Purification and partial characterization of the antiviral protein from Phytolacca americana which inhibits eukaryotic protein synthesis.

The antiviral protein from the pokeweed plant (Phytolacca americana) which inhibits eukaryotic protein synthesis has been purified to homogeneity and its molecular weight has been determined by two physical methods. The protein consists of a single polypeptide chain of an approximate molecular weight of 27,000. The inhibitory effect of this protein on the synthesis of polyphenylalanine in a cell-free system from Artemia salina suggests that this protein acts in an enzymatic manner on eukaryotic ribosomes. It is also demonstrated that polyphenylalanine synthesis on A. salina ribosomes is more sensitive to inhibition by this protein than on rabbit reticulocyte ribosomes.     

Inhibition of peptide chain initiation by a nonhemin-regulated translational repressor from Friend leukemia cells.

A nonhemin-regulated translational repressor protein has been purified partially from the postribosomal supernatant fraction of Friend leukemia cells grown in the absence of dimethylsulfoxide. This repressor inhibits protein synthesis in lysates from rabbit reticulocytes or Friend leukemia cells and in a fractionated system using Artemia salina ribosomes, reticulocyte mRNA, and soluble components from reticulocytes. In contrast, the hemin-controlled repressor from reticulocytes does not inhibit protein synthesis in lysates from Friend leukemia cells. The repressor from Friend leukemia cells has no effect on poly(U)-directed synthesis of polyphenylalanine using reticulocyte ribosomes nor on the extension and release of nascent globin chains that were initiated in intact reticulocytes. It does not block completion of peptides on ribosomes isolated from reticulocytes incubated with NaF nor does it inhibit initiation factor-dependent formation of methionylpuromycin, but it inhibits globin mRNA-dependent methionylvaline synthesis. The Friend leukemia cell repressor promotes peptide synthesis-dependent breakdown of polysomes in reticulocyte lysates that appears to involve inhibition of ribosome reattachment to mRNA during peptide chain initiation. It is concluded that the Friend leukemia cell repressor blocks peptide initiation at a point between the addition of methionyl-tRNA^fMet to the ribosomal initiation complex and the NaF-sensitive reaction.     

The inhibition of eucaryotic protein synthesis by procaryotic elongation factor Tu

The activity of elongation factor Tu (EF-Tu) from $\text{Escherichia}\text{coli}$ in eucaryotic protein synthesis systems was investigated. EF-Tu was found to inhibit polyphenylalanine synthesis when incubated with $\text{Artemia}$ 80S ribosomes, purified rabbit reticulocyte elongation factor Tu (eEF-Tu) and partially purified reticulocyte translocase enzyme, eEF-G. The inhibition could be overcome by supplying the system with additional eEF-Tu. EF-Tu also inhibited protein synthesis in rabbit reticulocyte lysates. Data presented in this report indicate that inhibition by EF-Tu results from the accumulation of ternary complexes of the protein factor, GTP and aminoacyl-tRNA which do not interact with the ribosomal A-site of 80S ribosomes under physiological conditions.     

Evidence for activities of rabbit reticulocyte elongation factor 1 analogous to bacterial factors EF-Ts and EF-Tu

Preparations have been obtained from rabbit reticulocyte elongation factor 1 (EF-1) that exhibit activities analogous to the heat stable and heat labile factors, EF-Ts and EF-Tu, of $\text{Escherichia coli}$. The heat stable fraction, prepared by heating EF-1 in the presence of GTP, has virtually no activity in poly (U)-directed polyphenylalanine synthesis. The fraction exhibiting activity similar to bacterial EF-Tu is obtained by the interaction of EF-1 with GTP and phenylalanyl-tRNA followed by passage of the solution through a nitrocellulose filter. The filtrate, which alone has low activity in polyphenylalanine synthesis, when combined with the heat stable fraction gives high activity suggesting that the heat stable preparation catalyzes recycling of the filtrate component.     

Inhibition of protein synthesis in mouse myeloma cells by Ricinus communis toxin.

The mechanism of inhibition of protein synthesis in mouse myeloma cells by Ricinus communis toxin was studied. No significant disaggregation of polysomes into monosomes was detected in the toxin-treated cells. The activity of the polysomes isolated from the cells treated with the toxin in protein synthesis was remarkably lower than that of the untreated cells, while the activity of the supernatant enzyme fraction was retained. The ribosomes derived from the polysomes of the toxin-treated cells were inactive in poly(U)-dependent polyphenylalanine synthesis. The activity of ribosomes reconstituted by hybridizing subunits derived from the ribosomes of normal and toxin-treated cells were measured in poly(U)-dependent polyphenylalanine synthesis, and the 60 S subunit was revealed to be inactive. These results indicate that the target of action of the toxin towards intact cells is the 60 S ribosomal subunit.     

