Inhibition Of Polypeptide Synthesis Research Articles

The Effect of Different Hormones and Antibiotics on Activity of AST Enzyme and its Isozymes in Wistar Rats

Background: One of the valuable tests for diagnosis of cardiovascular and liver diseases is measuring of AST activity. One of the main enzymes of transaminases group is aspartate aminotransferase. Previous Studies have shown that some alteration may occur in mitochondria function as the result of different disease or taking different medication; these changes in mitochondrial and cytosolic AST isozymes can be the sign of disorders. According to the role of steroid hormone in induction of its effects on protein synthesis genes, this study is conducted to shed some light on mechanisms and the interference of steroid hormones and antibiotics.Materials, Methods & Results: In this study, male Wistar rats were injected intramuscularly with Testosterone, progesterone and estradiol; while tetracycline and streptomycin injections were intraperitoneal. Testosterone, progesterone and estradiol injections were carried out in a short-term (15 days) and long-term (45 days) periods. Steroid hormones were dissolved in sesame in a way that by each injection, 0.2 mL sesame oil (containing specific amount of hormone) was injected to the rat. Control group was kept in the same condition except that their sesame oil injection contained no hormone. Tetracycline and Streptomycin injection was carried out for 5 days at 7 am and pm, at 50 mg/kg dosage intraperitoneally. In short- and long-term periods, rats were divided into four groups of 6-member. The concentrations were the same in the periods and 0.2 mL sesame oil was injected intramuscularly. 1 mg testosterone, 12 mg progesterone and 0.2 mg estradiol were intramuscularly injected to rats in group 2, 3 and 4, respectively [10]. Rats were divided into 9 six-member groups as follows: Group 1: intraperitoneal injection of 0.2 mL physiological serum; group 2: injection of 1 mg testosterone; group 3: injection of 1 mg testosterone + 50 mg/kg streptomycin; group 4: injection of 1 mg testosterone + 50 mg/kg tetracycline; group 5: injection of 0.2 mg estradiol; group 6: injection of 0.2 mg estradiol + 50 mg/kg streptomycin; group 7: injection of 0.2 mg estradiol + 50 mg/kg tetracycline; group 8: injection 50 mg/kg streptomycin; and group 9: injection of 50 mg/kg tetracycline. Serum concentration of AST enzyme was measured at the end of each period and the data were compared by SPSS software. all three steroid hormones had no significant impact on AST activity in short term. However, a significant effect was observed in long term in mean AST activities of the 4 groups. The group injected by testosterone exhibited 9% increases in comparison with the control group. Antibiotic-administrated groups showed lower activities as compared with hormone-injected groups. Steroid hormones and testosterone can enhance AST activity, in short-term and long-term, respectively by induction of protein enzyme. The second test confirmed this theory as the antibiotics decreased the AST activity enhancement by testosterone.Discussion: Based on the present study, steroid hormones can enhance the aspartate aminotransferase activity; and antibiotics can decrease the level of this liver enzyme by inhibition of polypeptide synthesis on related genes. This reaction could be due to interference of hormones and antibiotics effect which hinders the hormone effect along with the drug to activate the protein synthesis process.

Inhibition of polypeptide synthesis in cell-free systems by virginiamycin S and erythromycin. Evidence for a common mode of action of type B synergimycins and 14-membered macrolides

