The effects of zinc ions on activities of trna Leu and leucyl-trna synthetase of mice liver

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1 982 The effects of zinc ions on activities of trna Leu and leucyl-trna synthetase of mice liver Hiliaras Rodovičius, Dalė Vieželienė, 2, Ilona Sadauskienė, 2, Sandra Valentukonytė 2, Leonid Ivanov, 2 Department of Biochemistry, 2 Institute for Biomedical Research, Kaunas University of Medicine, Lithuania Key words: zinc, trna, leucyl-trna synthetase, mouse. Summary. The aim of this study was to examine the effects of zinc ions on activities of trna Leu and leucyl-trna synthetase of mice liver. Material and methods. White laboratory mice (2 25 g) were used for study purposes. Animals were intoxicated with ions of zinc by injection of.5 LD 5 dose of zinc sulphate solution (.56 mg Zn 2+ per kg of body weight) into abdominal cavity. After 8 hours, preparations of total trna and aminoacyl-trna synthetases were isolated from the intoxicated and normal (control) mice liver. Acceptor activity of trna Leu and activity of leucyl-trna synthetase were determined in trna aminoacylation reaction using [ 4 C]-labeled leucine. Actions of zinc ions on acceptor activity of trna Leu and on activity of leucyl-trna synthetase from liver of control animals in vitro were determined after addition into reaction mixture different concentrations of zinc sulphate solution. Results. It was determined that acceptor activity of mice liver trna Leu 8 hours after intoxication with zinc ions has increased by 29% and activity of leucyl-trna synthetase has increased by 2% as compared to control. Experiments in vitro have shown that 5 2 µm concentrations of zinc ions in reaction mixture stimulate the acceptor activity of mice liver trna Leu by 8 3%, higher concentration of zinc ions (4 µm) suppresses it by 26%. The study of leucyl-trna synthetase activity in vitro has shown that 5 µm concentrations of zinc ions in reaction mixture increase activity of this enzyme by 6%, higher concentrations of zinc ions (2 4 µm) decrease it by 3 2%. Conclusions. After 8-hour intoxication with zinc ions the activities of both studied components of the translation machinery trna Leu and leucyl-trna synthetase were increased. It may be connected with the stimulation of zinc-binding metallothionein synthesis which is involved in the detoxification of heavy metals. Low concentrations of zinc ions in reaction mixture increase trna Leu and leucyl-trna synthetase activities; higher concentrations of these ions decrease activity of those components of protein synthesis system. The results show that zinc ions directly act on the activities of both components of translation machinery. Introduction Zinc is one of the most abundant nutritionally essential elements in human body. This ubiquitous element is essential for normal enzymatic function in multiple metabolic pathways (). Zinc is essential for the structure and function of a large number of macromolecules and for over 3 enzyme reactions (2, 3). This metal exists primarily in the form of complexes with proteins and nucleic acids and participates in all aspects of intermediary metabolism, transmission and regulation of the expression of genetic information, storage, synthesis and action of peptide hormones and structural maintenance of chromatin and biomembranes (2, 4, 5). Zinc induces synthesis of metallothioneins (6 8), and prevents cells from apoptosis (5, 8, 9). On the other hand high doses of zinc are very toxic for the living organisms (, ). Chronic excessive zinc ingestion causes severe reversible anemia in humans; in animals zinc toxicity leads to anemia as well as physiologic and morphologic damage of pancreas, kidneys, and often, multisystem failure and death (). It is reported that zinc ions suppress ribonucleic acid (RNA) and protein synthesis due to direct zinc action on these biosynthetic pathways (2). Increasing concentrations of zinc ions induce progressive decrease Correspondence to H. Rodovičius, Department of Biochemistry, Kaunas University of Medicine, A. Mickevičiaus 9, 4437 Kaunas, Lithuania. hiliaras@med.kmu.lt