Temperature sensitivity of protein synthesis initiation in the reticulocyte lysate system. Reduced formation of the 40 S ribosomal subunit - Met-tRNAf complex at an elevated temperature.

Analysis of protein synthesis in the rabbit reticulocyte lysate system revealed the existence of a temperature-sensitive step in chain initiation which became irreversibly inactivated in the incubation at 42 degrees C. This inactivation of initiation was accompanied by a marked reduction in formation of the 40 S ribosomal subunit - Met-tRNAf complex. Decreased protein synthesis and a decrease in formation of the 40 S complex were also evident in unfortified lysates which had been prewarmed at 42 degrees C prior to protein synthesis. Hemin did not protect such lysates. The addition of supernatant fraction of a fresh lysate did not promote recovery of the reduced protein synthesis by such prewarmed lysates. Moreover, the addition of supernatant fraction prewarmed at 42 degrees C in the presence of added hemin caused little inhibition of protein synthesis by fresh lysate. The results indicate that the supernatant fraction is not involved in the inactivation.     

Evidence for an inhibitor of protein synthesis in rabbit reticulocytes activated by high pressure

Evidence is presented for an inhibitor of protein synthesis which is activated by subjecting rabbit reticulocyte postribosomal supernatant to high pressure (15,000 1b/in²). The inhibitor is antigenically and chromatographically distinct from the hemin-controlled repressor. The mechanism of inhibition by the pressure-treated postribosomal supernatant is not clear; however, the kinetics of inhibition seem to indicate that inhibition may be at the level of initiation.     

Inhibition of ribonucleases by ribonucleotides and transition state analogs in cell-free extracts from Ehrlich ascites tumor cells.

We investigated the ribonucleolytic breakdown of poly(U), poly(A), RNA trascribed from calf thymus DNA with E. coli RNA polymerase, ribosomal RNA, tRNA and mengovirus RNA by an enzyme fraction obrained from a postribosomal supernatant of Ehrlich ascites tumor cells. The single-stranded homopolyribonucleotides are preferentially degraded by the enzyme fraction with the production of ribonucleoside 5'-monophosphates. The RNase activity is completely dependent on the presence of Mg2+ ions and is highest at Mg2+ and K+ concentrations optimal for cell-free protein synthesis. Ribonucleoside 5'-monophosphates, ribonucleoside 2'(3')-monophosphates, ribonucleoside 2'(3'),5'-bisphosphates and transition state analogs consisting of vanadyl sulfate and either ribonucleosides or ribonucleoside 5'-monophosphates in a molar ratio 1:1 inhibit the ribonucleolytic activity of the enzyme fraction. The ribonucleoside 2'(3'),5'-bisphosphates and the transition state analogs are the most effective inhibitors. However, only in the presence of ribonucleoside 2'(3'),5'-bisphosphates a concomitant stimulation by 50 to 60% of poly(U)-directed polyphenylalanine synthesis is observed; all the other RNase inhibitors tested also inhibit polypeptide synthesis. The results of preliminary experiments show that poly(U) and ribonucleoside 2'(3'),5'-bisphosphates are well suited as ligands for affinity chromatography of ribonucleases from Ehrlich ascites tumor cells.     

Actin--an inhibitor of eukaryotic elongation factor activities

An inhibitor of diphtheria toxin- and endogenous transferase-dependent ADP-ribosylation of eukaryotic elongation factor 2 (eEF2) has been found in the cytoplasmic fraction from rat liver. We provide evidence that this cytoplasmic inhibitor corresponds to actin, which gives rise also to inhibition of polyphenylalanine (polyPhe) synthesis. Both globular monomeric (G-actin) and filamentous (F-actin) forms of actin appear to be inhibitory on the action of elongation factors 1 and 2 (eEF1 and eEF2) in polyPhe synthesis with the inhibitory effect of G-actin proving to be stronger. Some component(s) in the postribosomal supernatant (S-130) fraction and also DNase I prevent actin-promoted inhibition of polyPhe synthesis.     

