NONRIBOSOMAL INCORPORATION OF AMINO ACIDS INTO THE TROPONIN-LIKE PROTEIN FROM SYNAPTOSOMES

A preparation of synaptosomal cytoplasm was isolated from forebrain of young rats and incubated with various amino acids in vitro. Incorporation of amino acids into protein was observed. This incorporation did not occur by ribosomal protein synthesis. The amino acid incorporating system was not stimulated by ATP and was inhibited by calcium. The system incorporated amino acids enzymatically. An electrophoretic analysis of the synaptosomal preparation, following incubation in the presence of radioactive amino acids, showed only three labelled protein species (molecular weights 37,000, 26,000 and 20,000). This incorporation of amino acids was found to have a high degree of specificity for three protein species. Migration of the three protein species was found to be nearly identical to that of rabbit muscle troponin. The proteins incorporating amino acids were also found to have other characteristics of the troponin subunits. A possible role of troponin modification is discussed.     

Inhibition of aminoacyl-tRNA synthetases by p-chloroamphetamine and its role in protein synthesis inhibition

p-Chloroamphetamine inhibited to some degree all amino acid-dependent pyrophosphate-exchange activities which could be detected in a rabbit reticulocyte extract. A detailed kinetic analysis of the reaction catalyzed by reticulocyte leucyl-tRNA synthetase demonstrated that the inhibitor affected only amino acid binding. Less rigorous studies of other synthetases from both rabbit reticulocyte and Escherichia coli could be similarly interpreted, suggesting that this compound interacts in a common manner with these several enzymes. The contribution of such effects to the inhibition of protein synthesis by the drug was investigated using cell-free translation systems in which rates of amino acid incorporation were limited to varying degrees by the synthesis and availability of aminoacyl-tRNA. In a wheat germ system programmed with globin mRNA, in which levels of amino acids and aminoacyl-tRNAs were shown to limit the rate of protein synthesis, the inhibition produced by p-chloroamphetamine could be partially reversed by increasing the concentration of the limiting amino acid. In a reticulocyte lysate, in which amino acid concentrations were not limiting, inhibition failed to show an amino acid-reversible component. Thus, while the inhibition of aminoacyl-tRNA synthetases by amphetamines can be shown in some cases to play a role in the effects of these compounds on in vitro protein synthesis, other sites of interference with initiation and/ or elongation reactions may predominate, depending on the construction of the system under study.     

THE CELL-FREE SYNTHESIS OF ALKALOID-BINDING PROTEINS IN IMMATURE RAT BRAIN1

Abstract— cell-free amino acid incorporating system from immature rat brain, consisting of ribosomal and soluble fractions, has been investigated for its capacity to incorporate [¹⁴C]amino acids into specific soluble proteins that interact with vinblastine sulfate and colchicine. The soluble ¹⁴C-labeled proteins formed in the cell-free system during incubation were compared with similar soluble proteins from immature rat brain which had been labeled in vivo by the incorporation of ¹⁴C-labeled amino acids. Criteria for the formation of vinblastine-binding, ¹⁴C-labeled proteins were: (1) aggregation of ¹⁴C-labeled soluble protein by one mm -vinblastine sulfate and (2) immunoprecipitation of ¹⁴C-labeled soluble protein by an antiserum against vinblastine sulfate-precipitable material. Criteria for the formation of [³H]colchicine-binding, ¹⁴C-labeled protein were based upon: (1) co-precipitation of the ³H-and ¹⁴C-labeled materials by vinblastine sulfate and (2) the coincidence of ³H- and ¹⁴C-labeled elution peaks from columns of Sephadex G-200, DEAE-Sephadex A-50 and isoelectric focusing. Both in the in vitro and in the in vivo system, ¹⁴C-labeled amino acids were incorporated into soluble proteins of the post-microsomal supernatant fraction. Proteins labeled with ¹⁴C-labeled amino acids in vitro and in vivo yielded comparable and qualitatively identical results by the criteria tested, including the formation of immunoprecipitates. In the in vitro system, ¹⁴C-labeled amino acids were incorporated into protein with a molecular weight of approx 120,000, an isoelectric point of 5.3 and with a chromatographic mobility on Sephadex G-200 which is identical to [³H]colchicine-binding protein. The above experimental results are presumptive evidence for the synthesis of vinblastine-binding and colchicine-binding proteins in the in vitro cell-free system.     