2 The effects of zinc ions on activities of trna Leu and leucyl-trna synthetase of mice liver 983 in global protein synthesis and act on the initiation step of protein synthesis; they also strongly decreased the amount of polyribosomes (3). It is known that process of protein synthesis is very sensitive for influence of heavy metals (4, 5), but mechanisms of its toxicity remain understudied (6). The essential role in initial stages of protein synthesis assigns to transfer ribonucleic acids (trna) and aminoacyl-trna synthetases, which participate in specific reaction of aminoacylation of trna (7, 8). The objective of this study was to examine the effects of zinc ions on activities of trna Leu and leucyltrna synthetase of mice liver. Material and methods White laboratory mice (2 25 g) were used for study purposes (License of State Veterinary Service for Working with laboratory animals No. 28). Intoxication with ions of zinc was performed by injection of.5 LD 5 dose of sterile zinc sulphate (.56 mg Zn 2+ per kg of body weight) dissolved in deionized water into abdominal cavity of laboratory animals. The same volume of physiological solution was injected into abdominal cavity of control group animals. Eight hours after intoxication mice were sent to sleep by inhalation of anesthetic narcotine. The livers were quickly removed, washed, blotted, put to saucer and immediately cooled on ice bath. Total preparations of trna and aminoacyl-trna synthetases were isolated from intoxicated and control mice liver. Liver was weighted and homogenized with.5 volume (as compared with liver weight) of buffer ( mm Tris-HCl, ph 7.5;. M NaCl; 5 mm EDTA) for 3 min. Into homogenate was added.5 volumes (as compared with liver weight) 8% water saturated phenol and shake by shake machine for 5 min. Then the homogenate mixture was centrifuged for 5 min at 5 g. After centrifugation the upper aqueous phase was obtained and an equal volume of 8% phenol was added. Obtained mixture was shaken for 5 min and centrifuged again. After second centrifugation the upper aqueous phase was obtained, 3 volumes of 96% ethanol were added and the mixture was kept at 2 C for at least 3 hours. The precipitate formed was collected by centrifugation for 5 min at 8 g and dissolved in.3 M solution of sodium acetate, ph 7.4, (amount of sodium acetate in ml is equal to amount of grams of liver weight). To the obtained solution.54 volume of isopropanol was slowly added with constant stirring at room temperature. The resulting precipitate was immediately centrifuged at 8 g for 5 min, and discarded. To the supernatant was further added.44 volume of isopropanol (final volume of isopropanol was.98) and left over-night at 2 C. The obtained precipitate was collected by centrifugation at 8 g for 5 min and dissolved in twice-distilled water (preparation of total trna). For isolation of crude preparations of aminoacyltrna synthetases (postmitochondrial supernatant) liver was weighted and homogenized with 3 volumes (as compared with liver weight) of buffer ( mm Tris-HCl, ph 7.5; mm MgCl 2 ; mm KCl; 25 mm sucrose; mm dithiothreitol). The enzymes were extracted by constant stirring of homogenate in ice bath for 5 min. The fragments of cells, nucleus and mitochondria were removed by centrifugation of homogenate at 5 g for 5 min. Supernatant was filtrated through fourfold gauze bandage layers (crude preparation of aminoacyl-trna synthetases). Concentration of proteins in solution was determined according to Lowry (9). Acceptor activity of trna specific for leucine (trna Leu ) in vivo was determined in the µl of reaction mixture, consist of mm Tris-HCl (ph 7.5), mm MgCl 2, mm KCl, 4 mm ATP,.2 mm [ 4 C]-leucine, 25 µg of aminoacyl-trna synthetase and 5 µg of trna. The reaction mixture was incubated at 37 C for 2 min. The reaction was stopped by addition of.2 ml ice-cold % trichloracetic acid. Test tubes with reaction mixture were kept in ice bath for 2 min for formation of precipitate. The precipitate was collected on nitrocellulose filters. Radioactivity was measured with liquid scintillation counter Delta 3 (efficiency of countering 6%). About acceptor activity of trna Leu was decided by attachment of [ 4 C]-labeled leucine to trna Leu. Activity of leucyl-trna synthetase in vivo was measured by the initial rate of formation of leucyltrna in aminoacylation reaction with [ 4 C]-labeled leucine. Reaction mixture (final volume µl) consisted of mm Tris-HCl (ph 7.5), mm MgCl 2, mm KCl, 4 mm ATP,.2 mm [ 4 C]-leucine, 5 µg preparation of trna and 25 µg preparation of aminoacyl-trna synthetase. The reaction mixture was incubated at 37 C for 3 min. To evaluate the effects of zinc ions on activities of trna Leu and leucyl-trna synthetase in vitro different concentrations of zinc sulphate were directly added into the reaction mixtures. Reliability of the data was estimated by Student distribution coefficient (t). Changes were statistically significant when p<.5.

