Jorrriiul o/ NrirrochprrrrsrrJ. 1975 Vol. 24. pp. 1163-1 172.

Pergamon Press. Printcd in Grcat Britain

STRUCTURAL ALTERATIONS OF AMINO ACIDS AT THE LEVEL OF AMINOACYL-tRNAS: TRANSFORMATION OF DICARBOXYLIC AMINO ACIDS HUCUETTE Roux a n d M. R. V. MURTHY Department of Biochemistry, Faculty of Medicine, Lava1 University, Quebec, Canada (Received 18 November 1974. Accepted 23 December 1974) Abstract-Radioactive glutamate, glutamine,, aspartate and asparagine were incorporated into calf brain tRNA in the presence of homologous aminoacyl-tRNA synthetases. When the aminoacyl-tRNAs were deaminoacylated and the products chromatographed in the phenol solvent, the glutaminyland asparaginyl-tRNAs showed two products GnP, and AnP, respectively, in, addition to the original amino acids. These new substances moved close to the solvent front in contrast to glutamine and asparagine which had much lower RF values. Attachment of the amino acids to tRNA appeared to be a prerequisite for the formation of these substances, since they were not found in the reaction mixture used for aminoacylation in the absence of incubation or on omission of tRNA or when tRNA was degraded by RNase. Application of the deaminoacylation procedure to pure amino acids also failed to lead to their formation. In an other series of experiments, the dicarboxylic aminoacyltRNAs were hydrolysed with pancreatic RNase and then analysed by high voltage paper electrophoresis. Again, the glutaminyl- and asparaginyl-tRNAs showed two new components, GnE3 and AnE,, in addition to the expected glutaminyl- and asparaginyladenosines. GnE, and AnE, exhibited much faster electrophoretic mobilities in the direction of the cathode than the adenosine derivatives of the original amino acids and thus appeared to be more positively charged. The presence of these new compounds in the products of deaminoacylation or in RNase hydrolysates was specific to glutaminyl- and asparaginyl-tRNAs and did not occur in the case of either glutamyl or aspartyl-tRNAs, indicating that the amide group was probably involved in the transformation reaction.

THEDICARBOXYLIC amino acids, glutamate a n d aspartate, a n d their amides are found in very high concentrations in brain, both in the free state a s well as in peptide linkage (TALLAN,1962; MOORE, 1965; ROBERTS,1971). The presence of two carboxyl groups (or a carboxyl and an amide group) a n d an amino group permits these molecules to participate in a number of enzymatic reactions involving decarboxylation, deamination, deamidation, transamination etc. Some of these transformations have been shown to take place even a t the level of aminoacyl-tRNA where all the reactive groups, except the C,-carboxyl linked to tRNA, are presumably still available for chemical change under appropriate conditions. The few such reactions so far reported occur in bacteria, for example, conversions of glutaminyl-tRNA to pyroglutamyl-tRNA (BERNFIELD & NESTOR,1968) and glutamyl-tRNAG'" to glutaminyl-tRNAG'" (WILCOX,1969). In view of the possible implications of such reactions in the regulation of protein synthesis in the nervous tissue, we have undertaken studies to determine what types of structural modifications can occur in the aminoacyl moiety of dicarboxylic aminoacyl-tRNAs by enzymatic or physicochemical procedures. In an earlier communication (MURTHY& ROUX, 1974a), we Abbreviations used: BAW, butano1:acetic acid: water, 12:3:5, v/v/v; PW,Phenol:water, 10:2, w/w.

reported that new substances other than the original radioactive amino acids were found among the products of deaminoacylation of glutamyl and glutaminyl-tRNAs. The present papers extend these studies to aspartyl and asparaginyl-tRNAs a n d describe the results of experiments intended to identify these substances and determine the mechanisms of their formation. MATERIALS AND METHODS ~-[U-'~C]aspartate(197 mCi/mmol), ~-[U-'~C]asparagine (161 mCi/mmol), ~-[U-'~C]glutamate (217 mCi/ mmol) and ~-[U-'~C]glutamine(218 mCi/mmol) were purchased from New England Nuclear Co., Boston. L-Pyroglutamic acid (2-pyrrolidone 5-carboxylic acid) and monomethyl fumarate were obtained from K & K Laboratories, N.Y. Radioactive pyroglutamate was prepared by heating 1 ml of an aqueous solution containing 25 pCi (approximately 100 nmol) of ~-[U-'~C]glutamineand 5 mg of nonradioactive glutamine in boiling water for 30 min in a sealed tube (PUCHER& VICKERY,1940). The pyroglutamate was separated from the residual glutamine by extraction with ethyl acetate at pH 2.5. Aminoacylation of tRNA Aminoacyl-tRNA synthetases as well as tRNA were prepared from brains of 6-week-old male calves as described & Roux, 1974b). Aminoacylation of previously (MURTHY tRNA was carried out at 30°C for 45 min according to