Molecular correlates of tissue browning and deterioration in Scots pine calli

Callus cultures were initiated from the bud apices of 10–40-year-old Scots pines (Pinus sylvestris L.) at different seasons and maintained on modified MS medium without subculturing. Separate sets of experiments were used for analyses of carbohydrate content, ethylene production, amino acid composition, protein patterns andin vitro translation. In each case the change in the colour of the calli was recorded and the fresh mass of the samples measured. The onset of tissue browning was found to be associated with changes in protein pattern, amino acid content, ethylene production and the occurrence of sucrose and accumulation of starch.In vitro translation experiments using poIy(A)⁺ RNA isolated and purified from the calli indicated that the switch in metabolism accompanying browning is paralleled by activated protein synthesis. Thus, the development of brown colour does not as such seem to be harmful to the tissue. The later, more intense tissue browning and deterioration which is reflected in a reduced capacity for protein synthesis and changes in the free amino acid pool and protein pattern is probably a secondary phenomenon.     

Control of l-amino acid oxidase in Neurospora crassa by different regulatory circuits

l-Amino acid oxidase is synthesized in Neurospora crassa in response to three different physiological stimuli: (i) starvation in phosphate buffer, (ii) mating, and (iii) nitrogen derepression in the presence of amino acids. During starvation in phosphate buffer, or after mating, l-amino acid oxidase synthesis occurred in parallel with that of tyrosinase. Exogenous sulfate repressed the formation of the two enzymes in starved cultures, but not in mated cultures. Sulfate repression was relieved by protein synthesis inhibitors, suggesting that the effect of sulfate required the synthesis of a metabolically unstable protein repressor. With amino acids as the sole nitrogen source only l-amino acid oxidase was produced. Under these conditions enzyme synthesis was repressed by ammonium and was insensitive to sulfate. Biochemical evidence suggested that the l-amino acid oxidase formed under the three different conditions was the same protein. Therefore, the expression of l-amino acid oxidase appeared to be under the control of least two regulatory circuits. One, also controlling tyrosinase, seems to respond to developmental signals related to sexual morphogenesis. The other, controlling other enzymes of the nitrogen catabolic system, is used by the organism to obtain nitrogen from alternative sources such as proteins and amino acids.     

Abscisic-acid-induced turion formation in Spirodela polyrrhiza L III. Specific changes in protein synthesis and translatable RNA during turion development

Abstract The developmental process leading to the formation of the abscisic acid (ABA) induced turion of Spirodela polyrrhiza was accompanied by a repression of nucleic acid and protein synthesis. DNA synthesis in the developing lurion (induced by 10⁻⁴mol m⁻³ ABA) was inhibited within 3h of ABA addition, followed by a repression of protein synthesis after 24 h, while RNA synthesis was not inhibited until 3 d. The inhibitory effect of ABA on protein synthesis was found to be selective and the synthesis of several novel proteins appeared to be induced. These effects were specific to ABA-sensitive tissue. The relationship between the changes in the protein and mRNA profiles during the development of the turion was investigated. The rapid general inhibition of protein synthesis at early stages of lurion formation could not be accounted for by the level of translatable mRNA, indicating an effect of ABA at the translational level. The specific alteration to the pattern of in vivo labelled proteins could have resulted, however, from control of the level of specific mRNAs for those particular proteins. Only after 3 d in ABA, when the developing primordium is committed to the turion developmental pathway, is there a total inhibition in the production of mRNA leading to the shutdown of all primary processes and the onset of the irreversible events leading to the dormant state.     