Macrolides, lincosamides and type B synergimycins are powerful inhibitors of protein synthesis in vivo, but many of them were found to be inactive in vitro. In the present work, we confirm that virginiamycin S (a type B synergimycin) and erythromycin (a 14-membered macrolide) have no effect on poly(U)-directed poly(Phe) synthesis. However, the amino-acid polymerization reactions directed by poly(U,G), poly(U,C), poly(A,G) and poly(A,C) were increasingly inhibited (20–50%) by both antibiotics. The action of these inhibitors proved to be template-dependent and favored by the incorporation of proline and of basic amino acids into peptides. Under these conditions, virginiamycin S and erythromycin markedly stimulated a release of peptidyl-tRNA from the ribosomes. In the poly(A,C) model system, these antibiotics produced a 50% inhibition of amino-acid incorporation into total peptides, a 70% release of ribosome-bound peptidyl-tRNA, and a 95% repression of the synthesis of long peptide chains. The production of equivalent effects at saturating concentrations of these antibiotics in the four model systems examined is suggestive of a similarity in their mode of action. Our results indicate that 14-membered macrolides and type B synergimycins can act on ribosomes during the whole elongation process. The functional block produced by both antibotics is usually reversible, but may result in a premature release of peptidyl-tRNA when the stability of ribosomal complexes is lowered by the incorporation of basic amino acids.

Inhibition of HeLa cell protein synthesis under heat shock conditions in the absence of initiation factor eIF-2α phosphorylation

Subjecting a HeLa cell suspension culture to an increase in incubation temperature (from 37 degrees to 42 degrees C) results in the rapid cessation of polypeptide chain synthesis followed by a gradual increase in the synthesis of a class of polypeptides referred to as the heat-shock proteins. It has been proposed that the initial, rapid shutoff of protein synthesis (less than 20 min) is due to the phosphorylation of initiation factor eIF-2 in its alpha subunit, a modification known to result in the inhibition of polypeptide synthesis. Using an in vitro translation system derived from heat-shocked HeLa cells grown in suspension culture, we were unable to find any evidence implicating eIF-2 alpha phosphorylation in the initial shutoff of translation during the heat shock response. These results suggest that the rapid inhibition of protein synthesis observed under heat shock conditions is mediated by a mechanism(s) other than eIF-2 alpha phosphorylation.

Differential effects of defective interfering Semliki Forest virus on cellular and virus polypeptide synthesis

Defective interfering Semliki Forest virus (DI SFV) inhibited virus RNA and virus polypeptide synthesis in cells coinfected with standard virus but did not delay or alter kinetics of RNA synthesis. Inhibition of polypeptide synthesis was 20-fold greater than that of RNA synthesis which presumably reflected the amplification resulting from cumulative translation of mRNAs. At high concentration, DI virus p12e inhibited the shutoff of host protein synthesis and allowed no synthesis of structural or nonstructural polypeptides. Dilution of DI virus restored the inhibition of host protein synthesis but further dilution was necessary before virus-specified polypeptide synthesis could be demonstrated. Another DI virus (p20a) with the same interference titre as p12e also inhibited shutoff of host protein synthesis but synthesis of virus-induced polypeptides was inhibited differentially: significant amounts of polypeptides comigrating with the structural precursor polypeptide p62 and the nonstructural polypeptide nsp63 were present and the synthesis of nsp90 was little affected at any concentration of DI virus p20a tested. None of the DI viruses tested induced the synthesis of any viral or novel polypeptide. It was concluded that DI SFV preparations have qualitatively different interfering activities in relation to their effects on virus and host cell polypeptide synthesis.

Double-stranded RNA-stimulated enzyme activities isolated from human placental extracts

A (2′–5′)A n synthetase activity was isolated from human placental extracts by affinity chromatography on poly(rI)·poly(rC)-agarose. The oligonucleotide (2′–5′)A n was identified by (1) chromatography on PEI-cellulose and DEAE-cellulose, (2) inhibition of polypeptide synthesis in lysed rabbit reticulocytes (3) competition of the binding of pppA(pA)3,3′-[ 32P]pCp to rabbit reticulocyte lysates, and (4) alkaline phosphatase digestion. The synthetase activity in most placental preparations is activated by natural or synthetic dsRNA. However, in a few placental synthetase preparations, dsRNA is only marginally stimulatory and only becomes effective by prior treatment of the enzyme preparations with the calcium-dependent micrococal nuclease. This suggeststhat there is an endogenous placental dsRNA contaminant in the enzyme preparations. In some synthetase preparations, a second dsRNA-stimulated product, tentatively identified as the nucleotide 5′-IMP, is also observed. Because the specific AMP deaminase inhibitor coformycin (10 μM) blocks the formation of IMP from ATP and causes a quantitative accumulation of AMP, and because the formation of IMp becomes independent of dsRNA when ADP or AMP is used in plase of ATP, the presence of a dsRNA-stimulated ATP phosphohydrolase (ATPase) activity in human placenta is suggested.