1163

I164

HUGUETTE Roux and M . R. V. MURTHY

YANG & NOVELLI(1971) using the optimum ATP/MgZ+ ratios a s suggested by these authors. The volume of the final reaction mixture varied from 0.1 ml to 5.0 ml depending upon the quantity of aminoacyl-tRNA required for analysis. For the sake of convenience, the term 'free radioactivity' will he used in these papers to refer to all radioactive substances not attached to tRNA during the aminoacylation reaction, including the unreacted amino acid used as the tracer and all its transformation products, irrespective of whether the structural alteration was brought about enzymatically or otherwise. As will he described further on, the free radioactivity fraction was isolated from the reaction mixture either by extraction with trichloroacetic acid or by DEAE-cellulose chromatography. In a similar manner, the term 'tRNA hound radioactivity' will refer to all radioactive substances originally hound to tRNA under the conditions of the aminoacylation reaction and subsequently recovered from the tRNA by a procedure of deaminoacylation or by RNase hydrolysis. This will therefore include not only the original radioactive amino acid used as tracer for tRNA binding hut also any of its transformation products, irrespective of whether such transformation occurred prior to or after incorporation into tRNA, or a s a consequence of the procedures used in the recovery of radioactivity from the labelled tRNA. The term 'aminoacyl-tRNA' will be used to signify tRNA molecules labelled in the course of aminoacylation reaction and not yet subjected to deesterification or depolymerisation. Separation of Jive and t R N A hound radioactivity @om the reaction mixture

(a) For small volumes of reaction mixture: for 0.1-0.2 ml of reaction mixture, the following procedure was used (MURTHY& Roux, 1974a). Rectangular strips of Whatman No. 1 filter paper (2.5 cm x 3.0 cm for 0.1 ml and 3.0 cm x 3.5 cm for 0.2 ml) were folded in the form of wicks and were numbered for identification on the outer most surface of the folds by appropriate pencil marks. Following incubation, the reaction tubes were cooled in an ice bath and the contents of each tube were absorbed quantitatively by dipping one end of the paper wick into the solution. The wicks were then immediately transferred into a test tube containing 1.5-2.0 ml of cold 5% trichloroacetic acid + 1 mg per ml of the cold amino acid whose radioactive form was originally used for aminoacylation. The tubes were gently agitated at 2°C for 10 min and the filter papers were removed after draining the adhering liquid. After removing any fine particles of filter paper by light centrifugation, the solution was repeatedly extracted with ether in order to eliminate the last traces of trichloroacetic acid. The aqueous phase was lyophilized and the residue containing the acid soluble fraction (free radioactivity) was taken up in 50 pl of 50% ethanol for analysis by paper chromatography. The filter paper wicks from the above procedure, containing aminoacyl-tRNA, were freed of adsorbed low molecular weight substances according to NISHIMURA& NOVELLI(1964) by washing with 5% trichloracetic acid, Hokins reagent and ether. The tRNA on the paper was deaminoacylated by treatment with 0 3 M-ammonium carbonate and the radioactivity was released into solution & Roux, 39746). Water as described previously (MURTHY as well as ammonium carbonate were removed by lyophilizing the solution and the residue (tRNA hound radioacti-

vity) was taken up in 50 pI of 50% ethanol for paper chromatography. (b) For large volumes of reaction mixture: when the reaction was carried out in volumes greater than 0.2 ml. the aminoacyl-tRNA was separated from the rest of the constituents of the reaction mixture using a DEAE-cellulose column. DEAE-cellulose (Whatman DE-32, microgranular) was washed successively with 0.5 N-HCI, distilled water, 0.5 N-NaOH and again with distilled water. It was equilibrated with 0.25 M-NaCI, I mM-EDTA and 10 mMMgCI, (0.25 M-salt solution) and then transferred into a 10 ml disposable syringe to a bed volume of 5 ml. The reaction mixture was adsorbed on the column and then washed with 0 2 5 M-salt solution to remove the synthetases, ATP, the unreacted amino acids and other radioactive metabolites. The aminoacyl-tRNA remaining on the column was then eluted with a solution containing 0.7 M-NaCI, 1 mM-EDTA and 10 mM-MgCl, (0.7 M-Salt solution). The progress of the elution was monitored by collecting fractions at regular intervals, determining the absorbance at 260 n m of each fraction after appropriate dilution and measuring its radioactivity in small portions (Fig. 1). The radioactivity profile of 0.7 M-salt eluate consisted of only one peak whereas the optical density profile consistently showed two distinct peaks. Presumably, all isoacceptors of a single [14C]aniino acid eluted as a single family although mixed aminoacyl-tRNAs were separated into two groups under conditions of chromatography used here. The fractions of the radioactivity peak emerging with 0.25 M-salt solution (free radioactivity) and those of the second radioactivity peak eluting with 0.7 M-salt solution (aminoacyl-tRNA) were separately pooled. The 0.25 M-salt eluates were analysed directly by paper electrophoresis. Since the level of radioactivity in this fraction was quite high, only very small volumes were required for analysis. The

I

Voluma

O f e1Y.t.

Irnl)

FIG. 1. Separation of free radioactivity and ['4C]aminoacyl-tRNA by DEAE-cellulosechromatography.Aminoacylation of tRNA was carried out using ~-[U-'~C]asparagine Absorbance a t 260 nm; as the radioactive tracer. (+0) (o---o), radioactivity. The arrows indicate (I), starting of elution with 0.25 M-salt solution and (2), starting of elution with 0.7 M-salt solution. Other experimental details were as describcd in Materials and Methods.