METHYL MERCURY INHIBITION OF SYNAPTOSOME AND BRAIN SLICE PROTEIN SYNTHESIS: IN VIVO AND IN VITRO STUDIES

Subacute methyl mercury (MeHg) intoxication was induced in adult rats following the daily intragastric administration of 1 mg MeHg/100 g body weight. Decreased [¹⁴C]leucine incorporation into cerebral and cerebellar slice protein was found. Weight loss occurred during the latent and neurotoxic phases but pair feeding did not reveal a significant defect in amino acid incorporation into slice protein. There was no decline in synaptosome protein synthesis in vitro during the latent phase but a significant decline in cerebellar and cerebral synaptosome synthesis was found during the neurotoxic phase. MeHg in vitro inhibited cerebral slice and synaptosome protein synthesis at half maximal concentrations of 7.5 and 12.5 μM respectively. Inhibition of synthesis in synaptosomes was non-competitive with K₁ of 4 × 10^?6M. MeHg had no effect on [¹⁴C]leucine or [¹⁴C]proline uptake into synaptosomes. There was no significant inhibition of synaptosome basal ATPase or Na + K ATPase at concentrations of MeHg (12 μM) giving half maximal inhibition of protein synthesis. No preferential inhibition of the chloramphenicol (55S) or cycloheximide sensitive components of synaptosome fraction protein synthesis was found, suggesting that the inhibition is common to both mitochondrial and extramitochondrial protein synthesizing systems. Addition of nucleotides and/or atractylate failed to influence protein synthesis and did not reverse the MeHg inhibition. Mannitol, as a replacement for the predominant cation species of the incubation medium, gave 40% inhibition of protein synthesis in the control but protected against further inhibition by MeHg.     

Interaction of Melanin with Proteins – The Importance of an Acidic Intramelanosomal pH

Melanin is a highly irregular heteropolymer consisting of monomeric units derived from the enzymatic oxidation of the amino acid tyrosine. The process of melanin formation takes place in specialized acidic organelles (melanosomes) in melanocytes. The process of melanin polymerization requires an alkaline pH in vitro, and therefore, the purpose of an acidic environment in vivo remains a mystery. It is known that melanin is always bound to protein in vivo. It is also seen that polymerization in vitro at an acidic pH necessarily requires the presence of proteins. The effect of various model proteins on melanin synthesis and their interaction with melanin was studied. It was seen that many proteins could increase melanin synthesis at an acidic pH, and that different proteins resulted in the formation of different states of melanin, i.e., a precipitate or a soluble, protein‐bound form. We also present evidence to show that soluble protein‐bound melanin is present in vivo (in B16 cells as well as in B16 melanoma tissue). An acidic pH appeared to be necessary to ensure the formation of a uniform, very high molecular weight melano–protein complex. The interaction between melanin and proteins appears to be largely charge‐dependent as evidenced by zeta potential measurements, and this interaction is also increased in an acidic pH. Thus, it appears that an acidic intramelanosomal pH is essential to ensure maximum interaction between protein and melanin, and also to ensure that all the melanin formed is protein‐bound.     

Inhibition of protein synthesis by products of lipid peroxidation

Effects of lipid peroxidation products on in vivo and in vitro protein synthesis have been studied. Malondialdehyde (MDA), a product, and a routinely used index of lipid peroxidation, inhibits in vivo protein synthesis in the two mosses, Tortula ruralis and Cratoneuron filicinum, and in pea (Pisum sativum) leaf discs. When wheat germ supernatant or poly(A)-rich mRNA of T. ruralis was incubated with MDA its subsequent activity in a cell-free protein-synthesizing system was reduced. When MDA was added directly to the in vitro protein-synthesizing mixture containing moss polyribosomes, the inhibition of amino acid incorporation was small. However, when simultaneous lipid peroxidation was allowed to occur along with in vitro protein synthesis there was a strong inhibition of amino acid incorporation and MDA accumulated in the reaction mixture indicating that products of lipid peroxidation other than, and apparently more toxic than, MDA were involved. It was concluded that lipid peroxidation inhibits protein synthesis probably by releasing toxic products which may react with and inactivate some components of the protein-synthesizing complex.     

The effects of phenylpyruvate and hyperphenylalaninemia on incorporation of (6- 3 H)glucose into macromolecules of slices of rat cerebral cortex