Control of protein synthesis in rabbit reticulocytes. Inhibition of polypeptide synthesis by ethanol.

Ethanol (0.21-0.84 M) added to rabbit reticulocyte lysates results in a 42-89% decrease of polypeptide synthesis following a 60-min incubation at 30 degrees C. The onset of inhibition is preceded by a 5-15-min lag. Eukaryotic initiation factor 3 (eIF-2) (85% pure, 0.42-4.2 micrograms added/30-microliters assay) or high concentrations of cAMP, MgGTP, and glucose 6-phosphate (0.55-5.0 mM) partially reverse the inhibition was observed with fructose 6-phosphate (0.55 mM). Ethanol was also shown to directly inhibit ternary complex formation between eIF-2, GTP, and initiator Met-tRNA (50% inhibition with 0.5 M ethanol). The inhibitory effect of ethanol on polypeptide synthesis, however, appears to be independent of its effect on ternary complex formation, and may be related to the activation of a translational inhibitor. This tentative conclusions is based on the following results. First, when the postribosomal supernatant or unfractionated lysate is incubated with ethanol, the supernatant or lysate becomes inhibitory to polypeptide synthesis following a 60-min incubation with ethanol at 30 degrees C. Second, when ethanol-treated postribosomal supernatant is chromatographed on DEAE-cellulose, an inhibitory activity is observed. The peak of inhibition coincides with the elution position of a translational inhibitor termed heme-controlled repressor. Third, pro-heme-controlled repressor becomes more inhibitory to protein synthesis when incubated with ethanol. These findings suggest that ethanol may activate a translational inhibitor by affecting the proper conformational change of a dormant inhibitor.

Inhibition of polypeptide synthesis by a factor isolated from ribosomes of resting human lymphocytes: Studies on the mechanism of action

Peripheral blood lymphocytes are non-dividing cells which synthesize small amounts of RNA and protein. These resting cells are stimulated when incubated in the presence of mitogens, and after a variety of metabolic changes which include a marked increase of RNA and protein synthesis, they start DNA replication and reach a state of active proliferation [ 1,2]. The enhancement of protein synthesis occurring during the first hours after the addition of PHA to a lymphocyte culture does not require the synthesis of RNA 13-51, indicating that during this period preexisting mRNA molecules and ribosomal particles are being utilized. Thus lymphocyte activation may involve regulation at the translational level besides other controls which might be occurring on transcription and DNA replication. Experiments with cell-free systems have indicated that the low rate of protein synthesis observed in unstimulated lymphocytes is probably due to the absence of initiation factors [6] and/or to the presence of cytoplasmic inhibitors which block the formation of initiation complexes [7,8]. The possibility of a more complex control of protein synthesis in lymphocytes has been reinforced by the isolation of a new ribosome-bound translational inhibitor (TI) obtained from resting human lymphocytes [9].

The interaction of fusidic acid with peptidyl-transfer-ribonucleic-acid - ribosome complexes.

