT B. LOFTFIELD
Department of Biochemistry, University of New Mexico, Albuquerque, New Mexico 87131 Received November 23. 1976; accepted April 25, 1977 A rapid and convenient method is presented by which one may determine the extent of formation of Enz.(AA - AMP) in the presence of isotopic amino acid, isotopic nucleotides, tRNA, enzyme, etc. Separation of the components of the reaction mixture is achieved by chromatography on tic cellulose. Aminoacyl adenylate separates from other solutes and is characterized by its chemical and enzymatic reactions. The method may be used to determine the equilibrium constant for the synthesis of Enz.(AA - AMP) which is very sensitive to salt concentration.
As early as 195.5, aminoacyl adenylates were proposed as intermediates in the activation of amino acids for protein biosynthesis (1,2). Aminoacyl adenylates have been synthesized chemically and enzymatically and have the properties that would be expected of intermediates in the formation of aminoacyl-tRNAs (3-6). Several assay methods have been developed for estimation of the amount of enzyme-bound aminoacyl adenylate in a reaction mixture (8- 11). We describe an assay that permits the estimation of total aminoacyl adenylate in the presence of amino acids, aminoacyl-tRNA, ATP. etc. MATERIALS
AND METHODS
Amino acid:tRNA ligases specific for isoleucine, valine, leucine, and phenylalanine were prepared from Escherichia co/i (12). The enzymes were estimated to be 40-50% pure by Sephadex isolation of Enz. [14C]-Ile - AMP under saturating conditions (85% pure as estimated by gel electrophoresis). The tRNA”” was about 90% pure (1700 pmoYOD,,,J. 14C-labeled amino acids and [14C]ATP were purchased from New England Nuclear Corporation and AmershamKearle and were used at maximal specific ’ Supported by Grant Np 103 from the American Cancer Society and by Grant CA 08000 from the United States Public Health Service. * Permanent address: Institute of Biochemistry, Agricultural University, Wolynska 35. Poznan 60-637, Poland. 29 Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
ISSN 0003-2697
30
JAKUBOWSKI,
PASTUSZYN,
AND LOFTFIELD
activities (280-556 mCi/mmol). Precoated tic plates on transparent plastic supports were purchased from Brinkman (cellulose, 0.1 mm; silica gel, 0.2 mm) or from Eastman (cellulose, 0.16 mm). All other chemicals and solvents were commercial products of reagent quality. Enzyme-bound aminoacyl adenylate was formed by reacting 20-30 PM 14C-labeled amino acid, 1 mM ATP, 10 mM MgCl,, and ca. l-2 PM ligase for 5 min at 15°C in 100 ~1 of 0.1 M Tris, pH 7.4, containing 1 mM mercaptoethanol and 0.2% bovine serum albumin. The mixture was applied to the top of a 0.5 x 16-cm column of Sephadex G-100 that had been equilibrated at 4°C with 10 mM Tris, pH 7.4, containng 10 mM mercaptoethanol. Elution with the Tris buffer yielded all of the enzyme-bound aminoacyl adenylate in less than 200 ~1, well separated from the unreacted free amino acid (11). For tic chromatography, aliquots of 3 or 4 ~1 are applied to the origin of 20-cm plates, occasionally using warm air to accelerate drying. Development is always with 4 butanol: 1 acetic acid: 1 water (13). The solvent rises through 8 cm of cellulose in 60 min at 21°C or in 90 min at 4°C. Development of the silica gel plates is slower: 2 hr at 21” C and 3 hr at 4°C. Some Eastman cellulose plates were satisfactory, but more recent purchases containing an improved binder develop so slowly with this solvent that they cannot be used. The tic plates are dried, cut into l- or 0.5-cm strips, and assayed for radioactivity by immersion in scintillation cocktail (0.05% POPOP:O.5% PPO:toluene) followed by counting in a scintillation spectrophotometer at, room temperature. The efficiency of counting 14C-labeled compounds on the tic plates was 57-60% and the backgrounds were between 30 and 55 cpm. Cellulose plates have an advantage over silica gel plates in that the 14C-labeled substances are eluted from the silica gel into the toluene. This introduces some variability in the counting and precludes the use of the scintillation cocktail for more than a single assay. RESULTS
Figure 1 shows the behavior of enzyme-bound [14C]isoleucyl adenylate chromatographed on tic cellulose at 4°C with butanol/acetic acid/water. Essentially, no radioactivity remains at the origin, while 90-95% of the isotope is found in a symmetrical peak about 2 cm from the origin. As shown in Fig. 1, this peak is well separated from free isoleucine as well as from other components of the reaction mixture. The results for the phenylalanine, leutine, and valine systems are similar except for slight changes in the migration, as shown in Table 1. If silica gel tic plates are used, there tends to be somewhat greater decomposition of aminoacyl adenylate to amino acid. Similarly, if cellulose plates are developed at 21°C phenylalanyl adenylate and leucyl adenylate partially decompose. In other respects, silica gel plates and development at 4 or 21°C give comparable results.