Reactions of dicarboxylic amino acids attached to tRNA amounts of NaCl and other salts present in such an aliquot were too little to interfere with electrophoresis and therefore no prior desalting or concentration of this fraction was performed. The aminoacyl-tRNA was precipitated from the 0.7 Msalt solution by adding 2.5 vol of ethanol and keeping the mixture at -20°C overnight. The precipitate was recovered by filtration through a millipore filter and washed 3-4 times with 70% ethanol to remove any adsorbed free radioactivity. The millipore filter was placed in a scintillation vial and incubated with 0.51.0 ml of a nonbuffered aqueous solution (pH 7.0) of pancreatic RNase (Worthington, 5 x crystallised, 10 pg per ml) at room temperature for 40 min, with occasional shaking. Following incubation, the solution was recovered by means of a suction pipette and lyophilized. The residue (tRNA bound radioactivity) was taken up in a small volume of 50% ethanol and used for paper electrophoresis. Paper chromatography and paper electrophoresis

Paper chromatography was carried out on Whatman No. 1 filter paper by the descending technique using butanol :acetic acid:water (12:3:5, v/v) (BAW Chromatography) or pheno1:water (10:2, w/w) (PW Chromatography) as described previously (MURTHY & Roux, 1974b). After chromatography, the filter paper was dried by a t

1 I65

current of air a t room temperature. The strip corresponding to each sample was cut into narrow bands and radioactivity in each band was determined by suspending in 15 ml of Toluene-POPOP and counting in a Mark I1 liquid scintillation counter. Electrophoresis was carried out on Whatman No. 3MM filter paper sheets (46 cm x 92 cm). Samples were spotted at a distance of 26 cm from one end of the paper (+ end) with a separation of 5 cm between neighbouring spots. The paper was moistened with the electrophoresis buffer (0.05 M-ammonium acetate, pH 3.5) and was then placed in a Savant high voltage electrophoresis tank. Approximately 6 cm of paper at each end was allowed to dip into the reservoirs containing the buffer. Electrophoresis was carried out at 3000 V (0.033 W/cm2) for 1 h a t room temperature. The papers were then dried in a draft of air and examined by a U.V.lamp (Mineralight, model-SL 2537) for the presence of ultraviolet absorbing or fluorescent spots. The strip corresponding to each sample was then cut into narrow bands and counted as mentioned in the previous paragraph. In some experiments, the radioactive components separated by paper electrophoresis were further analysed by paper chromatography either in BAW or PW solvents or in each of the two solvents successively. In such procedures, the peaks on the electrophoregram or the chromatogram were first visualized by passing each of the strips through a Packard Radioachromatogram Scanner, Model 7200. The regions of the paper corresponding to each of the peaks was then cut into small rectangular pieces (0.2 cm x 1 cm) and then extracted 3 times successively with distilled water in a conical flask. Enough water was added to cover the pieces of paper and the flask was slowly shaken each time for 30 min. The extracts were pooled, centrifuged to remove the disintegrated particles of paper and then lyophilized. The residue was taken up in a small volume of 50% ethanol for further analysis. RESULTS

bl

Rf

FIG.2. Paper chromatography of products of deaminoacyland glutaminyl-tRNA ation of (a) glutamyl (+O) (*--*), and of (b) aspartyl-)+ .( and asparaginyltRNA ( * - 0 ) using butano1:acetic acid: water as the solvent system. The positions of reference compounds on the chromatogram are indicated at the top horizontal lines of figures (a) and (b) by an arrow head accompanied by the appropriate symbol of the compound: G, glutamate; Gn, glutamine; PG, pyroglutamate; A, aspartate; and An, asparagine. The suffixes B,, Bz and B, (for the butanol solvent system) after the amino acid symbols signify different radioactivity peaks resulting from deaminoacylation of corresponding aminoacyl-tRNAs. Other experimental details were as in Materials and Methods. \ c 24Jh

I

B A W Chromatography of products of deaminoacylation of glutamyl-, glutaminyl-, aspartyl- and asparaginyltRNAs Deaminoacylation of glutamyl-tRNA gave rise to a major component (GB,) with approximately 85% of total radioactivity a n d 2 minor components (GB, and GB,) each containing 3-5% of radioactivity originally present in the deaminoacylated products (Fig. 2a). Comparison with standard compounds showed that GB,, GB2 and GB, had the same R , values as glutamine, glutamate a n d pyroglutamate. Analysis of the deaminoacylation products of glutaminyltRNA also showed 3 peaks, a major constituent (GnB,) with approx 80% total radioactivity and two minor components (GnB, and GnB,) each making u p for 5 7 % of radioactivity. GnB,, GnB, and GnB, had R P values very similar t o glutamine, glutamate and pyroglutamate. The above results indicated that deaminoacylation of glutamyl and glutaminyl-tRNAs yielded predominantly the labelled amino acid originally incorporated into tRNA. There was a small apparent interconversion of glutamate and glutamine and a slight formation of pyroglutamate from b o t h the amino acid