The inhibitory action of fusidic acid on peptide-chain elongation was studied with systems in vitro directed by either polyuridylic acid or endogenous messenger (Escherichia coli polysomes washed with 1 M NH4Cl) or R17 RNA, and supplemented with either crude or purified elongation factors. In all cases strong inhibition of synthesis required high concentrations of the antibiotic (approx. 1 mM), while a similar inhibition of the EF-G-plus-ribosome-dependent GTP hydrolysis required between 10 and 100 times less antibiotic. Since most of the GTP hydrolysis observed was presumably due to free ribosomes (without aminoacyl-tRNA or peptidyl-tRNA), fusidic acid seemed to interact far more easily with these ribosomes than with ribosomes engaged in peptide-chain elongation. The role of the GDP-EF-G-ribosome-fusidic acid complex in the inhibition of polypeptide synthesis was assessed by measuring formation of this complex on polysomes engaged in peptide-chain elongation. Using purified elongation factors the complex formed on only 25-35% of ribosomes, as measured either by retention of [3H]GDP or by hydrolysis of [3H, gamma-32P]GTP. In contrast, with crude factors (S 100 extract) it formed on more than 70% of ribosomes. The results are compatible with the postulated role of the complex in polypeptide synthesis inhibition (blockade of the ribosomal acceptor site and subsequent inhibition of aminoacyl-tRNA binding) and indicate that formation of the complex takes place by overriding the control that prevents interaction of EF-G when the donor site is occupied by peptidyl-tRNA. In the polyuridylic-acid-directed system for synthesis of oligophenylalanine the antibiotic inhibits every round of peptide elongation, including dipeptide formation, to roughly the same extent.

Inhibition of Polypeptide Synthesis by tRNA Fractions in Rat Liver Cell-free Systems

The mechanism of inhibition of polypeptide synthesis by the addition of a tRNA fraction in a rat liver cell-free system was studied. The inhibition was found to occur at the step of aminoacyl-tRNA binding to ribosomes, in which aminoacyl-tRNA's were mainly responsible for the inhibition. The addition of EF-1 decreased the inhibition by the tRNA fraction. The tRNA fraction inhibited polypeptide synthesis in a polysome-S100 system under conditions in which poly U- and poly A-dependent polypeptide syntheses were not inhibited. The possibility that the aminoacyl-tRNA inhibitory activity functions through improper binding to the ribosomes in the polysome-S100 system is discussed.

Comparison of rat liver and Escherichia coli cell-free systems in inhibition of polypeptide synthesis by deoxycholate

1. Polyphenylalanine synthesis by rat liver and Escherichia coli ribosomes was not affected by the addition of non-ionic detergents (Tween 40, Brij 58, and Triton X-100), while the addition of deoxycholate (DOC) produced strong inhibition. On the other hand, the rat liver polysomes prepared with the use of DOC showed no significant difference in either activity or yield as compared with those prepared with non-ionic detergents. 2. In the case of rat liver, S100 fraction was inactivated irreversibly by DOC, while no irreversible inactivation by DOC was observed with ribosomes. With respect to the components of S100 fraction, it was observed that phenylalanyl-tRNA synthetase and the elongation factor(s) (EF1 and/or EF2) were both inactivated irreversibly by DOC. 3. In the case of E. coli ribosomes, slow but irreversible inactivation by DOC was observed. E. coli S100 fraction was not inactivated irreversibly by DOC, but phenylalanyltRNA synthetase in E. coli S100 fraction was found to be inhibited reversibly by DOC. 4. Non-enzymatic poly(U)dependent binding of phenylalanyl-tRNA to ribosomes was inhibited by DOC in both rat liver and E. coli systems.

Chloroquine and primaquine inhibition of rat liver cell-free polynucleotidedependent polypeptide synthesis

Chloroquine and primaquine inhibited polypeptide formation directed by poly U and random poly A,G,U in a rat liver cell-free system. It was necessary to preincubate the drug and polynucleotide before addition to the cell-free system to observe this inhibition. These antimalarials also decreased the binding of poly U to ribosomes under these conditions. No inhibition of polypeptide syntheses or poly U binding to ribosomes was observed when the drugs and polynucleotides were added to the cell-free system separately. Chloroquine and primaquine apparently interacted with the free polynucleotide and prevented the subsequent formation of an active polynucleotideribosome complex.