DETERMINATION
OF AMINOACYL
31
ADENYLATE
AMP
CPM. 5oo-
ATP
-
ISOLEUCINE
-RNA 400
-
PROTEIN -
L0
300-
ZOO-
loo-
, .I 0
i
,\.
II -\r
I
2
,
,._,_
5
6
_,
,,c=--1. 3 4 CENTIMETERS
7
8
FIG. 1. tic chromatography of enzyme. isoleucyl adenylate complex. An aliquot of 4 ~1 of Sephadex G-50 (fine)-purified isoleucine:tRNA ligase.isoleucyl adenylate complex was applied to the origin of a cellulose tic plate and dried immediately. The chromatogram was developed with 4 butanol: 1 acetic acid: 1 water at 0°C. Strips of the tic plate (0.5 cm) were cut and radioassayed by immersion in scintillation counting liquid. The location of protein was determined with Coomassie blue stain (20), isoleucine was determined with ninhydrin, nucleotides were determined by quenching, and [i4C]Ile-tRNA was determined by radioassay.
Figure 2 illustrates the experimental data that establish that the major peak in Fig. 1 is isoleucyl adenylate. A mixture of [14C]isoleucine, ATP, MgC&, and isoleucine;tRNA ligase was incubated for 5 min at 15°C and pH 7.4, after which 20 ~1 was pipeted as a strip at the origin of a cellulose tic plate. Trace A in Fig. 2 is the result. About two-thirds of the [14C]isoleutine moves as free isoleucine, and one-third moved only 2.0 cm. The presumed isoleucyl adenylate was eluted from the cellulose by scraping the powder into 0.5 ml of 0.1 M sodium cacodylate, pH 5.4. TABLE
1
tic CHROMATOGRAPHY AND THEIR
OF ISOLEIJCINE, VALINE, LEUCINE, AND PHENYLALANINE ADENYLATES ON CELLULOSE USING 4 BUTANOL: 1 WATER: 1 ACETIC ACID AT 4”Ca R, (AA
Isoleucine Valine Leucine Phenylalanine
- AMP) 0.25 0.19 0.25 0.22
R, (AA) 0.69 0.53 0.70 0.56
n Aliquots of 4 ~1 of a solution of Enz. [14C]AA - AMP were applied to the origin line of a cellulose tic plate, dried, and developed at 4°C as described under Materials and Methods. W-labeled amino acid was applied and developed separately.
32
JAKUBOWSKI,
PASTUSZYN,
AND LOFTFIELD
C.p.M.lI
CP.M -20000
-0
- 1000
-0
CENTIMETERS
FIG. 2. Chemical characteristics of isoleucyl adenylate. (A) An ahquot (20 ~1) of a reaction mixture containing 22 PM [‘*C]isoleucine, 1 mM ATP, 1 mM mercaptoethanol, 0.1% bovine serum albumin, 10 mM MgCl,, and 6 PM iso1eucine:tRNA ligase was applied to the origin of tic cellulose, dried, and developed. The cellulose carrying isoleucyl adenylate (1 .O- 1.5 cm) was scraped from the tic plate and extracted at 0°C with 500 ~1 of pH 5.4,O. 1 M sodium cacodylate buffer. (B) After standing for 4 hr at room temperature, 40 ~1 of the extract of A was rechromatographed. (C) Thirty microliters of the extract of A was combined with 10 ~1 of 1.0 M KOH and chromatographed after 10 min. (D) One hundred fifty microliters of the extract of A was combined with 1 PM tRNA lie, 10 mM MgCl,, 1 mM mercaptoethanol, and 0.5 pM isoleucine enzyme. Aliquots of 30 ~1 were removed at 2, 5, 10, and 20 min and assayed for [14C]iso1eucyl. tRNA formation by trichloroacetic acid precipitation. Reaction was almost complete in 10 min at 0°C. A final aliquot of 30 ~1 at 20 min was chromatographed and yielded chromatogram trace D. A control omitting enzyme yielded no [i4C]Ile-tRNA and gave a chromatogram similar to B.