HUGUETTE Roux and M. R. V. MURTHY

1166

A A A and its amide. Pyroglutamate is known to be formed 0 Gn PG a) from both glutamate and glutamine by enzymatic as well as nonenzymatic cyclization of these amino acids (MESSER& OTTESEN,1964; GILBERT et al., 1949). Although pyroglutamate is found to be in the amino i l terminal positions of several proteins (CROFT,1973), it is not incorporated into brain tRNA under conditions of aminoacylation which favor attachment of other amino acids (Table 1). It is also reported to be not directly incorporated into protein (KITOS & WAYMOUTH, 1966; MOAV& HARRIS, 1967). It is possible that pyroglutamate could be formed at the level of aminoacyl-tRNA by an enzyme present in the reaction mixture, by a mechanism similar to that found in E . coli (BERNFIELD & NESTOR,1968), but it appears A” to be more probably a physicochemical consequence of the deaminoacylation procedure. As for the possibility of interconversion between glutamate and glutamine, even assuming the presence of some glutamine synthetase activity in the enzyme preparation used for aminoacylation of tRNA, the reaction mixture used for aminoacylation was not optimum for amidation of glutamate since no ammonium salt was present. Treatment of glutamyland aspartyl-tRNAs with strong methanolic ammonia has been reported to give rise to isoglutamine and isoasparagine respectively by ammonolysis (COLESet Rf al., 1962). Since glutamine and isoglutamine have very FIG.3. Paper chromatography of products of deaminoacylclose R F values in BAW solvent, it was deemed prob- ation of (a) glutamyl- ( t - 0 ) and glutaminyl-tRNAs able that GB, peak was due to isoglutamine produced (O---O) and of (b) aspartyl- (-0) and asparaginylby deaminoacylation of glutamyl-tRNA using tRNAs (=--a), using phenol :water as the solvent system. ammonium carbonate, as was done in our studies. The position of reference compounds on the chromatoThat this was indeed so was confirmed by high volt- gram are indicated at the top horizontal lines of figures age paper electrophoresis of GB1 in sodium diethyl (a) and (b) by an arrow head and the appropriate symbol of the compound as in Fig. 2. The suffixes P I and P2 barbiturate buffer at pH 8.5 (COLESet al., 1962). No deamidation of glutamine was observed under (for the phenol solvent system) after the amino acid symbols signify different radioactivity peaks resulting from the reaction conditions used for aminoacylation either deaminoacylation of corresponding aminoacyl-tRNAs. in the presence or absence of tRNA (MURTHY& Other details were as described in Materials and Methods. Roux, 1974~).The appearance of the small glutamate peak in the chromatogram of deaminoacylated glutaminyl-tRNA could be a n artifact of chromatography in the acidic BAW solvent, since, as will be shown later, no analogous peak was observed when aqueous phenol was used as solvent system (Fig. 3a). Deaminoacylation of aspartyl-tRNA and subsequent chromatography gave rise to 2 peaks, of TABLE1. INCORPORATION OF DICARBOXYLIC AMINO ACIDS which AB, containing more than 90% of total radioactivity had the same R , . value as pure aspartate INTO TRNA* (Fig. 2b). The minor peak AB1 with less than 5% Radioactivity in of radioactivity migrated in a manner very similar to aminoacyl-tRNA asparagine in the BAW solvent. However, by high Amino acid (c.p.m.) voltage paper electrophoresis, it was possible to iden12,560 [ ‘‘C]glutamate tify this compound as isoasparagine presumably 8420 [ I ‘C]glutamine formed by ammonolysis of aspartyl-tRNA in the 202 [ I ‘C]pyroglutamate course of deaminoacylation, analogous to the forma9560 [’‘Claspartate tion of isoglutamine form glutamyl-tRNA, as noted 7120 [ “Clasparagine above. The products of deaminoacylation of aspara* Aminoacylationwas carried out as described in Mater- ginyl-tRNA showed only one component AnB, with ials and Methods in 0.1 ml of final reaction mixture. The an R,; value very similar to pure asparagine. indicating ATP/Mg* ratio for pyroglutamate was arbitrarily fixed at the same level as for glutamate (YANG & NOVELLI, 1971). that no measurable transformation of this amino acid The aminoacyl-tRNAs were processed on filter paper-strips had occurred either prior to or after attachment to for counting. tRNA. +

1167

Reactions of dicarboxylic amino acids attached to tRNA

P W Chromatography of products of deaminoacylation of glutamyl-, glutaminyl-, aspartyl- and asparaginylt RN A s When glutamyl-tRNA was deaminoacylated and then chromatographed in the PW solvent, 1 major peak (GP,) and 1 minor peak (GP,) were obtained (Fig. 3a). The high proportion of radioactivity in GP, and its R F value indicated that it was ["C] glutamate originally used for esterification of tRNA. Comparison of the R F value of GP, with those of isoglutamine and pyroglutamate suggested that it could represent a mixture of these 2 compounds. This distribution of radioactivity was thus in accord with the results obtained by BAW chromatography (Fig. 2a). When the products of deaminoacylation of glutaminyltRNA were analysed by PW chromatography, 1 major peak (GnP,) and 1 minor peak (GnP,) were found. Using criteria similar to those described above, GnP,, was identified as [14C]glutamine possibly contaminated with a small amount of pyroglutamate. There was no peak corresponding to glutamate, as in BAW chromatography, indicating that its formation in the latter case was probably an artifact of the solvent system. One striking feature of the radioactivity profile obtained by PW chromatography was the appearance of the fast moving (RF0.9) component GnP, which could not be equated with any of the substances resolved previously with BAW chromatography. Following deaminoacylation, PW chromatography of aspartyl-tRNA resulted in the separation of two radioactive fractions, AP, and AP, (Fig. 3b). The relative proportion of radioactivity in these two fractions and their R F values indicated that they corresponded to aspartate and isoasparagine respectively, thus confirming the results obtained by BAW chromatography. The PW chromatogram of deaminoacylated asparaginyl-tRNA, again revealed in addition to all the components previously separated by BAW chromatography a new fast moving (RF 0.85) radioactive peak AnP,. In view of the frequent observation that paper chromatography under certain conditions could produce multiple spots for the same compound (SMITH, 1952; OVENSTON,, 1952; KELLER& GIDDINGS,1960; MURTHY& Roux, 1974b), the appearance of AnP, and GnP, were examined after subjecting asparagine and glutamine to a number of different reaction conditions. Table 2 shows that these peaks were found only in the products of deaminoacylation of asparaginyl- and glutaminyl-tRNAs. No significant radioactivity appeared at these RF values when chromatography was performed using the acid soluble fractions of a variety of reaction mixtures (incubated or unincubated, with intact tRNA, with depolymerized tRNA or without tRNA) or untreated ['4C] amino acid or ['"C] amino acid treated with ammonium carbonate under conditions identical to those used for deaminoacylation. This indicated that AnP, and GnP, were not artifacts of chromatography, but were

