Biochemical Characterization of a Mutant Asparaginyl ...

THE JOURNAL OF BIOI.OGICAL CHEMISTRY Vol. 253, No. 1, Issue of January 10, pp. 58-62, Printed LIZ 1J.S.A

1978

Biochemical Characterization of a Mutant AsparaginyltRNA Synthetase from Chinese Hamster Ovary Cells* (Received IRENE L. ANDRULIS,?’ $ C. S. C!~IANG,& AND G. WESLEY HATFIELDI, /I

From the Department College of Medicine,

y STUART

of Biological Chemistry,? University of California,

M. ARFIN,~

TWYLA

for publication,

13, 1977)

A. MINER,~

and Department of Medical Microbiology,% Irvine, Irvine, California 92717

California

molecules (5, 6). The isolation of specific, mammalian cell conditional mutants defective in their ability to aminoacylate tRNA has provided a more powerful tool for analyzing these possible roles of unacylated tRNA in control mechanisms in animal cells. In our laboratory, studies with a Chinese hamster ovary cell mutant which has a temperature-sensitive defect in the aminoacylation of tRNA with asparagine has suggested a role for asparaginyl-tRNA in the regulation of the biosynthetic enzyme, asparagine synthetase (7). Further analysis of the role of tRNA in metabolic regulation will require a variety of physiologically and biochemically characterized aminoacyl-tRNA synthetase mutants. The isolation of a number of such mutants from CHO1 and other cell lines has been described (8-111, but only in the case of a leucyl-tRNA synthetase mutation have the biochemical properties of the enzyme from wild type and mutant cells been compared (12, 13). In this report, we describe the biochemical and physical properties of the asparaginyl-tRNA synthetase from cells of wild type CHO and from CHO Zys 65a, a mutant whose growth rate is dependent upon both temperature and the asparagine concentration in the medium.

The biochemical and physiological analysis of bacterial mutants with altered aminoacyl-tRNA synthetases has demonstrated that tRNA is involved in the repression control of a number of amino acid biosynthetic operons (l-3). Further, a general regulatory role in which any given tRNA molecule can affect multiple cellular processes, by regulating the intracellular level of the nucleotides guanosine 3’-diphosphate, 5’diphosphate, and guanosine 3’-diphosphate, 5’-triphosphate has also been established (4). Evidence is accumulating that tRNA may play a comparably important role in regulating a variety of metabolic events in mammalian cells. Treatment of cells with amino acid analogs which inhibit the aminoacylation of specific tRNAs has been found to decrease the intracellular ATP and GTP pools, block the initiation of protein synthesis, inhibit stable RNA synthesis and decrease the uptake of a number of small

EXPERIMENTAL

PROCEDURES

Materials-ATP, CTP, and Tris were obtained from Sigma. [‘*ClAsparagine (180 mCi/mmol) was purchased from New England Nuclear, yeast tRNA from Calbiochem, and Sephacryl S-200 from Pharmacia. a-ME medium was purchased from K-C Biologicals and dialyzed fetal calf serum from Irvine Scientific. Penicillin, streptomycin, and Fungizone were purchased from Grand Island Biological Co. CHO tRNA was prepared by the method of Hampel and Enger (14).

Growth of Cells-The wild type CHO cell line (15) and the temperature-sensitive mutant cell line (Zys 65a) were kindly provided by Dr. L. H. Thompson, Lawrence Livermore Laboratory. Cells w&e grown in suspension culture in a-ME medium containing 10% dialyzed fetal calf serum (heat-inactivated at 56” for 30 min), 150 units/ml of penicillin, 100 pg/ml of streptomycin, 0.25 pg/ml of Fungizone, and adenosine and thymidine, each at 10 fig/ml. Unless otherwise specified, the growth temperature was 34”. Cell number determinations were made with a Coulter counter. Cells were harvested during log phase growth (2 to 4 x lo5 cells/ml) by centrifugation, and washed two times in isotonic saline. Enzyme Assay -Asparaginyl-tRNA synthetase activity was determined by measuring the esterification of [14Clasparagine to tRNA. The reaction mixture contained in a final volume of 0.2 ml: 0.1 M Tris/HCl, pH 7.7; 5 rnM disodium ATP; 2.5 rnM disodium CTP; 40