(i) After 10 min at 0°C) the suspension was filtered and 40 ~1 was rerun after 4 hr at 4°C on tic cellulose; approximately half the material behaved liked isoleucyl adenylate and half now ran with isoleucine (curve B, Fig. 2). (ii) Thirty microliters was combined with 10 ~1 of 1 .O N KOH and, after 10 min at room temperature, 30 ~1 was applied to tic cellulose; all of the radioactivity moves with isoleucine (curve C, Fig. 2). (iii) Two 150~~1 aliquots were incubated at 0°C with 10 mM MgC&, 1 mM mercaptoethanol, 1.0 PM tRNAie, and 0.5 or 0.0 PM Enzlie. At intervals of 2 to 20 min ahquots were removed, precipitated with trichloroacetic acid, and assayed for [14C]Ile-tRNA1Le. Transfer was almost complete in 10 min and was complete in 20 min with a total yield of 80%. An aliquot taken at 20 min and chromatographed on cellulose gave curve D in Fig. 2 showing about 90% of the total radioactivity associated with the origin as Ile-tRNA. The
DETERMINATION
OF AMINOACYL
ADENYLATE
33
yM ENZYME
FIG. 3. Relationship of yield of isoleucyl adenylate to enzyme concentration; 22.5 PM [14C]isoleucine, 10 mM MgC12, 1 mM ATP, 1 mM mercaptoethanol, and the indicated concentration of isoleucine:tRNA ligase were combined in 0.1 M Tris, pH 7.4, and permitted to react for 5 min at 16°C. Aliquots of 4 ~1 were transferred to tic cellulose and analyzed as described. Above 2 f&M enzyme, the yield of Ile - AMP is 80% and independent of enzyme concentration, white the yield falls to abodt 20% at 0.2 PM enzyme.
parallel run in the absence of Em+ transferred no isoleucine to tRNA as determined by either the trichloroacetic acid assay or the tic assay (which resembled curve B). This confirms our impression that the isoleucyl adenylate peak contains no undenatured enzyme. (iv) When the original incubation mixture contained [14C]ATP as well as P4C]isoleucine, hydrolysis of the presumed isoleucyl adenylate yielded equimolar amounts of AMP and isoleucine. The amount of radioactivity appearing in the isoleucyl adenylate peak is exactly proportional to the size of the aliquot applied to the origin. However, in a series of reactions all of which would be expected to have enzyme 80% saturated with isoleucyl adenylate, the yield of isoleucyl adenylate activity is not proportional to the amount of enzyme below 0.5 PM (Fig. 3). We cannot explain this defect, which may be a consequence of denaturation at low enzyme concentrations. Alternatively, it is possible that the equilibrium is displaced as very small amounts of enzyme bind to relatively large amounts of cellulose. Regardless of the explanation, caution is in order and a modification of our technique may be necessary when low concentrations of enzyme are used. In general, however, it seems that no substantial changes in the reaction mixture occur after application of the aliquot to the tic plate. For instance, in an effort to determine the equilibrium constant for the reaction, at 0°C and pH 5.4, Enz. [14C]Ile-AMP
+ PPi s Enz + ATP + [14C]Ile,
34
JAKUBOWSKI,
PASTUSZYN,
AND
LOFTFIELD
