Characterization of tRNA-nucleotidyltransferase from the Moss Ceratodon purpureus

Biochem. Physiol. Pflanzen 111, S. 239-248 (1977)

Characterization of tRNA-nucleotidyltransferase from the Moss Ceratodon purpureu8

z. SCHNEIDER and J. SCHNEIDER Institute of Biochemistry, Academy of Agricultural Sciences, and Institute of Biology, Adam Mickiewicz University, Poznan, Poland Key Term Index: tRNA-nucleotidyltransferase, enzyme purification and characteristics, moss protonema; Ceratodon purpureus.

Summary The tRNA-nucleotidyltransferase (EC 2.7.7.25) from the protonema of the moss Ceratodon purpureus has been highly purified by ammonium sulphate fractionation and chromatography by means of Sepharose 6B. The Km values for CTP (2.8 x 10- 5 M), ATP (3.3 x 10- 5 M) and tRNA (23.8 x 10- 7 M) were determined. The molecular weight of the enzyme is 63,000 daltons as estimated by gel filtration on Sephadex G-100 column. The enzyme is stabilized by tRNA and CTP but not ATP. Its properties with those of the corresponding enzymes from higher plants, bacteria and animals are compared.

IntroductioR

One of the grratly specific enzymes of the nucleotidyl polymerases group is tRNAnucleotidyltransferase (EC 2.7.7.25) which in the presence of both CTP and ATP catalyzes the synthesis/reparation of 3' terminal C-C-A sequence common to all active tRNA molecules. Since the biological activity of tRNA is limited by its intact 3'end, the enzyme may playa significant role in the regulation of the protein synthesis. The present paper is a continuation of our previous work on the tRNA-nucleotidyltransferase from the protonem a of the moss Ceratodon purpureus (SC;HNEIDER and SZWEYKOWSKA 1975). Enhancement of the activity of this enzyme subsequent to cytokinin treatment of the proton em a (as found previously in the crude extract), inclined us to carry out investigation on the purification and chara.cterization of the enzyme. It seems more so valid that most so far available data concerning tRNA-nucleotidyltransferase originate from animal (DEUTSCHER 1973; HECHT et 801. 1958; CHUNG et 801. 1960; DANIEL and LITTAUER 1963) and microbial (FURTH et a1. 1961; PREISS et a1. 1961; CARE et 801. 1970; MILLER and PHILIPS 1971) materials. In regard to higher plants, only two papers appeared recently describing tRNA-nucleotidyltransferase from Lupintts seeds (CUDNY et 801. 1975) and wheat embryos (DULLIN et 801. 1975). It was thus interesting to compare properties of enzymes isolated from plants belonging to completely different taxonomic groups. 17 Bioohem. Physiol. Pflanzen, Bd. 171

240

z. SCHNEIDER and J. SCHNEIDER Material and Methods

Sources of chemicals

[14C] CTP (specific activity 53 mCi/mmol) and (l4C] ATP (specific activity 50 mCi/mmol) were from Radiochemical Centre Amersham, England. ATP, CTP, 5 AMP, 5', 3' cAMP, enzymes and standard proteins from Boehringer, W. G. Tris from Koch-Light Laboratory Ltd., England. Sepharose 6B from Pharmacia Fine Chemicals, Sweden. Yeast specific tRNA and phenylalanyl-tRNA were received from Dr. W. KEDZIERSKI. Other chemicals were from Polskie Odczynniki Chemiczne Gliwice, ' Poland. Plant material and isolation of the enzyme

The enzyme was isolated from sterile, vegetatively propagated culture of the protonema of the moss Ceratodon purpureus (L. ap. Hedw.) Brid. cultivated in vitro. The growing conditions and the method of isolation of the crude enzyme was described previously (SCHNEIDER and SZWEYKOWSKA 1975). Purification of the enzyme