Heat inhibition of reticulocyte protein synthesis. Evidence for a mechanism independent of the hemin-controlled repressor

Incubation of rabbit reticulocytes at 45 degrees C results in a prompt but reversible decrease in protein synthesis and a concomitant conversion of polyribosomes to smaller aggregates. These effects occur even in the presence of 100 micrometer hemin in the incubation medium. There is also inhibition of heme synthesis but this occurs at a later time than the effect on protein synthesis. The inhibtion of heme synthesis results from a decrease in activity of beta-aminolevulinic acid synthetase. This decrease of heme synthesis appears to be secondary to the inhibition of protein synthesis with resultant accumulation of intramitochondrial heme (which will decrease beta-aminolevulinic acid synthetase activity). An inhibitor of reticulocyte cell-free protein synthesis formed in the postribosomal supernatants of cells incubated at both 45 and 37 degrees C but not at 0 degrees C. No temporal or quantitative differences in the amount of this inhibitor from cells treated at either 37 or 45 degrees C was apparent. The inhibitor was not found in the fraction where the hemin-controlled repressor is isolated. It is concluded that heat inactivation of intact reticulocyte protein synthesis does not depend upon a decrease in heme synthesis, heme concentration or generation of the hemin-controlled repressor. Furthermore, it appears that the inhibitor formed in the post-ribosomal supernatant cannot be the sole cause of the heat inhibition of protein synthesis.     

Localization of glycollate dehydrogenase in Dunaliella salina

Glycollate dehydrogenase of the halotolerant green alga Dunaliella salina, isolated from a brine pond, was found associated with the membrane fraction which exhibited complete photosynthetic activity. Highest enzyme activity was found in cells grown in the presence of 5% NaCl. Any increase in NaCl concentration led to a decrease in specific enzyme activity.Abbreviations PSI(II) photosystem I(II)     

The termination of the synthesis of proteins in vitro with extracts from the undeveloped cyst of Artemia salina

Soluble fractions (S-100) from both undeveloped cysts and developing embryos of Artemia salina promoted elongation of polypeptides initiated in vivo on polysomes of developing embryos or nauplius larvae. The ability of the extract from the undeveloped cyst to terminate correctly the synthesis of polypeptides has been determined indirectly from the distribution of polysomes before and after in vitro translation and, more directly, from the nature of the protein product released from rabbit reticulocyte polysomes. The extract from the undeveloped cyst and also, as expected, that from the developing embryo catalyzed a reduction in the amount of the polysomes of larger size and an increase in the amount of 80 S ribosomes. The soluble extract from the undeveloped cyst can terminate the synthesis of rabbit globin on reticulocyte polysomes. The major polypeptide product released from the polysomes had an electrophoretic mobility identical with that of the subunit of isolated rabbit globin. This indicated that the cyst contained the components necessary to complete and terminate the synthesis of polypeptides correctly and that the released protein product was not predominantly as a result of premature chain termination. The size distribution of Artemia salina proteins released from polysomes from developing embryos was similar when the synthesis was directed by the S-100 at each stage of development.     

Localization of polyamine enhancement of protein synthesis to subcellular components of Escherichia coli and Pseudomonas sp. strain Kim.

At 5 mM Mg2+, spermidine stimulation of polyphenylalanine synthesis by cell-free extracts of Escherichia coli was found to be about 30 times greater than that by extracts of Pseudomonas sp. strain Kim, a unique organism which lacks detectable levels of spermidine. By means of reconstitution experiments, the target of spermidine stimulation was localized to the protein fraction of the highspeed supernatant component (S-100) of E. coli and was absent from, or deficient in, the S-100 fraction of Pseudomonas sp. strain Kim. The spermidine stimulation did not appear to be due to the presence in the E. coli S-100 fraction of ribosomal protein S1, elongation factors, or E. coli aminoacyl-tRNA synthetases. The failure to observe spermidine stimulation by the Pseudomonas sp. strain Kim S-100 fraction was also not due to a spermidine-enhanced polyuridylic acid degradation. The synthesis of polyphenylalanine by Pseudomonas sp. strain Kim extracts was stimulated by putrescine and by S-(+)-2-hydroxyputrescine to a greater degree than was synthesis by E. coli extracts. The enhancement by putrescine and by S-(+)-2-hydroxyputrescine with Pseudomonas sp. strain Kim extracts was found to be due to effects on its ribosomes.     