TABLE2. PAPER CHROMATOGRAPHY DEAMINOACYLATION OF ASPARAGINYL;

'

OF PRODUCTS OF AND GLUTAMINYL-

TRNAs* Reaction

Total radioactivity found (%I

I . Free radioactivity (a) complete reaction, no incubation (b) complete reaction. incubation, 40 mins (c) RNA omitted (d) RNase added

GnP, 2.3 2.1

AnP, 16 22

23 2-4

21 I .4

2. tRNA-bound radioactivity

14.3

29-6

2.9 3.4

I -6 33

3. Radioactive amino acids (a) ["C] amino acid, untreated (b) [ ' T I amino acid, after treatment with ammonium carbonate

* Aminoacylation and deaminoacylation of tRNA were carried out as described in Materials and Methods using 0.1 ml of reaction mixture. Two pg of pancreatic RNase was added to reaction Id. Free radioactivity in the reaction mixture was analysed at zero min (la), or at 40 min (Ib, lc & Id) of incubation. Aminoacyl-tRNA for experiment 2 was obtained by incubation of the reaction mixture for 40 min. The radioactive amino acids (glutamine and asparagine) used in these reactions were also chromatographed directly (3a), or after treatment with ammonium carbonate (3b) in a manner identical to that used for deaminoacylation of aminoacyl-tRNA. GnP, and AnP, refer to radioactivity peaks in Fig. 3. The total radioactivity recovered from the chromatogram of each sample was taken as 100 for calculating the per cent radioactivity found in the indicated peaks.

new compounds formed at the .level of the corresponding aminoacyl-tRNAs and not directly from the free amino acids. It is also seen from Table 2 that the amount of AnP, formed was approximately twice that of GnP, under similar conditions of reaction and analysis. The observation that the reaction giving rise to the above new compounds occurred a t the level of aminoacyl-tRNA does not, of course, resolve the question whether the transformation occurred in the course of aminoacylation of tRNA by a n enzyme present in the aminoacyl-tRNA synthetase preparation or whether it was produced as a result of subsequent non-enzymatic manipulations. Although the treatment used for deaminoacylation had no effect on the structure of pure amino acids (Table 2), it could have a n effect when the latter are esterified to tRNA. As a n alternate procedure, therefore, the aminoacyl-tRNAs were hydrolysed enzymatically under very mild conditions by incubating a t room temperature and in unbuffered solution at neutral pH in order to avoid any effects due to high ionic strength. Pancreatic RNase was selected for hydrolysis, because of the specificity of this enzyme and the unique structure of the tRNA molecule. which would assure that the amino acid or its transformation product, if still linked to tRNA, would be liberated as the adenosine derivative. The RNase digests were examined by high voltage paper electrophoresis taking only 1-2 h for separation as compared to 18-24 h for paper chromatography, thus reducing

1 I68

HUGUETTE Roux and M. R. V. MURTHY

TAIjLI 3 MIGRATION CHARACTI RlSTlCS OF

THI PRODUCTS OF RNASt HYDROLYSIS OF GLUTAMYL- A N D GLUTAMINYL-tRNAS IN P A P t R ELFCTROPHORtSlS A N D PAPER CHROMATOGRAPHY*

Radioactivity found in each peak Electrophoretic peaks

2. Glutaminyl-tRNA Gnf

GnE, GnE, GnE,

(%I

EP(cm)

Migration BAW (RF)

PW(RF)

0.28(54) 0.63(29) 0.29 0.28 0.26(70) 0.34( 12)

0.22 0.60 0.23 0.23 0.24 -

0.51 0.52 0.52

8.5

+ 6.0

88.6 19.7 7 61

- 2.0 - 1.8 - 7.0

91.2 26.1 46. I 19.2

- 4.0 - 3.7 - 6.0 - 18.0

0.16 0.17 0.17 0.17

diffuse

96.5 95.8 90.2 90.6

- 2.0

0.28 0.16

0.22 0.5 1

-

-

-

-

3. Standards

['4C]Glutamate

[14C]Glutamine ['4C]Glutamatet [ l 4C]Glu taminet

- 4.0 - 2.0 - 4.0

* RNase hydrolysates of glutamyl- and glutaminyl-tRNAs were first subjected to paper electrophoresis (EP) as in Fig. 4, from which % radioactivity and electrophoretic mobility (EP) values for the various peaks were calculated. Paper strips corresponding to each of the electrophoretic peaks were then extracted with distilled water and the extracts were chromatographed as described in Materials and Methods using butanol :acetic acid:water (BAW). The radioactivity peaks obtained in this manner were reextracted with distilled water and each fraction was rechromatographed using pheno1:water (PW) as solvent system. The electrophoretic peaks Gfl and GE2 were each resolved into two components after BAW chromatography. The figures in parenthesis indicate the % of radioactivity found in these subcomponents. The total % does not add up to 100 since some radioactivity was distributed all along the chromatogram with no identifiable peaks. t ['4C]glutamate and [14C]glutaminewere treated with RNase under conditions identical to those used for hydrolysis of aminoacyl-tRNA. the time of exposure to possible degradation by solvents. The free radioactivity fractions (isolated by DEAE-cellulose chromatography from the aminoacylation reaction mixtures) were also simultaneously analysed by paper electrophoresis.