Inhibition of polypeptide synthesis in E. Coli cell free system by influenza viral protein

Inhibition of polypeptide synthesis in E. Coli cell free system by influenza viral protein

Mechanism of inhibition of protein synthesis by fusidic acid and related steroidal antibiotics.

Fusidic acid and related steroidal antibiotics were demonstrated to inhibit ribosomc-dependent GTPase activity of G factor in the presence and absence of sRNA, phenyl-alanyl-sRNA and/or poly U. They also inhibited polypeptide synthesis, and the grade of inhibition of both reactions was parallel. The inhibition of polypeptide synthesis by fusidic acid was reversed by excess of G factor, but not by excess of T factor or ribo-somes, Puromycin-dependent release of peptide from the ribosomes was not significantly affected by fusidic acid in the absence of GTP and G factor; but the puromycin reaction, enhanced by GTP and G factor, was inhibited by the antibiotic. The results indicate that fusidic acid and related antibiotics affect polypeptide synthesis by inhibiting GTPase activity of G factor and by inhibiting translocation of peptidyl-sRNA on the ribosomes. The results also seem to support the assumption that GTP split reaction, linked to the ribosomes and G factor, is essential for translocation of peptidyl-sRNA on the ribosomes. The ribosome-associated GTPase activity of G factor was not significantly affected by blasticidin S, mikamycin B, bottromycin A2, chloramphenicol, thiophenicol, and erythromycin.

Streptomycin and the genetic code.

It is presently accepted that streptomycin (Sm) and the related aminoglycoside antibiotics (these include neomycin (Nm), Kanamycin (Km), paromomycin, and gentamycin) can suppress mutant phenotypes by virtue of their interaction with the ribosome (Gorini and Kataja, 1964; Davies, Gilbert, and Gorini, 1964; Anderson, Gorini, and Breckenridge, 1965; Gorini and Beckwith, 1966); it also seems likely that this interaction is the primary event in the lethal action of these drugs, although this point has not been completely resolved. Sensitivity to both Sm-misreading and inhibition of polypeptide synthesis has been shown to be a property of the 30 S subunit of the ribosome (Davies, 1964a; Cox, White, and Flaks, 1964; Davies, unpublished results), and has been shown to affect specifically the recognition process (Pestka, Marshall, and Nirenberg, 1965; Pestka, 1966b; Kaji and Kaji, 1965) presumably by disturbing the normal messenger RNA-ribosome-transfer RNA interaction in such a way that incorrect base pairing can...

Stimulation and inhibition of polypeptide synthesis by streptomycin in ribosomal systems of Escherichia coli, programmed with various messengers

1. The action in vitro of streptomycin on polypeptide synthesis has been studied with cell-free extracts from streptomycin-sensitive and resistant Escherichia coli. Various messengers, such as turnip yellow mosaic virus RNA, alfalfa mosaic virus RNA, polyuridylic acid and polyadenylic acid were employed, and ribosomes still associated with their endogenous messenger(s) were also included. 2. Varying effects of streptomycin on ribosomes from streptomycin-sensitive cells with messengers of viral origin and endogenous messenger(s) have been recorded. Depending on the Mg 2+ concentration either stimulation or inhibition was found. 3. Polyuridylic acid-directed polyphenylalanine synthesis and polyadenylic acid-induced polylysine formation were generally inhibited by streptomycin at all Mg 2+ concentrations studied. 4. The effects exerted by streptomycin on ribosomal systems from streptomycin-resistant cells were usually small or negligible. 5. Stimulation of “sensitive” systems was exerted on a ribosomal site. 6. Streptomycin-action became apparent during protein synthesis immediately after the antibiotic was added. The degree of inhibition or stimulation was the same whether the antibiotic was added before or during the process of protein synthesis. 7. The results are discussed in relation to current concepts of streptomycin action.

Stimulation and inhibition of polypeptide synthesis by streptomycin

Stimulation and inhibition of polypeptide synthesis by streptomycin

Inhibition of polypeptide synthesis by streptomycin

Inhibition of polypeptide synthesis by streptomycin