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Inhibition of Eukaryotic Protein Chain Initiation by Vanadate

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Inhibition of Eukaryotic Protein Chain Initiation by Vanadate Proc. Nati Acad. Sci. USA Vol. 80, pp. 3148-3152, June 1983 Biochemistry Inhibition of eukaryotic protein chain initiation by vanadate (protein synthesis) RAjINDER SINGH RANU Department of Microbiology and the Graduate Program in Cellular and Molecular Biology, Colorado State University, Fort Collins, Colorado 80523 Communicated by Raj C. Bose, December 30, 1981 ABSTRACT Vanadate inhibits protein chain initiation in rab- grade) from Baker; cetyltrimethylammonium bromide and bit reticulocyte lysates. The evidence that supports this conclusion poly(uridylic acid) from Sigma; purified 9S globin mRNA from is as follows: (i) the biphasic kinetics of inhibition in which protein Searle (Wycombe, England); and [3S]methionine (1,200 Ci/ synthesis is maintained at the control rate for 1-2 min is followed mmol; 1 Ci = 3.7 X 1010 Bq), [14C]leucine (320 mCi/mmol), by an abrupt decline in the rate of synthesis; (ii) inhibition is as- and [14C]phenylalanine (270 mCi/mmol) from New England sociated with a marked disaggregation of polyribosomes and a Nuclear. Sparsomycin (NSC 59729) was provided by Natural concomitant increase in 80S ribosomes; and (iii) vanadate concen- Products Branch, Division of Cancer Treatment, National Can- trations that inhibit protein chain initiation do not inhibit poly- cer Institute. The noncapped satellite tobacco necrosis virus peptide chain elongation or the aminoacylation of tRNA. In par- was Clark of tial reactions of protein chain initiation, vanadate concentrations (STNV) RNA provided by J. (University Illinois, that inhibit protein synthesis have no detectable effect on the for- Urbana, IL). The sources of other reagents have been de- mation of eukaryotic initiation factor eIF-2-promoted ternary scribed (12). The following procedures also have been de- complex with Met-tRNAf and GTP and on the assembly of 40S scribed: preparation of rabbit reticulocyte lysates and protein- ribosomal subunit-Met-tRNAf complexes. On the addition of synthesis reaction mixtures, assay of protein synthesis, the mRNA, the 40S ribosomal subunit-Met-tRNAf complexes also are preparation of purified eIF-2 (12), and the preparation of [3S]- transformed into 80S ribosome-mRNA-Met-tRNAf complexes, Met-tRNAf (100,000 cpm/pmol) (12, 13). termed 80S initiation complexes. In vanadate-treated samples, Inhibition of Protein Synthesis by Vanadate. Rabbit retic- however, these 80S initiation complexes are defective and unable ulocyte lysate-based protein-synthesis reaction mixtures (25 .ul) to proceed beyond this step. containing 10 puM hemin were incubated at 30°C with various concentrations of vanadate. At intervals, aliquots (5 ,ud) were The requirement of vanadium in trace amounts as an essential removed and protein synthesis was assayed (12). The vanadate nutrient has been recognized for some time (1, 2). The vana- solutions used in this study were prepared fresh each day in dium compounds in moderately high levels can be highly toxic deionized distilled water. (1, 2). Although the biological role of vanadium at the molecular Assay of Poly(uridylic Acid)-Dependent Polyphenylalanine level is not known, recent interest in vanadate has arisen as a Synthesis in Lysates. Rabbit-reticulocyte-lysate reaction mix- result of the findings of Cantley et aL (3) that vanadate is a po- tures (25 u1) containing 10 puM hemin were incubated at 30°C tent inhibitor of membrane Na+,K+-ATPase. Vanadate also in- with (25 ,ug) or without poly(uridylic acid) in the presence of hibits a variety of other enzymes-e.g., myosin ATPase, dy- 8 mM Mg2+. Under these conditions, the endogenous natural nein ATPase, Ca2+-ATPase (sarcoplasmic reticulum), Mg2+- mRNA-dependent protein synthesis is completely suppressed, ATPase, and adenylate kinase (4, 5). Almost all of these en- and maximal poly(uridylic acid)-dependent polyphenylalanine zymes are phosphohydrolases, and frequently a phosphoen- synthesis is observed. At intervals, aliquots were removed and zyme intermediate is involved in the mechanism of action of protein synthesis was assayed (12). these enzymes. Current evidence suggests that vanadate com- Assay of Aminoacylation of tRNA. The aminoacylation assay petes with phosphate for the enzyme-binding site (4). was carried out under conditions of protein synthesis (12). Ed- The protein biosynthesis is dependent on a series of reac- eine (5 ,uM) was added to the reaction mixture to block initia- tions that require ATP and GTP hydrolysis-e.g., aminoacyl- tion of protein synthesis (14). At intervals, aliquots (5 ,ul) were ation, GTP- and ATP-dependent initiation of polypeptide, and removed and transferred to 1 ml of 10% cold trichloroacetic the GTP-dependent elongation and termination of polypeptide acid containing 0.5 mM methionine or leucine (see Fig. 4 leg- (6-8). The eukaryotic protein synthesis also is regulated by ATP- end). The precipitate was collected on Millipore filter and was dependent