we varied initial concentrations of isoleucine, pyrophosphate, and salt over wide ranges. In every case, the formation of Enz. Be - AMP had reached equilibrium at 1 min and did not change for at least 8 min. The constancy of K,, (Table 2) over large changes in concentrations of substrates and products suggests that the equilibrium was not shifted significantly during application of the sample or during development. The presence of the neutral, “indifferent” salt, KCl, changes the K,, dramatically. We previously reported that KC1 has an almost insignificant effect on the rate of ATP:PP, exchange (14). The interacting charges on ATP. Mg and Ile are probably - 1 and -0 and those on Ile - AMP and PPi* Mg are about 0 and -2, respectively, at pH 5.0. From this, one would anticipate almost no simple Debye-Hiickel dependence on ionic strength. Presumably addtional charges are involved if one considers the enzyme, but it seems unlikely that 0.3 M KC1 could account for a 20-fold change in the K,,. If one postulates the formation of an inert Enz*(KCl), complex, Enz + Be + ATP % Enz. Ile - AMP + PPi, + Kw nKC1 11 K, Enz*(KCl), one derives the relationship log(2--
lj=nlog[KCl]-nlog(K,),
where K’,, is the apparent K,, measured in the presence of KC1 and K, is the dissociation constant for Enz*(KCl),. Figure 4 shows that there is a linear relationship between [(K’,,IK,J - l] and log [KC11 over a IO-fold change in K’eq. If this interpretation is correct, n is 2.9 and K, is 0.11 M. DISCUSSION Yarus and Berg (15) reported that isoleucine:tRNA ligase was adsorbed tightly to nitrocellulose filters. They used the technique to determine binding constant, specificity, kinetic constants, etc., for the specific complex Enz. tRNA1le (16). Thiebe and Hirsch (10) have improved the binding and washing procedures and extended the assay to the determination of phenylalanine and phenylalanyl adenylate bound to phenylalanine:tRNA ligase (yeast). Interestingly, Thiebe (17) found that, under some conditions, one molecule of ligase binds one molecule of phenylalanine and one molecule of the Phe - AMP, both of which are transferable to tRNA. Bartmann et al. (9) developed a similar assay based on the affinity of phenylalanine:tRNA ligase (E. coli) for DEAE-cellulose. They established a stoichiometry of binding of phenylalanine to enzyme of 2:1, exactly
DETERMINATION
OF AMINOACYL TABLE
DETERMINATION
2
K,, ATDIFFERENTINITIALCONCENTRATIONSOF
OF
ISOLEUCINE,
35
ADENYLATE
AND POTASSIUM
PYROPHOSPHATE,
CHLORIDE"
[Enzl [Enz’Ile
- AMP]
(FM)
(PM)
WI (PM)
3.55 2.70 0.75 0.09
3.55 13 101 loo0
19 19.8 21.8 22.4
0.65 1.50 3.45 4.1
9.79 8.46 9.92 10.22
-
[ppilo (M) 0 10-5 10-4 10-Z
Wil
PM
x 10VM
Welo(PM) 7.5 22.5 75
2.7 3.5 4.0
2.7 3.5 4.0
4.8 19 71
1.5 0.7 0.2
9.87 10.85 8.87
3.5 3.2 2.4 1.6
3.5 3.2 2.4 1.6
19 19.3 20.1 20.9
0.7 1.0 1.8 2.6
10.85 18.85 62.81 212.26
Wllo (M) 0 0.1 0.2 0.3
(2Unless otherwise indicated, the reaction mixture included, in 40 ~1 of 0.1 M sodium cacodylate buffer, pH 5.4: 1 mM ATP, 10 mM MgCl,, 22.5 FM [**C]isoleucine, 4.2 PM Enz’ie, 1mM mercaptoethanol, and 0.02% bovine serum albumin. After mixing and incubation at WC, 4pl aliquots were removed at intervals of from 1 to 10 min and chromatographed. There was no time-dependent change in the amount of [r4C]isoleucyI adenylate, from which we infer that enzymatic or nonenzymatic hydrolysis of adenylate is not significant in these determinations. [PPJ, [Enz], and [Ile] were estimated by adding the [Enz’lle - AMP] to initial [PP,],, or subtracting it from initial [Enz], or [isoleucine],, respectively.