Comparison of cell-free protein synthesis by different regions of chicken brain

The cell-free protein synthesis by the postmitochondrial supernatant from chicken cerebrum was twofold greater than protein synthesis by the cerebellum or optic lobes. Ribosomal aggregation of mRNA and ribonuclease activity of the postmitochondrial supernatant from the three brain regions was not statistically different. The higher protein synthetic activity of the cerebral postmitochondrial supernatant was associated with both the postribosomal supernatant (cell sap) and microsomal fractions. Cerebral monomeric ribosomes were more active in polyuridylic acid directed polyphenylalanine synthesis than monomeric ribosomes from either the cerebellum or optic lobes. The ability of cerebral cell sap to support polyuridylic acid directed polyphenylalanine synthesis was 1.6 to 2 times greater than cell sap from the other two regions. Cell sap factors other than tRNA^phe or phenylalanyl-tRNA synthetases appear to be responsible for the higher protein synthetic activity of the cbr cell sap.     

Regulation of catecholamine-responsive adenylate cyclase activity in rat reticulocyte membranes by endogenous factors General characteristics and resolution into protein and nucleotide components

A 100 000 × g soluble, supernatant fraction obtained from the hemolysate of rat reticulocytes was studied for its effect upon catecholamine-sensitive adenylate cyclase activity in reticulocyte membranes. The supernatant material, devoid of adenylate cyclase activity itself, amplified isoproterenol-dependent activity in responsive membranes and was an essential requirement for the expression of hormone sensitivity in membranes rendered unresponsive to isoproterenol alone. The increment in catecholamine-associated activity conferred upon reticulocyte membranes by the supernatant material was β-adrenergic because it did not affect basal or fluoride-related activity and was completely inhibited by propranolol. Guanine nucleotides were present in the supernatant but could account for only a fraction of the total activity because the supernatant was able to cause greater stimulation than maximal concentrations of GTP and when specified concentrations of exogenous GTP were compared with equivalent nucleotide concentrations in the supernatant, the supernatant always led to greater activity. The supernatant was resolved into protein- and nucleotide-containing components by ion-exchange chromatography. Each component was approximately one-half as active in amplifying catecholamine-dependent adenylate cyclase as the unresolved, crude supernatant material. The activity eluted in the first peak of the DEAE chromatogram was resistant to alkaline phosphatase, sensitive to trypsin, not dialyzable and contained no detectable concentrations of GTP or GDP. In contrast, the activity eluted in the second peak of the DEAE chromatogram was sensitive to alkaline phosphatase, resistant to trypsin, completely dialyzable and contained both GTP (30 μM) and GDP (10 μM) in significant concentrations. Neither the crude supernatant not its two active components affected the binding of [¹²⁵I]-iodohydroxybenzylpindolol to reticulocyte membranes. These observations establish in rat reticulocytes the presence of protein and guanine nucleotide constituents which have independent influences upon the catecholamine-responsive adenylate cyclase of reticulocyte membranes.     

Immunological detection of the messenger RNA cap-binding protein

The 24-kilodalton messenger RNA cap-binding protein (CBP) was purified from the rabbit reticulocyte postribosomal supernatant fraction using an affinity resin consisting of the p-aminophenyl gamma-ester of m7GTP coupled to Sepharose. The affinity-purified CBP was used to raise a goat antiserum. Anti-CBP antibodies were purified by adsorption to CBP coupled to either Controlled-Pore Glass or diazobenzyloxymethyl paper. The affinity-purified antibodies reacted specifically with only the 24-kilodalton polypeptide in whole reticulocyte lysate and in initiation factors prepared from the same source. During a conventional (nonaffinity) purification of CBP from a high salt extract of the ribosomal pellet, immunological reactivity paralleled the ability to reverse cap analogue inhibition of translation, indicating that the 24-kilodalton polypeptide present in the postribosomal supernatant fraction is immunologically cross-reactive with the CBP purified from ribosomes. Fractionation of whole reticulocyte lysate by sucrose gradient sedimentation followed by immunoblotting revealed that CBP was present in the supernatant fraction and the region of the gradient corresponding to ribosomal subunits but not in mono- or polysomes. The CBP to ribosome ratio was found to be approximately 0.02, assuming that the m7GTP-Sepharose retains all of the protein. This is considerably lower than that of other initiation factors and suggests that CBP may be the limiting polypeptide factor involved in the initiation of protein synthesis. The antibodies also inhibited the translation of a capped messenger RNA (globin). Inhibition of the translation of an uncapped RNA (satellite tobacco necrosis virus) was also observed, but to a lesser degree than with globin mRNA.     