* This investigation was supaorted bv United S+Ttes Public Health Service &ant GM ZlOOZ-is. M. A&n) and grrnts from the National Science Foundation (BMS 73-06711 A02) and Lne American Cancer Society (NP XC) (G. W. Hatfield). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Reciuient of United States Public Health Service Predoctoral Trainee Fellowship GM 07134. I Recipient of United States Public Health Service Postdoctoral Trainee Fellowship GM 07307. /I Recipient of United States Public Health Service Research Career Development Award GM 70530.

1 The abbreviations used are: CHO, Chinese hamster ovary; ME medium, an enriched Eagle’s minimal essential medium.

58

(Y-

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The biochemical and physical properties of asparaginyltRNA synthetase from wild type Chinese hamster ovary cells and a temperature sensitive mutant strain (Zys 65a) are compared. The asparaginyl-tRNA synthetase in the mutant strain exhibits a greater temperature lability in uitro, a higher temperature-independent K, for asparagine, and a lower temperature-dependent catalytic capacity than the enzyme from the wild type strain. The mutant enzyme shows no differences in its molecular weight, its K, for tRNAAS”, or its ability to aminoacylate tRNAAS” isoacceptor species compared to the wild type enzyme. These observations, as well as the growth properties of the mutant cells as a function of temperature and exogenous asparagine concentrations, are consistent with their decreased ability to aminoacylate tRNA*“” in uiuo.

June

Mutant

Asparaginyl-tRNA

rn~ KCl; 10 rn~ Mg(C,H,O,),; 0.4 rn~ dithiothreitol; 0.1 rn~ asparagine; 0.4 mg of yeast tRNA. Enzyme, as specified, was added to start the reaction. Al reaction velocities were determined under linear assay condition by transferring 0.05-ml aliquots directly from the reaction vessel into 3 ml of cold 10% trichloroacetic acid at 0-, 2.5-, 5, and lo-min intervals. The resulting suspension was chilled in ice for 20 min. The precipitate was collected on a glass fiber disc, washed three times with 6 ml of cold 10% (w/v) trichloroacetic acid, dried, and counted in a toluene-based scintillation mixture in a Beckman LS-230 liquid scintillation counter. Protein concentration was determined by the method of Lowry et al. (16) with bovine serum albumin as the standard. Extracts were prepared in Buffer A (0.1 M KCl, 10 rnM Tris/HCl, 0.1 rnM dithiothreitol, pH 7.5) with 0.1 rnM MgCl, except where indicated. Cells were disrupted using Nonidet P40 (l%, v/v) as described by Hampel and Enger

(14).