3 984 Hiliaras Rodovičius, Dalė Vieželienė, Ilona Sadauskienė ir kt. Acceptor activity of trna (nmol of leucine / mg of trna) 2,5 2,5,5 control Zn Leucyl-tRNA synthetase activity (pmol of leucine / min / mg of protein) control Zn Fig.. Acceptor activity of trna Leu from mice liver in norm and after 8-hour intoxication with.5 LD 5 dose of zinc ions Results The activities of trna Leu and leucyl-trna synthetase isolated from control mice liver and mice liver 8-hour after intoxication with zinc ions were compared in vivo. The results of study have shown that acceptor activity of mice liver trna Leu 8 h after intoxication with zinc ions increased by 29% (p<.5) (Fig. ) and activity of leucyl-trna synthetase increased by 2% (p<.5) (Fig. 2) as compared to control. For the purpose to examine action of zinc ions directly on trna Leu and on leucyl-trna synthetase experiments in vitro were performed. The solutions of zinc sulphate were directly added into reaction mixture with preparations of trna and aminoacyltrna synthetases isolated from control mice liver. Final concentration of zinc ions in reaction mixture was, 5,, 5, 2, 3 and 4 µm. Acceptor activity of trna Leu and activity of leucyl-trna synthetase were estimated. Experiments in vitro showed that 5 Fig. 2. Activity of leucyl-trna synthetase from mice liver in norm and after 8-hour intoxication with.5 LD 5 dose of zinc ions 2 µm concentrations of zinc ions in reaction mixture increased acceptor activity of mice liver trna Leu by 8 3% (p<.5), higher concentration of zinc ions (4 µm) decreased it by 26% (p<.5) (Fig. 3). Determination of leucyl-trna synthetase activity in vitro has shown that 5 mm concentrations of zinc ions in reaction mixture increased activity of this enzyme by 6% (p<.5), higher concentrations of zinc ions (2 4 µm) decreased it by 3 2% (p<.5) (Fig. 4). Discussion The data of our experiments suggested that acceptor activity of trna Leu, isolated from mice liver 8 hours after intoxication with.5 LD 5 dose of zinc ions in vivo, was increased as compared to control. Increasing of acceptor activity of trna under action of zinc ions could be associated with increasing synthesis of trna molecules (2, 2) Acceptor activity of trna (%) Leucyl-tRNAsynthetase activity (%) Concentration of Zn iones (µmol /l) Concentration of Zn iones (µmol /l) Fig. 3. Acceptor activity of trna Leu from mice liver in vitro under action of different concentrations of zinc ions Acceptor activity of trna Leu of control group is equated to percent. Fig. 4. Activity of leucyl-trna synthetase from mice liver in vitro under action of different concentrations of zinc ions Activity of leucyl-trna-synthetase of control group is equated to percent.