Crude extract of the enzyme was fractionated with ammonium sulphate. About 70% of activity has been found in fraction precipitated within the range of 35-75% of saturation. The precipitate was dialysed against 50 mM Tris-HCl buffer, pH 8.4, 5 mM MgCl z and 10% glycerol for 1 hour and next purified by gel filtration on Sepharose 6B column. The procedure of this step was described in "Results" section (Fig. 3). The most active fractions after Sepharose chromatography were free of endogenous tRNA, which has been ascertained on the basis of the lack of acceptor activity towards eMP (without addition of exogeneous tRNA) and the high ratio ~280 (1.45). The preparation obtained 260

with the procedure described above was about 500 times purified. Electrophoretic separation of this preparation on 7% polyacrylamide gel showed 3 bands. The enzyme is highly unstable at temperatures above 0 °C and without glycerol, however, if freezed at -18°C in the presence of 10% glycerol it may be stored for a year without less of activity even if highly diluted. A further purification of the enzyme tried by several methods was unsuccessful, also the active enzyme could not be located among the 3 bands of polyacrylamide gel after electrophoretic separation. Moreover, limited amounts of the protonema source of the enzyme made it impossible to carryon further purification. Preparation of tRN A-X deprived of 3' terminal nucleotides

Purified preparation of phenylalanyl tRNA (60 % purity) was digested for 1 hour by snake venom phosphodiesterase in an 1 ml incubation mixture containing: 200 units (5 mg) tRNA, 14 mM MgCl 2 , 96 mM Tris-HCl pH 8.4 and 100 fig snake venom phosphodiesterase. Because of the limited amounts of specific tRNA, conditions for the digestion wue worked out by the use of an unpurified tRNA preparation from yeast. Fig. 1 shows the effect of the time of digestion on acceptor capacity of tRNA with regard to CMP incorporation. As can be seen from the plot, the optimal time for digestion is 60 minutes and next a slow decrease of acceptor capacity of tRNA has been observed, probably due to the hydrolysis of the fourth terminal nucleotide. The results seem to confirm the reports of other authors in this respect. Enzymatic preparation of the tRN A-XCC

This substrate was enzymatically prepared by incorporation of two CMP residues to tRNA-X (obtained by the method described above) with the use of purified tRNA-nucleotidyltransferase. The reaction mixture contained in 300 fil: 20 optical units of yeast tRNA_xphenylalanyl, about 20 fig purified tRNA-nucleotidyltransferase, 1 mM CTP, 15 mM MgCl 2 and 50 mM Tris-HCl buffu, pH 8.4. The mixture was incubated at 30°C for 2 hours, followed by heating at 60 °0 for 3 minutes. The mixture was dialyzed against 50 mM Tris-HCl buffu, pH 7.5 for 2 hours. Because of limited amounts of specific yeast tRNAphenylalanyl the paramet~rs for this procedure were worked out in a pilot experiment (described in the legende of Fig. 2) with the use of a mixture of tRNAs from yeast.

tRNA-nucleotidyltransferase from Moss

241

Determination of the tRN A-nucleotidyltransferase activity

Activity of the enzyme was estimated in incubation mixture containing: 50 mM Tris-HCl buffer, pH 8.0, 5 mM MgCl 2 , 5-10,tg tRNA-X, 0.1 mM 14C CTP and 0.1-5Itg purified enzyme's prepara-

tion. The incubation was carried out at a temperature of 30°C for times suitable for each kind of experiment (see "Results"). The volume of the incubation mixture was according to needs: 25, 60, or 100 Itl. The reaction was stopped by cooling following addition of 10 Itl of mixture containing: 70 Itg CTP, 0.15 M Na 4 P2 0 7 and 0.05 M Tris-HCl buffEr, pH 8.0. The whole sample was collected on a filter paper disk (What man 3 MM) and immErsed inicecold 10 % trichloroacetic acid with 0.9% Na4 P 2 0 7 • The acid insoluble product retained on the filter paper disk was washed by a cool 5% trichloroacetic acid containing 0.9 % Na4 P 2 0 7 and finally with 96 % ethanol. The dry filters were placed in toluene scintillator (0.1 g of POPOP and 1 g of PPO in 1 litre of toluene) and radioactivity was measured in Packard Tri-Carb scintillation counter. Estimation of the mJlecular weight of the tRN A-nucleotidyltransferase

This was carried out by the use of a Sephadex G-100 column at conditions described in diagram of Fig. 4. Prokin concentration was determined by the method of LOWRY et al. (1951).