The effects of phenylpyruvate and hyperphenylalaninemia on the incorporation of [6-³H]glucose into lipids, proteins and nucleic acids were examined in differentiating and adult rat brain. Foetal brain was most sensitive to inhibition by phenylpyruvate in vitro, with significant effects occurring at 2·5 mM for labelling of lipids and proteins and at 5 mM for labelling RNA and DNA. Older age groups were less affected, and cortical slices from adult brain were slightly or not at all affected by phenylpyruvate. The inhibition by phenylpyruvate of incorporation of [6-³H]glucose into nucleic acids, proteins, and lipids could be further distinguished by the reversibility of the effect on nucleic acid and protein synthesis at high levels of glucose and the irreversibility of the effect on lipid synthesis. Lipid synthesis was most sensitive to inhibition by phenylpyruvate at the stage of fatty acid synthesis, with lesser effect on the formation of glyceride glycerol. Exposure in utero of the foetal brain to maternal hyperphenylalaninemia resulted in reduction of 26–38 per cent in the subsequent incorporation in vitro of [6-³H]glucose into lipids, proteins, RNA and DNA of brain slices from foetal animals. Feeding hyperphenylalaninemic pregnant rats a high-glucose diet significantly protected the foetal brain from the neurotoxicity accompanying the hyperphenylalanemia.     

Protein Synthesis in Plant Microsomal System

An active microsomal system from 48-h germinating seeds of Vigna sinensis (L.) Savi has now been developed. It can incorporate amino acids into protein under both in vitro and in vivo conditions, provided dithiothreitol (a protective reagent for SH groups) and phenylthiourea (an inhibitor of phenol oxidase) are present in the buffer system for extraction; and provided the assay mixture contains added dithiothreitol. The system consists of microsomes or ribosomes, tRNA or pH 5 fraction and 20 natural amino acids, ATP and an ATP-generating system and GTP with requirement for Mg ions. The cell fractions possess aminoacyl-RNA synthetase activity as indicated by the aminoacylhydroxamate formation. Microsomal synthesis is stimulated by exogenous tRNA from Escheriehia coli or rat liver and sensitive to various inhibitors such as cyclo-heximide, chloramphenicol, fusidic acid. The ribosomal transfer reaction has absolute dependence on the microsomal wash, on the crude enzyme from the same participate source, and on a synthetic messenger. It is greatly suppressed by fusidic acid and by cycloheximide.     

Effect of glutamine on penicillin formation in Penicillium chrysogenum

Summary Resting-cell studies in Penicillium chrysogenum have indicated that penicillin formation is inhibited by glutamine concentrations higher than 1 mM. Total inhibition was obtained with 10 mM glutamine. This action was neither reverted by the amino acid precursors of the antibiotic moiety nor glutamin affected the in vitro activity of the first enzyme of the penicillin formation pathway. The inhibition was prevented by 1 mM glutathione by mechanisms not related to limitation in the glutamine incorporation nor connected with degradation of the tripeptide.     

Studies on the incorporation of ( 14 C)amino acids into protein by isolated rat brain nuclei

—Various parameters of the in vitro incorporation of [¹⁴C]amino acids into protein by cell nuclei isolated and purified from rat brain and liver were investigated. Nuclei purified through 2.2 m sucrose solution were capable of amino acid incorporation in vitro; and washing procedure to eliminate hypertonic sucrose before incubation was essential since sucrose in high concentration was inhibitory. Microbial contamination was found to be a serious source of error and the use of sterile conditions for incubation were necessary to obtain reproducible and valid results. Using completely sterile conditions, Na ⁺, K⁺, RNase, DNase, puromycin, cycloheximide and chloramphenicol were without any effect on the ability of brain and liver nuclei to incorporate labelled amino acids into protein. Results of time-course and preincubation experiments revealed that some factors essential for amino acid incorporation pass out of the nucleus into the medium. In addition, approximately 15 per cent of the labelled nuclear proteins with higher specific radioactivity was recovered in the incubation medium. Incorporation of [¹⁴C]leucine was proportional to the concentration of labelled amino acid and to the number of nuclei, and it is suggested that carefully controlled conditions of incubation are essential to obtain valid comparisons between different types of nuclei in terms of their relative abilities to incorporate amino acids in vitro. No evidence was obtained indicating isotope dilution phenomenon in these experiments. Whether or not in vitro incorporation of amino acid by nuclei represents protein synthesis is discussed.     

Ethanol-induced changes in lipid composition of Escherichia coli: Inhibition of saturated fatty acid synthesis in vitro

Growth of Escherichia coli in the presence of ethanol results in the synthesis of lipids containing elevated proportions of unsaturated fatty acids. Previous in vivo experiments indicated that the ethanol-induced changes in fatty acid composition result from a preferential inhibition of saturated fatty acid synthesis. In this study, the inhibition of saturated fatty acid synthesis by ethanol was confirmed in vitro. This inhibition was not membrane mediated and resulted from a direct action of ethanol on the soluble enzymes of fatty acid synthesis. The addition of ethanol resulted in a decrease in chain length of both saturated and unsaturated acyl products in vitro. Experiments with enzymes prepared from several fatty acid synthesis mutants of E. coli indicate that β-hydroxydecanoyl-acyl carrier protein dehydrase is not the site of the ethanol inhibition of saturated fatty acid synthesis. The two condensing enzymes are the probable sites for inhibition by ethanol.     