Electrophoresis of the RNase hydrolysate of [14C]glutamyl-tRNA revealed two radioactivity peaks at distances of - 1.8 cm (GE,) and - 7.0 cm (GE,) from the origin (Fig. 4a). Approximately 20% of total radioactivity was found in GE, (Table 3), which showed no absorption when viewed under U.V. Paper electrophoresis of tR N A bound (RNase digests) light. This peak had a n electrophoretic mobility and and free radioactivities following aminoacylation with R , values in BAW and PW solvents very similar ['4C]glutamate and ['4C]glutamine to those of pure glutamate. GE1 could, therefore, When [I4C]glutamate was used for aminoacyl- correspond to glutamate produced from glutamylation, the free amino acid fraction in the reaction mix- tRNA by spontaneous deaminoacylation. We have ture gave 1 minor (Gf,) and one major (Gf,) radioac- observed that even a short time in aqueous solution tivity peak at + 6.0 cm and -2.0 cm respectively at room temperature produces measurable deaminofrom the origin (Fig. 4a). The occurrence of the large acylation of dicarboxylic aminoacyl-tRNAs (MURTHY This question will be further discussed majority of total radioactivity in Gf, (88.6%) and & Roux, 1974~). et al., 1975). similarity of its chromatographic and electrophoretic in the following communication (MURTHY mobilities with pure glutamic acid (Table 3), would The peak GE, was attributed to glutamyladenosine identify this peak as being mainly due to [I4C]gluta- because of ultraviolet absorption in this area, its faster mate. Chromatography of peak Gfl using the BAW migration toward the cathode (- 7.0 cm as compared to solvent system showed the presence of two products -1.8cm for GE,) and the presence of the majority with R , values of 0.28 and 0.63 (Table 3). The slower of radioactivity (76.1%) in this peak. There were a moving component had the majority of radioactivity number of fluorescent and U.V. absorbing spots (54%) and R F values identical to that of glutamate migrating toward the anode due to oligonucleotides in both solvent systems. It, therefore, probably repre- of various chain length present in the RNase digest. sented a tailing of the main glutamate peak. The The small area circumscribing GE2 with U.V. absorpR , values of the faster moving component with 29% tion would not obviously be entirely due to [14C]gluof the radioactivity of Gf, were very similar to that tamyladenosine which would be expected to be presof pyroglutamic acid in both BAW and PW solvents. ent in very minute amounts, but due to a mixture

Reactions of dicarboxylic amino acids attached to tRNA

1169

ined under U.V.light. Based on arguments similar to those followed above, GnE,, was attributed to glutamine formed as a result of spontaneous deaminoacylation of glutaminyl-tRNA, approximately Gf2 A 26% of the total radioactivity being liberated in this E 2 manner (Table 3). The peak GnE, constituting about " 46% of radioactivity was identified as being mainly I glutaminyladenosine. Glutamine and glutaminyl0 adenosine, like glutamate and glutamyladenosine, had Gnf I identical R , values when chromatographed in BAW or PW solvents. One striking difference between the electrophoretic profiles of RNase degrdation products of glutamyl-tRNA and glutaminyl-tRNA was the appearance in the latter of a rapidly moving third peak GnE, (-18.0 cm from the origin). When the material in this peak was extracted and chromato2 graphed in the BAW solvent, it migrated in a manner I identical to glutamine and glutaminyladenosine (Table 3). However, when chromatographed using the 20 10 0 10 20 30 cmr Migrotion from oriqin lcmr) PW solvent, it exhibited an uneven distribution of FIG.4. Paper electrophoresis of RNase hydrolysates of radioactivity throughout the length of the chromatogram without any well defined peaks. (a) glutamyl-tRNA and (b) glutaminyl-tRNA. ( t - O ) ,

I

st

0)

GEz

I

I

1I

free radioactivity; (O -o), tRNA bound radioactivity. The free radioactivity and the [14C]aminoacyl-tRNAs were separated by DEAE-cellulose chromatography as in Fig. 1. The aminoacyl-tRNAs were then hydrolysed with RNase prior to electrophoresis (Materials and Methods). The following symbols are used: G, glutamate; Gn, glutamine. The suffixes f l and fi after the amino acid symbols signify different radioactivity peaks separated from the free amino acid fraction of the reaction mixture. The suffixes E,, Ez and E3 (for electrophoresis) following the amino acid symbols indicate the radioactive components separated from the RNase hydrolysates of the respective aminoacyl-tRNAs.

of a number of nonradioactive aminoacyladenosines produced by RNase hydrolysis of the mixed aminoacyl-tRNAs. When the radioactive substance in peak GE, was extracted and chromatographed in the BAW solvent, one major component ( R , 0.26) with 70% of radioactivity and one minor component ( R , 0.34) with 12% of radioactivity were observed (Table 3). The identity of the minor component has not been investigated because of quantitative limitations. The major component, presumably glutamyladenosine, had the same R , values in both BAW and PW solvents as pure glutamate. We have observed that adenosine derivatives of not only glutamate, but also of glutamine, aspartate and asparagine are inseparable from the corresponding free amino acids by either BAW or PW chromatography. Paper electrophoresis has proved to be the best means for their separation. When ["C]glutamine was used for aminoacylation, the free amino acid fraction gave only one sharp peak (Gnf) corresponding to glutamine (Fig. 4b). RNase hydrolysis of ['4C]glutaminyl-tRNA, on the other hand, revealed 3 different radioactive substances GnE,, GnEz and GnE,. Of the 3, only the area encompassing GnE, showed absorption when exam-