Results and Discussion

The present work supports our previous finding (SCHNEIDER and SZWEYKOWSKA 1975) that the enzyme isolated from the protc,nema of the moss Ceratodon purpnreus is a typical tRNA-nucleotidyltransferase. Because of limited amount of plant material available, methods of purification were selected which yielded high recovery of the enzyme. Fractionation with ammonium sulphate of the crude enzyme followed by the chromatography on Sepharose 6B at temperature 8 °C in 10 % of glycerol proved to be very useful for this purpose (Fig. 3). Almost 60% of highly purified enzyme was recovered from Sepharose 6B gel filtration. Fractions of the highest specific activity, free of RNA polymera'le, phosphatases and ribonucleases were used for kinetic characterization of the enzyme. The molecular weight of the enzyme (63,000 daltons) estimated on Sephadex G-100 is close to the molecular weight of corresponding enzyme from E. coli (MILLER and PHILIPPS 1971). The estimation of kinetic constants of the enzyme require substrates tRNA-X and tRNA-XCC commercially not available. These substrates were obtained by enzymatic degradation of specific phenylalanyl-tRNA from yeast of about 60 % purity. As can bee seen from Fig. 1. one hour digestion at the chosen conditions is optimal to get a tRNA of the highest acceptor ability. The preparation so obtained deprived of three terminal nucleotide residues wa3 used to an enzymatic preparation of the tRNA-XCC. Investigations of DEUTSCHER (1973) and CARRE et CHAPEVILJ,E (1974) show that tRNA-nucleotidyltransferase incorporates in vitro three or more CMP residues however, the rate of abnormal incorporation residues are slower than the normal synthesis of the first two CMP. Data given in Fig. 2 show two phases of the incorporation of CMP residues as a function of time: fast incorporation proceeds within two first hours and slow one after longer times. Twohour-incubation time was considered to be optimal to get tRNA-XCC. Investigation of the yield of AMP incorporation after different times of preincubation of tRNA-X with CTP confirmed this 17"

z. SCHNEIDER and J. SCHNEIDER

242

Table 1. Effect of some reagents on the activity of tRN A-nucleotidyltransferase from the moss Ceratodon purpureus The volume of incubation mixture- was 100 ftl. Other conditions were standard. 2iP concentrations used were 0.5 1, 10, 100 and 1000 I'M respectively Reagent added

Concentration

Activity in of control

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Fig. 1. Time-course of tRN A digestion by phosphodiesterase on the acceptor capacity of tRN A from yeast. Incubation mixture for tRNA digestion contained in a volume 12501'1: 200 units (5 mg) tRNA, 14 roM MgCl2 , 96 mM Tris-HCl buffer, pH 8.4, and lOO.ug snake venom phosphodiesterase. After various incubation times, carried out at 37°C, 1001'1 samples of incubation mixture were heated 5 minutes at 80°C (for enzyme inactivation), diluted 20 times with H 2 0 and used for acceptor capacity estimation. Fig. 2. Time-course of (14C] CMP incorporation into tRN A-X. The incubation mixture contained in 5001'1: 5 units of tRNA-X, 0.1 mM [14C] CTP and 20 .ug of the purified tRN A-nucleotidyltransferase. Other conditions were standard. At various times of incubation 30 1'1 sampl~s were withdrown and the incorporation of [14C] CMP into tRNA-X measured.