4 The effects of zinc ions on activities of trna Leu and leucyl-trna synthetase of mice liver 985 Results of the investigation in vitro have shown that low concentrations of zinc ions (5 2 µm) in reaction mixture stimulated acceptor activity of trna Leu and high concentration of these ions (4 µm) suppressed. Decreasing of trna acceptor activity under action of high concentrations of heavy metals ions may be associated with cleavage of certain bonds between nucleotides in trna molecule (22 24) or with deacylation of aminoacyl-trna (24). Eight hours after intoxication with zinc ions activity of leucyl-trna synthetase of mice liver in vivo increased as compared to control group. This effect may be associated with regulation of enzymatic processes by phosphorylation. This opinion confirms data that some heavy metal ions have got activating action on protein kinase cascade (25, 26). It also may be associated with increasing synthesis of aminoacyl-trna synthetase molecules (2, 2). Investigations in vitro have shown that zinc ions in concentrations of 5 µm in reaction mixture increased activity of leucyl-trna synthetase and in higher concentrations (2 4 µm) decreased. The decreasing of leucyl-trna synthetase activity may be due to strong zinc-thiolate interaction with zinc ions of the mercapto groups of cysteines (3, 27). The stimulating action of zinc ions on the of the acceptor activity of trna and activity aminoacyl-trna synthetases and may be related with increased synthesis of protein metallothionein which take part in detoxication of this metal (5, 7, 8) and with increased synthesis of heat shock proteins, which protect proteins of organism under extreme conditions (28). Conclusions Acceptor activity of mice liver trna Leu was increased after 8-hour intoxication with.5 LD 5 dose of zinc ions. Activity of mice liver leucyl-trna synthetase was increased after 8-hour intoxication with.5 LD 5 dose of zinc ions. Low concentrations (5 2 µm) of zinc ions in vitro increased acceptor activity of trna Leu from control mice liver, and higher concentration (4 µm) decreased. Low concentrations (5 µm) of zinc ions in vitro increased activity of leucyl-trna synthetase from control mice liver, and higher concentrations (2 4 µm) decreased. Cinko jonų poveikis pelių kepenų trnr Leu ir leucil-trnr-sintetazės aktyvumui Hiliaras Rodovičius, Dalė Vieželienė, 2, Ilona Sadauskienė, 2, Sandra Valentukonytė 2, Leonid Ivanov, 2 Kauno medicinos universiteto Biochemijos katedra, 2 Biomedicininių tyrimų institutas Raktažodžiai: cinkas, trnr, leucil-trnr-sintetazė, pelė. Santrauka. Darbo tikslas. Ištirti ūmaus intoksikavimo cinko jonais poveikį pelių kepenų trnr Leu ir leuciltrnr-sintetazės aktyvumui. Tyrimo medžiaga ir metodai. Tyrimams naudotos baltos laboratorinės pelės, sveriančios 2 25 g. Intoksikavimui cinko jonais į pelių pilvo ertmę sušvirkšta cinko sulfato tirpalo (,56 mg Zn 2+ vienam kg kūno masės, o tai atitinka,5 LD 5 dozę). Praėjus 8 val. po injekcijos, iš intoksikuotų ir cinko jonais nepaveiktų (kontrolinės grupės) pelių kepenų buvo išskirti suminiai trnr ir aminoacil-trnr-sintetazių preparatai. trnr Leu ir leucil-trnr-sintetazės aktyvumui įvertinti atlikta trnr aminoacilinimo reakcija, kuriai vartotas [ 4 C]-leucinas. Cinko jonų poveikis trnr Leu ir leucil-trnr-sintetazės aktyvumui in vitro vertintas įpylus į reakcijos terpę skirtingos koncentracijos cinko sulfato tirpalo. Rezultatai. Nustatyta, kad po 8 val. intoksikavimo cinko jonais pelių kepenų trnr Leu aktyvumas padidėja 29 proc., o leucil-trnr-sintetazės aktyvumas 2 proc. Atliekant eksperimentus in vitro, nustatyta, kad reakcijos terpėje esant 5 2 µm cinko jonų koncentracijai, trnr Leu aktyvumas padidėja 8 3 proc., esant didesnei šių jonų koncentracijai (4 µm), sumažėja 26 proc. Tiriant leucil-trnr-sintetazės aktyvumą in vitro, nustatyta, kad reakcijos terpėje esant 5 µm cinko jonų koncentracijai, šis aktyvumas padidėja 6 proc., o esant didesnei šių jonų koncentracijai (2 4 µm), sumažėja 3 2 proc. Išvados. Praėjus 8 val. po pelių intoksikavimo cinko jonais, trnr Leu ir leucil-trnr-sintetazės aktyvumas padidėja. Reakcijos mišinyje in vitro esant mažoms cinko jonų koncentracijoms, padidėja tiek trnr Leu, tiek leucil-trnr-sintetazės aktyvumas, o esant didesnėms šių jonų koncentracijoms, abiejų baltymų sintezės sistemos komponentų aktyvumas mažėja. Tai rodo, kad cinko jonai tiesiogiai veikia abiejų tirtų transliacijos sistemos komponentų aktyvumą. Adresas susirašinėti: H. Rodovičius, KMU Biochemijos katedra, A. Mickevičiaus 9, 4437 Kaunas El. paštas: hiliaras@med.kmu.lt

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