Paper electrophoresis of t R N A bound (RNase digest) and p e e radioactivities following aminoacylation with [ 14C]asparratr and [14C]asparagirw The free radioactivity in the reaction mixture after aminoacylation of tRNA with ['4C]aspartate gave, on electrophoresis, a single peak (Af) at + 2.0 cm from the origin (Fig. 5a). Its electrophoretic migration and R F values in BAW and PW solvents were identical to those of pure [14C]aspartate (Table 4). This peak contained 96.2% of all radioactivity recovered

4 c)

0

x

3

52 U I

0

Miprotion from origin (cms)

FIG. 5. Paper electrophoresis of RNase hydrolysates of (a) aspartyl-tRNA and (b) asparaginyl-tRNA. (*a), free radioactivity; ( O - O ) , tRNA bound radioactivity. The experimental protocol was as in Fig. 4 and in Materials and Methods. The following symbols are used: A, aspartate; An, asparagine. The suffixes f, El, Ez and E3 have the same significance as in Fig. 4.

HUCUETTE Roux and M. R. V. MURTHY

1170

TABLE 4.

MIGRATION CHARACTERISTiCS OF THE PRODUCTS OF RNASEHYDROLYSIS OF ASPARTYL A N D IN PAPER ELECTROPHORESIS AND PAPER CHROMATOGRAPHY*

Radioactivity found in each peak Electrophoretic peaks

(77)

EP(cm)

96.2 14.1 83.2

+ 2.0 + 1.8

I A.V/~a~tpl-tR NA I

Af

AE, AE2

- 7.0

ASPARAGINYL-TRNAS

Migration BAW(RF)

PW(RF)

0.17 0.17 0.17

0.12 012 0.13

010 0.10 0.10 0.10(47) 0.33 (18)

0.30 0.31 0.3 1 0.28

2. Asparayinyl-tR N A

Anf AnE AnE, AnE,

90.6 22.7 24.0 48.2

- 4.0

- 4.0 - 8-0 -21.0

diffuse

3. Standards [ '4C]Aspartate [ I4C]Asparagine [ 14C]Aspartatet [ 14C]Asparaginet

96.0 95.3 95.8 92.5

i2.0

- 4.0 + 2.0 - 4.0

0.17 010

0.12 0.3 I -

-

* Experimental details were as in Table 3. The nomenclature of electrophoretic peaks were as in Fig. 5. t [ I4C]Aspartate and [14C]asparaginewere treated with RNase under conditions identical to those used for hydrolysis of aminoacyl-tRNA.

from the chromatogram indicating that aspartate had not undergone measurable structural alterations in the course of incubation. In contrast to this, RNase hydrolysis of aspartyl-tRNA gave 2 electrophoretic peaks, one of which (AE,) was identified as aspartate, based again on its electrophoretic and chromatographic properties (Table 4). AE1 occurred to the extent of approximately 14%. The peak AE, was identified as aspartyladenosine due to its U.V.absorption, high proportion of radioactivity (83.2%) and relatively rapid migration toward the cathode ( - 7.0 cm for AE2 as compared to $2.0 for pure aspartate). Although AE, & AE, were distinctly separable by paper electrophoresis, when they were extracted and then rechromatographed in the BAW or PW solvents, they were both found to have the same R , value (Table 4). Free [I4C]asparagine in the aminoacylation reaction mixture did not appear to be significantly transformed, since more than 90% of radioactivity not bound to tRNA was recovered in a single peak migrating in electrophoresis and chromatography (both BAW and PW solvents) in a manner identical to pure [I4C]asparagine (Fig. 5b and Table 4). Treatment of asparaginyl-tRNA with RNase, on the contrary, released 3 different radioactive substances migrating at -40 cm (AnE,), -8.0 cm (AnE,), and -21.0 (AnE,) respectively from the origin. Of these, only AnE, exhibited U.V. absorption. Based on reasoning similar to those employed previously for radioactivity peaks of other amino acids, AnE, and AnE, were identified as asparagine and asparaginyladenosine. The rapidly migrating peak AnE, was present in the RNase hydrolysate of only ['4C]asparaginyltRNA and not aspartyl-tRNA (Fig. 5). This difference was similar to that found between RNase hydroly-

sates of glutamyl and glutaminyl-tRNAs (Fig. 4). However, the relative. proportion of AnE, was much higher (48.2% of all the radioactive products liberated from asparaginyl-tRNA) than that of the analogous glutaminyl derivative GnE, (19.2% of all the radioactive products liberated from glutaminyl-tRNA). The proportion of asparaginyladenosine, AnE, represented only 24% of the total radioactivity and approximately half of the radioactivity found in AnE,. When AnE, was extracted and chromatographed in BAW solvent, it was resolved into two main components: a slow moving substance (RF 0.10) containing 47% of radioactivity and a faster moving material (RF 0.33) with 18% of radioactivity (Table 4). When these 2 substances were reextracted and rechromatographed, this time in P W solvent, the fast component gave no distinct peaks, but was distributed diffusely along the length of the chromatogram. The slow component had an R , value which was similar to that of asparagine or of asparaginyladenosine (AnE,). DISCUSSION A single species of radioactive aminoacyl-tRNA should theoretically give, after treatment with pancreatic RNase, only one labelled aminoacyladenosine. If more than one radioactive substances are detected in the RNase digest, as was the case in the present experiments, the following may be considered among possible explanations. (1) The radioactive amino acid used for aminoacylation of tRNA was not pure, but was contaminated with one or more other amino acids which could also be simultaneously incorporated into tRNA. Since both the tRNA and the aminoacyl-tRNA synthetases in these studies were mixtures and not homogeneous preparations specific for a given amino acid, the number of different radioactive