243

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a3sumption. The above described substrates tRNA-X and tRNA-XCC (after thermal inactivation of the enzymatic proteins and exhaustive dialysis) were used to determine kinetic constants of the enzyme. As reported in fig. 5 the Km for tRNA-X was estimated as 23.8 x 10-7 M. This value is close to the data cited for other tRNA-nucleotidyltransferases (CARRE et al. 1974). Also the Km for CTP 2.8 x 10-5 M and ATP 3.3 X 10-5 M (Fig. 6 and 7, respectively) are of the same magnitude as for enzymes from other organisms (CARRE et al. 1970). The purified enzyme preparation was also examined with respect to the effect of some inhibitors and of cytokinin (6-Ll2-isopentenylaminopurine). No change in enzyme activity was found in the presence of the wide range cytokinin (2iP) concentrations in the incubation mixture (Table 1). Thus the result excludes an allosteric mechanism of the cytokinin-induced increa3e in tRNA-nucleotidyltransferase activity (SC;HNEIDER and SZWEYKOWSKA 1975). Besids several nucleotides vitamin B12 revealed a small inhibitory property in the investigated enzymatic preparation (Table 1). In further investigation on the effect of salt (Fig. 4) it has been found that NaCI has inhibitory effect already at low concentration. KCI below 0.10 M cause a slight stimulation whereas higher salt concentrations inhibit CMP incorporation although to a less degree than NaCl. Similar results !:ave been obtained for tRNA-nucleotidyltransferase from E. coli (CARRE and CHAPEVILLE 1974). Results of experiments concerning the protection of the enzyme by the substrates against thermal inactivation are also in agreement with tho~e

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column. The column, 1.5 x 91 cm, cooled to 5°C, was equilibrated with 50 mM Tri- HOI buffer, pH 8.8, in 10 % glycerol. Purified or crude preparations of nucleotidyltransferase were applied in 1.5 ml volumes. Aliquots, 20 pI from each of 2 ml fraction were taken for enzyme activity determination. The column was calibrated in a separate run with alkaline phosphatase from calf intestine (100,000 daltons), bovine albumine (67,000 daltons) and horse myoglobin (17,500 daltons). Kav value of 0.175 was determined for tRNA-nucleotidyltransferase which corresponds to a molecular weight of 63,000 daltons. 70

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tRNA-nucleotidyltransferase from Moss

247

reported for the E. coli enzyme (MILLER and PHILIPPS 1971a; CARRE et al. 1974). It can be seen in Fig. 9 that only tRNA and CTP, in contrast to ATP, stabilize the enzyme against thermal inactivation. In such properties as requirement of divalent ion Mg+2 or Mn+ 2 and pH (estimated previously by SCHNEIDER and SZWEYKOWSKA 1975), the enzyme isolated from the moss protonema is similar to those obtained from Lupinus luteus seeds (CUDNY et al. 1975) and from R. coli (CARRE et al. 1970) but differes (especially in respect to pH) from wheat embryos tRNA-nucleotidyltransferase, showing two pH optima (DULLIN et al. 1975) and from rat liver enzyme (DANIEL and LIT TAUER 1963). Small amounts of the plant material available and lability of the enzyme preparation limited a further purification and more precize characterization of the tRNA-nucleotidyltransferase from the proton em a of the moss Ceratodon purpureus. Since the partialy purified enzyme is free of nucleases and phosphatases it has been successfully used in our laboratory as a reagent to restore amino acyl acceptor ability of some specific lupin tRNAs. Acknowledgements ' The authors are thankful to Prof. Dr. ALIOJA SZWEYKOWSKA for her interest and critical reading of the manuscript. Grateful thanks are also due to Dr. WOJCIECH KEDZIERSKI for a gift of phenylalanyl tRN A from yeast. This work was supported by the Polish Academy of Sciences within the project 09.3.1. , ,