from

CHO Cells

59

medium. At 34”, a decrease from the normal medium asparagine concentration (0.33 mM) to one-half that amount or less increases the doubling time and decreases the maximum cell density. At 37”, lys 65a cells grow more slowly and achieve lower maximum cell densities at normal medium asparagine concentrations than at 34” under the same conditions. At 39.5”, these cells undergo less than one doubling in 72 h at normal medium asparagine concentrations but grow as well as wild type of asparagine is __ cells when the concentration increased lo-fold. Similar changes in growth temperature and asparagine concentration in the medium have little effect on the doubling time or the maximum cell density of wild type cells (data not shown). Temperature Lability of Wild Type and Mutant Asparaginyl-tRNA Synthetases -When crude extracts of wild type and lys 65a cells prepared in Buffer A are incubated at 37” in the absence of substrates, a dramatic difference in stability is observed (Fig. 2). Although the wild type enzyme is stable under these conditions, the mutant enzyme is irreversibly inactivated. The half-life of the mutant enzyme at 37” is 4.8 min. Only one species of asparaginyl-tRNA synthetase appears to be present in crude extracts of lys 65a since the inactivation is complete and follows first order kinetics. Although the lys 65a asparaginyl-tRNA synthetase is temperature-labile at 37” under the conditions described, the activity of this enzyme is linear for at least 10 min under optimal assay conditions at 37”, suggesting that some component(s) in the assay mixture exerts a stabilizing effect. Therefore, the heat inactivation experiment was repeated in the presence of the various components of the assay mixture. Table I shows that ATP and Mg*+ are both required for this stabilization of the enzvme. Determination of Kinetic Constants-The K,,l value for tRNA*“” of the wild type and lys 65a asparaginyl-tRNA synthetases was determined from the data shown in Fig. 3. A value of approximately 6 x 10mR M was obtained for the __ enzyme from both sources. Initial velocities as a function of asparagine concentration were determined at both 30” and 37”. Reciprocal plots of the data (Fig. 4) show that the K, value for asparagine for the Zys 65a enzyme (1 x 10.” M) is approximately 3-fold higher than the K, for asparagine for the wild type enzyme (3 x 10m5 M). These K, values are independent of the assay temperature, but the relative V,,,

RESULTS

Effect of Temperature and Asparagine Concentration on Growth of CHO Wild Type and lys 65a Cells-Previous studies with temperature-sensitive mutants of CHO cells having defective aminoacyl-tRNA synthetases have shown that in some cases, the effect of temperature on growth is dependent upon the concentration of the cognate amino acid in the medium (9-12). The growth of lys 65a cells at 34”, 37”, and 39.5” with several asparagine concentrations is shown in Fig. 1. As the growth temperature is increased, both the initial growth rate and the maximum cell density is significantly influenced by the concentration of asparagine in the culture

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Time (h) FIG. 1. Growth of Zys 65a cells as a function of temperature and asparagine concentration. a-ME medium prepared without asparagine was supplemented with the following concentrations of asparagine: X, 0.033 m&r; 0, 0.099 mM; a, 0.165 mM; A, 0.330 mM; 0, 3.30 mM.

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Aminoacylation of tRNA and Reverse Phase Column Chromatography -Reaction mixtures for the aminoacylation of tRNAAS” were similar to that described above for the aminoacylation assay except that the [14C]asparagine (180 mCi/mmol) concentration was 30 FM, the bulk CHO cell tRNA concentration was 225 ugiml and sufficient extract from wild type or lys 65a cells was added to achieve complete aminoacylation within 10 min. The reaction was terminated by the addition of 0.10 volume of 2 M sodium acetate, pH 5.0, and RNA was extracted with phenol. tRNA was precipitated from the aqueous phase with 2 volumes of ethanol, dissolved in a small volume of Buffer B (10 rnsi sodium acetate, 10 m&i MgCl,, 1 rnM EDTA, 5 mM mercaptoethanol, pH 4.51, and passed over a column of Sephadex G25 (1.5 x 20 cm) equilibrated with the same buffer. Fractions containing aminoacylated tRNA resolved from free [“‘Clasparagine were reprecipitated with ethanol, dissolved in 0.5 ml of Buffer B, and subjected to RPC-5 column chromatography at 37” as described by Holmes et al. (17) with the indicated linear NaCl gradient. Aminoacylation of tRNA in Viva -The procedure for determining aminoacylation of tRNA Asn in uivo as a function of temperature and of asparagine concentration was a slight modification of the method of Thompson et al. (10). Wild type and mutant cells were grown in a-ME medium containing 0.03 rnsi asparagine (one-tenth the normal medium concentration) at 39.5” for6 h.Samples containing 1 x lo7 cells were harvested by centrifugation and resuspended in tubes containing 1 ml of fresh u-ME medium supplemented with either 0.033 mM, 0.099 mM, 0.165 mM, or 0.330 rnM asparagine and incubated at 39.5”. After 10 min, 0.05 ml of [“Clasparagine was added to each tube. Following the addition of the label,