protein kinases that are activated in the presence of washed extensively with cold 5% trichloroacetic acid (15). The double-stranded RNA or by heme deficiency (9-11). These filters were dried and radioactivity was assayed. considerations and the apparent selective inhibition of the The assay of eukaryotic initiation factor eIF-2-dependent ATPases by vanadate prompted the examination of the effect ternary complex (eIF-2'GTP'Met-tRNA) formation and the as- of vanadate on protein synthesis in eukaryotes. The results pre- say of the formation of 40S ribosomal subunit-Met-tRNAf com- sented in this report show that vanadate preferentially inhibits plexes in lysates have been described (12, 13, 16). protein chain initiation. Analysis of the Distribution of Polyribosomes. The polyri- bosome distribution in protein-synthesis reaction mixtures was MATERIALS AND METHODS analyzed in sucrose density gradients (10-45%) in buffer A (20 The materials utilized in these studies were obtained from the mM Tris HCl, pH 7.6/80 mM KCl/2 mM magnesium acetate). following sources: ammonium metavanadate (analytical reagent Aliquots of reaction mixture (25 ,ul) were removed and trans- ferred to 100 1,u of ice-cold buffer A. The sample was layered The publication costs of this article were defrayed in part by page charge over a sucrose density gradient and then was centrifuged at 38,000 payment. This article must therefore be hereby marked "advertise- ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: eIF, eukaryotic initiation factor. 3148 Downloaded by guest on October 2, 2021 Biochemistry: Ranu Proc. Natd Acad. Sci. USA 80 (1983) 3149 rpm in a Spinco SW-50. 1 rotor for 2 hr at 4TC. The absorbance The radioactive peptides were stripped of tRNA by exposure profile was monitored in a ISCO density gradient monitor at to 1% trimethylamine. The samples were applied to Whatman 254 nm. The fractions from the gradients were analyzed for the 3 MM filter paper strips along with internal standards [methi- radioactivity associated with the nascent polypeptide chains ac- onine (10 gg) and methionylvaline (20 pg)] and were subjected cording to Darnbrough et aL (13). to ascending chromatography at room temperature in butanol/ Assay of Formation of 80S Initiation Complexes. The for- acetic acid/H20, 45:5:12.5 (vol/vol) (15). The positions of the mation of 80S ribosome-mRNA-Met-tRNAf complexes, termed markers (methionine and methionylvaline) were located with 80S initiation complexes, was determined by shift assay in ly- ninhydrin. The paper was cut into 1.5-cm pieces and assayed sate protein-synthesis reaction mixture (33 y1) containing 20 for radioactivity. ,M hemin. Incubation with or without 20-30 AuM vanadate was at 30TC for 5 min. Sparsomycin (40 ,M) was then added, RESULTS AND DISCUSSION and incubation was continued for another 3.5 min, at which time The effect of vanadate on eukaryotic protein synthesis was ex- [35S]Met-tRNAf (100,000 cpm) and 2 Ag of globin mRNA were amined in rabbit reticulocyte lysates because in this system added. After 2 min of incubation, the sample was diluted with the in vitro rates of protein chain initiation and elongation ap- 130 td of ice-cold buffer B (10 mM Hepes, pH 7.6/80 mM KCV proach the in vivo rates (12). Moreover, the requirement of heme 2 mM magnesium acetate). The sample was layered over a 5.2- for the maintenance of protein synthesis, first observed in in- ml 10-35% sucrose density gradient in buffer B. The samples tact cells, is preserved in lysates (12). were centrifuged at 45,000 rpm in a Spinco SW-50. 1 rotor for Vanadate strongly inhibited protein synthesis (Fig. 1A). The 2 hr at 2°C. The absorbance profile of the gradients was mon- inhibition of synthesis with 10-40 ,uM vanadate showed a con- itored at 254 nm. The fractions were then analyzed for radio- centration dependence. Beyond these concentrations, the in- activity as described by Darnbrough et aL (13). hibition reached a plateau. The kinetics of inhibition in the Assay of the Formation of Initiation Dipeptide (Methionyl- presence of 10 ,uM vanadate showed that protein synthesis dur- valine) of Globin. The micrococcal nuclease-treated lysate (16) ing the first 4-6 min was maintained at the control rate, fol- protein-synthesis reaction mixtures (40 Al) containing 24 ,uCi lowed by a progressive decline in the rate of synthesis. In the of [3S]methionine and 0.7 Ag of purified globin mRNA were presence of 20 ,uM vanadate, synthesis at the control rate was incubated at 30°C in the presence of sparsomycin (0.2 ,uM) or maintained only for the first 1-2 min, and then there was an vanadate (30 ,AM). A control without added mRNA also was in- abrupt decline in the rate of synthesis. However, after this sharp cluded [the micrococcal nuclease-treated lysate system itself decline, a synthesis at5-10% of the control rate was preserved. shows a high rate of formation of 80S initiation complexes be- These biphasic kinetics of inhibition of protein synthesis cause of the presence of mRNA fragments; these fragments suggest that vanadate inhibits protein chain initiation.
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