,I

References CORRE, S. D., LITVAK, S., and CHAPEVILLE, F., Purification and properties of Escherichia coli CTP (ATP)-tRNA nucleotidyltransferase. Biochim. Biophys. Acta 224, 371-381 (1970). - CHAPEVILLE, F., Study of the Escherichia coli tRNA nucleotidyltransferase. Specificity of the enzyme for nucleoside triphosphates. Biochimie 06,1451-1457 (1974). - LITVAK, S., and CHAPEVILLE, F., Study of the Escherichia coli tRNA nucleotidyltransferase. Interaction of the enzyme with tRNA. Biochim. Biophys. Acta 361,185-197 (1974). CUD NY, R., PIETRZAK, M., and BARTKOWIAK, S., tRNA-nucleotidyltransferase activity in Lupinus luteus seeds. Phytochemistry 14, 85-87 (1975). CHUNG, C. W., MAHLER, R. R., and ENRIONE, M., Incorporation of adenine nucleotide into ribonucleic acid by cytoplasmic enzyme preparation of chick embryos. J. BioI. Chern. 230, 1448 (1960); DANIEL, V., and LITTAUER, Z. U., Incorporation of terminal ribonucleotides into soluble ribonucleic acid by a purified rat liver enzyme. J. BioI. Chern. 2JJ8, 2102-2112 (1963). DEUTSCHER, M. P., Reactions at the 3'terminus of transfer ribonucleic acid. VI. Properties of the poly (C) polymerase activity associated with rabbit liver transfer ribonucleic acid nucleotidyltransferase. J. BioI. Chern. 248, 3108-3115 (1973). DULLlN, P., FABISZ-KIJOWSKA, A., a,nd WALERYCII, W., Isolation and properties of tRNA nucleotidyltransferase from wheat embryos. Acta Bioch. Pol. 22, 279-2E9 (1975). FURTH, J ..J., HURWITZ, J., KRUG, R., and ALEXANDER, M., The incorporation of adenylic and cytidylic acids into ribonucleic acid. J. BioI. Chern. 236, 3317-3322 (1961). HECHT, L. I., ZAMECNIK, P. C., STEPHENSON, M. L., SCOTT, F. J., Nucleoside triphosphates as precursors of ribonucleic acid end groups in a mammalian system. J. Biol. Chern. 233, 954-964 (1958). LOWRY, O. R., ROSEBROUGH, N. J., FARR, A. L., a::<1 R.\ND.\LL, J. R., P~'otein 'measurement with the Folin phenol reagent; J. BioI. Chem. 193, 265-275 (1951).

248

Z. SOHNEIDER and J. SOHNEIDER, tRNA-nucleotidyltransferase from Moss

MILLER, J. P., and PHILIPPS, R. G., Transfer ribonucleic acid nucleotidyltransferase from Escherichia. coli. II. Purification, physical properties and substrate specificity. J. BioI. Chern. 246, 1274-1279 (1971). - and PHILIPPS, R. G., Studies on thermal inactivation of transfer ribonucleic acid nucleotidyltransferase from Escherichia coli. Biochemistry 10, 1001-1007 (1971a). PREISS, J., DIEOKMANN, M., and BERG, P., The enzymic synthesis of amino acyl derivatives of ribonucleic acid. IV. The formation of the 3' -hydroxyl terminal trinucleotide sequence of aminoacid-acceptor ribonucleic acid. J. BioI. Chem. 236 (1748-1757 (1961). SOHNEIDER, J., and SZWEYKOWSKA, A., A tRNA-nucleotidyltransferase from moss protonema and promotion of its activity by cytokinin. Biochem. PhysioI. Pflanzen 167,207-217 (1975). Received November 30, 1976. Authors' address: Dr. ZENON SOHNEIDER, Institute of Biochemistry, Academy of Agriculture, Wolynska 35,606-37 Poznan, Poland, and Dr. JOLENTA SOHNEIDER, Laboratory of General Botany, Institute of Biology, Adam Mickiewicz University, Stalingradzka 14,61-713 Poznan, Poland.