Baciphelacin: a new eukaryotic translation inhibitor

Baciphelacin an antibiotic produced by Bacillus thiaminolyticus was a potent inhibitor of protein synthesis in HeLa cells and other mammalian cell lines. It had no effect on DNA or RNA synthesis. Concentrations of baciphelacin around 10⁻⁷ M inhibited protein synthesis by 50% in intact cells. The antibiotic had no effect on protein synthesis in Saccharomyces cerevisiae or Escherichia coli, but inhibited the protozoan Trypanosoma brucei. In vitro protein synthesis in a rabbit reticulocyte cell-free system was blocked by baciphelacin. However, translation of globin mRNA in a wheat cell-free system was not affected by this antibiotic. Baciphelacin had no activity against a number of cell-free systems used to measure different steps of translation, including binding of substrates to the ribosome, peptide bond formation and polyphenylalanine synthesis. Therefore, it is assumed that it affects the initiation of translation or the charging of tRNA. Finally, the inhibition of protein synthesis by compounds structurally related to baciphelacin was tested and their effects compared to baciphelacin.     

Functional Properties of Lactate Dehydrogenase from Dunaliella salina and Its Role in Glycerol Synthesis

The dependence of the catalytic properties of lactate dehydrogenase (LDH, EC 1.1.1.27) from a halophilic alga Dunaliella salina, a glycophilic alga Chlamydomonas reinhardtii, and from porcine muscle on glycerol concentration, medium pH, and temperature was investigated. Several chemical properties of the enzyme from D. salina differentiated it from the LDH preparation obtained from C. reinhardtii and any homologous enzymes of plant, animal, and bacterial origin. (1) V _max of pyruvate reduction manifested low sensitivity to the major intracellular osmolyte, glycerol. (2) The affinity of LDH for its coenzyme NADH dropped in the physiological pH region of 6–8. Above pH 8, NADH virtually did not bind to LDH, while the enzyme affinity for pyruvate did not change considerably. (3) The enzyme thermostability was extremely low: LDH was completely inactivated at room temperature within 30 min. The optimum temperature for pyruvate reduction (32°C) was considerably lower than with the enzyme preparations from C. reinhardtii (52°C) and porcine muscle (61°C). (4) NADH greatly stabilized LDH: the ratio of LDH inactivation constants in the absence of the coenzyme and after NADH addition at the optimum temperature in the preparation from D. salina exceeded the corresponding indices of LDH preparations from C. reinhardtii twelve times and from porcine muscle eight times. The authors believe that these LDH properties match the specific metabolism of D. salina which is set at rapid glycerol synthesis under hyperosmotic stress conditions. The increase of cytoplasmic pH value produced in D. salina by the hyperosmotic shock can switch off the terminal reaction of the glycolytic pathway and thus provide for the most efficient utilization of NADH in the cycle of glycerol synthesis. As LDH is destabilized in the absence of NADH, this reaction is also switched off. In the course of alga adaptation to the hyperosmotic shock, glycerol accumulation and the neutralization of intracellular pH stabilize LDH, thus creating the conditions for restoring the complete glycolytic cycle.     

Mechanism of inhibition of hepatic protein synthesis in rats by the carcinogen, methylazoxymethanol acetate.

Treatment of rats with the carcinogen, methylazoxymethanol acetate, results in a rapid, marked inhibition of hepatic protein synthesis and disaggregation of polysomes. Studies were undertaken to learn the mechanism by which this carcinogen induces these effects in rat liver. The data show that the inhibition of endogenous protein synthesis is not due to an effect on the high speed supernatant 'factors' but rather at the level of the polysome, and that both free and membrane-bound polysomes are affected. Poly(U)-directed polyphenylalanine synthesis by native ribosomal subunits is greater in preparations isolated from rats treated with carcinogen than it is in controls. Moreover, the native ribosomal subunit fraction from treated livers in response to added rabbit globin mRNA is able to synthesize a protein similar in molecular weight to globin. These studies show that methylazoxymethanol acetate does not induce significant alterations of ribosomal subunits or of initiation factors and suggest that the inhibition of protein synthesis and disaggregation of polysomes may be the results of an alteration of cytoplasmic mRNA, or its association with ribosomes.