Reactions of dicarboxylic amino acids attached to tRNA

1171

aminoacyl-tRNAs formed would be determined by electrophoretic mobility between GnE, and GnE, the number of radioactive protein amino acids pres- and between AnE, and AnE,; (2) the rapidly migratent in the reaction mixture; (2) the radioactive amino ing peaks were found only for glutamine and asparaacid used in the reaction could be partly converted gine and not for glutamate and aspartate; (3) more into one or more other amino acids in the course than one radioactive peak were obtained not only of aminoacylation reaction, with the result that both when glutaminyl- and asparaginyl-tRNAs were the parent and the newly formed amino acids would treated with RNase, but also when they were debe incorporated into tRNA. Such a possibility could aminoacylated. In the latter case, isomerism due to be very tangible in the case of dicarboxylic amino adenosine would not be involved. The possibility could be considered that glutamate acids and their amides because of the presence of a number of reactive sites in these molecules; (3) and glutamine as well as aspartate and asparagine the treatment used for the hydrolysis of aminoacyl- could undergo a small degree of interconversion tRNA could cause structural alterations of the through amidation or deamidation reactions cataaminoacyl or aminoacyladenosine moieties giving rise lysed by brain extracts. If a significant amount of to substances with different chromatographic or elec- such interconversions occurred in the course of trophoretic mobilities; (4) the amino acid could have aminoacylation reaction, two different radioactive undergone structural alterations at the level of the aminoacyladenosines could be formed after subaminoacyl-tRNA catalysed by an enzyme present in sequent RNase hydrolysis of aminoacyl-tRNAs. In some experiments, a very small amidation of glutathe extracts used for aminoacylation. Paper chromatography as well as paper electro- mate was noticed in the absence of tRNA (MURTHY phoresis of radioactive glutamate, glutamine, aspar- & Roux, 1974a), possibly due to absorption of tate and asparagine used in these experiments for atmospheric ammonia since no added ammonium aminoacylation of tRNA gave a single major peak salts were present in the reaction mixture. However, for each of these amino acids, with more than 95% no deamidation of glutamine or asparagine were of total radioactivity when examined by electrophor- observed. Even assuming such an interconversion, esis (Tables 3 and 4). Treatment of these amino acids electrophoretic analysis of the radioactive comby ammonium carbonate or by RNase under condi- ponents in the RNase digests of asparaginyl- and glutions identical to those employed for releasing the taminyl-tRNAs showed that the unidentified products amino acids or aminoacyladenosines from aminoacyl- present in each case (AnE, and GnE, respectively) tRNAs did not lead to significant formation of deriva- were different from any of the adenosine derivatives tives or degradation products (Tables 2-4). On the of the dicarboxylic amino acids and their amides (Fig. other hand, when the same treatments were applied 4 and 5). Similarly, the unidentified products of deto [ 4C]glutaminyl- and 4C]asparaginyl-tRNAs, aminoacylation (AnP, and GnP,) had R F values in several radioactive substances were found for each PW solvent much different than those of any of the tRNA when examined by chromatography or by elec- dicarboxylic amino acids or their amides (Fig. 3). New radioactive compounds other than the oritrophoresis. Two of the products of RNase hydrolysis could be due to the appropriate amino acid and ginal amino acids were liberated in measurable aminoacyladenosine, but at least one other substance amounts by deaminoacylation of only asparaginyland glutaminyl-tRNAs and not of aspartyl- and glutain each case remained to be identified It has been reported that aminoacyladenosines iso- myl-tRNAs (Fig. 3). Experiments using RNase instead lated from aminoacyl-tRNA contained a mixture of of ammonium carbonate for recovering the radioac3‘ and 2 esters of amino acids (FRANK & ZACHAU, tive components confirmed the above observation 1963; WOLFENDEN et al., 1964; FELDMAN & ZACHAU, that the amides of the two dicarboxylic acids were 1964). This has been attributed to acyl migration dur- specifically involved in such transformations (Figs. ing isolation procedures; however, the rate of acyl 4 and 5). This specificity by itself, however, is not migration even under physiological conditions is sufficient to indicate whether the new compounds found to be so rapid that it has been suggested that were formed as a result of an enzymatic reaction all free aminoacyl-tRNA could exist as an equilibrium and remained attached to tRNA prior to deaminoacylmixture of 2 - and 3’ isomers (MCLAUGHLIN & ation, or whether they were formed from the normal INGRAM,1965; GRIFFINet al., 1966). If this were the aminoacyl-tRNAs by a physiochemical mechanism case for the dicarboxylic amino acids, one would subsequent to their isolation from the aminoacylation expect that RNase hydrolysis of the aminoacyl- medium. This question and the identification of the tRNAs would produce two isomeric aminoacyl- new products will form the subject matter of the adenosines for each of these amino acids. The follow- following communication (MURTHY rt a/., 1975). ing arguments can be cited against the possibility that the peaks GnE,, and GnE, of Fig. 4b and peaks Acknowledgements-The authors wish to thank the MediAnE, and AnE, of Fig. 5b were not isomeric forms of cal Research Council of Canada, the Ministry of Educaglutaminyladenosineand asparaginyladenosine respec- tion, Government of Quebec and Le Conseil de la tively. (1) The difference in charge between the 2 and recherche en sante du Quebec for research grants to carry 3’ isomers of the above aminoacyladenosines would out this work. The excellent technical assistance of Mrs. not be enough to produce such large differences in UMAMANI RAO is appreciated.

[’

1 I72

HUGUETTE Roux and M. R. V. MURTHY

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