equivalent to Thiebe’s observation. Bartmann did not use labeled ATP so it is not possible to confirm in E. coli Thiebe’s conclusion in yeast that the activated complex contains twice as much amino acid as adenylate. Both of these techniques appear to be at least 90% quantitative and are certainly useful for the phenylalanine system. Each depends on finding conditions where the ligase is bound quantitatively to the filter and, as such, may not be generally applicable. The vast majority of amino acid:tRNA ligases form stable complexes with aminoacyl adenylates which can be isolated by Sephadex chromatography (11). This has been used to quantitate the amount of enzyme, the major problem being that synthesis of the adenylate may be reversed as amino acid and ATP concentrations are reduced by the Sephadex filtration. Our technique resembles the Sephadex isolation in simplicity of principle and generality of application, but without the complication of reversal of synthesis. Furthermore, it becomes a simple matter to determine K,, and
36
JAKUBOWSKI,
-1.0
PASTUSZYN,
AND LOFTFIELD
-.8 Log [KC11
-.6
-.4
FIG. 4. Variation of apparent equilibrium constant with potassium chloride concentration. The data of Table 2 are replotted to determine the order of reaction of KC1 with enzyme and its binding constant on the assumption that KC1 competitively inhibits formation of Enz.Ile - AMP. K,, is the average of eight values ofK ,gderived from a total of 40 determinations of Be - AMP under different conditions. Each estimate of K’,, is based on four or five determinations of IIt - AMP is the presence of various KCI concentrations after incubations of 0.5 to 8 min.
therefore to establish what concentrations of ATP and amino acid will be required to assure an almost quantitative formation of Enz*(AA - AMP). We emphasize that the yield of Enz *(AA - AMP) is not quantitative at low concentrations of enzyme: We find the same deficiency in the assay using Sephadex columns. The Thiebe-Hirsch (10) assay is shown to be linear from about 10 to 150 nM enzyme, while the Bartmann assay was evaluated only at relatively high concentrations of enzyme. The enzymatic activity of amino acid:tRNA ligase preparations has been measured by numerous assay techniques (18). Kinetic assays based on the rate of ATP:PP, exchange or the rate of formation of amino acid hydroxamate measure an activity that may persist when the ability to aminoacylate tRNA has been lost (17,19). Kinetic assays based on aminoacylation of tRNA measure the physiologically important enzymatic activity but are unpredictably sensitive to assay condition and changes in the nature of the buffer, resulting in a 40-fold change in rate. Static assays such as the one described here as well as those of Thiebe and Bartmann have an advantage in being precisely quantitative if reaction conditions assure quantitative formation of Enz . (AA - AMP). Since these assays might measure enzyme that lacks aminoacylation competence, it is probably desirable to use a combination of assays to determine ligase activity. REFERENCES 1. 2.
Hoagland, M. B. (1955) Biochim. Biophys. Acta Berg, P. (1956) J. Biol. Chem. 222, 1025-1034.
16, 288-289.
DETERMINATION
OF AMINOACYL
37
ADENYLATE
3. DeMoss, J. A., Genuth, S. M., and Novelli, G. D. (1956) Proc.
Nut.
Acad.
Sci.
Chem.
Sot.
USA
42,32.5-332. 4.
Castelfranco,
P., Moldave,
K., and Meister,
A. (1958) J. Amer.
80,
2335-2336.
5. Moldave, K. Castelfranco, P., and Meister, A. (1959) J. Biol. Chem. 234, 841-848. 6. Berg, P. (1958)J. Biol. Chem. 233, 608-611. 7. Kingdon, H. S., Webster, L. T., and Davie, E. W. (1958) Proc. Nut. Acad. Sci. USA 44, 757-765.
8. 9. 10. 11. 12.
Norris, A. T., and Berg, P. (1964) Proc. Nat. Acad. Sci. USA 52, 330-337. Bartmann, P., Hanke, T., and Holler, E. (1976) Anal. Biochem. 70, 174- 180. Thiebe, R., and Hirsch, R. (1975) FEBS Left. 60, 338-341. Allende, J. E., and Allende, C. C. (1971) Methods in Enzymol. 20,210-220. Liivgren, T. N. E., Heinonen, J., and Loftfield, R. B. (1975) J. Biol. Chem. 250,38543860.
Randerath, K. (1%6) Thin Layer Chromatography, p. 110, Academic Press, New York. 14. Pastuszyn, A., Liivgren, T. N. E., and Loftfield, R. B. (1974) Fed Proc. 33, 1433. 15. Yarus, M., and Berg, P. (1967) .I. Mol. Biol. 28, 479-490. 16. Yarus, M., and Berg, P. (1969)J. Mol. Biol. 42, 171-189. 17. Thiebe, R. (1975) FEBS L&r. 60, 342-345. 18. Eigner, E. A., and Loftfield, R. B. (1974) Methods in Enzymol. 29, 601-619. 19. Cassio, D. (1968) Eur. J. Biochem. 4. 222-224. 20. Weber, K., and Osbom, M. (1972) Methods in Enzymol. 26C, 3-27. 13.