Tryptophan Overload in the Pregnant Rat: Effect on Brain Amino Acid Levels and In Vitro Protein Synthesis

The concentration of most amino acids was higher in the brains of 19- and 21-day rat fetuses than in their respective mothers. After an intraperitoneal load of tryptophan to the mother, the intracerebral concentration of several amino acids (including leucine) decreased not only in the mothers, but also in their fetuses. The in vitro incorporation of pHJleucine into proteins in brain postmitochondrial supernatant fractions was enhanced in both the mothers and fetuses after tryptophan administration, but this effect disappeared when protein synthesis was calculated by using specific activities corrected for the amount of unlabeled leucine in the preparation. By this criterion, protein synthesis activity appeared similar in the brains of 19- and 21-day pregnant rats but was higher in their fetuses, especially in the 21-day subjects. Thus, protein synthesis in the brain was not altered by marked changes in the amino acid pool and more profound and prolonged metabolic disturbances must occur to cause permanent damage in the developing brain.     

PROTEIN SYNTHESIS IN EHRLICH ASCITES CELLS

Rapidly dividing Ehrlich ascites cells have a higher rate of protein synthesis than slowly-growing cells. This is true whether protein synthesis is measured in vivo or in a cell-free amino acid incorporating system. A lack of endogenous messenger RNA or a ribosomal defect in stationary cells is suggested as a likely cause of the decreased rate of protein synthesis. The soluble proteins synthesized by rapidly-dividing cells are qualitatively different from those synthesized by slowly-growing cells. The latter also have a larger intracellular pool of several amino acids and a slower protein turnover than rapidly-dividing cells.     

Mammalian cells do not have a stringent response

A key attribute of the stringent response of bacteria is the rapid inhibition of ribosomal RNA synthesis mediated by unusual nucleotides in response to uncharged tRNA. The question as to whether mammalian cells show a stringent response analogous to that of bacteria was critically tested by the effective rapid amino acid starvation of both normal and transformed cells. Rapid starvation giving a high proportion of uncharged tRNA for leucine was produced within 7 minutes of expression of a nonleaky ts leucyl tRNA synthetase mutation in transformed CHO cells (tsH1) and in its normal growth control revertant (L-73). To control for the effect of temperature alone, tsrevertants of tsH1 and L-73 were included in the study, and to control for effects due simply to the inhibition of protein synthesis, the translational elongation inhibitor cycloheximide was used. In addition, rapid starvation for histidine was effected by incubation of both the CHO cell lines and of freshly explanted normal Chinese hamster embryo fibroblasts in histidine-free medium containing high concentrations of histidinol. The rate of preribosomal RNA synthesis and the extent of its maturation to mature rRNA was measured using (³H-methyl) methionine as a donor of methyl groups during synthesis and methylation of pre-rRNA. There was no effect on pre-rRNA synthesis of the rapid generation of uncharged tRNA for 45 minutes for any of the cell types tested. A nonspecific inhibition of maturation of 18S rRNA and late (3 hour) inhibition of pre-rRNA synthesis was observed, but could be mimicked by the inhibition of protein synthesis to comparable levels with cycloheximide. Less severe amino acid starvation resulting in a more physiological inhibition of protein synthesis to 30% also had no specific effect on pre-rRNA synthesis and maturation. Intracellular nucleotide pools were also examined for the appearance of unusual nucleotides such as guanosine tetraphosphate or pentaphosphate and for changes in the levels of normal nucleotides after severe amino acid starvation. No such changes could be detected. We conclude that although mammalian cells may have some biochemical reactions which respond to uncharged tRNA, they do not possess a macromolecular control system analogous to the stringent response of bacteria.     

Effect of amino acid starvation on UV sensitivity ofLactobacillus acidophilus cells

InLactobacillus acidophilus cultures UV irradiated in the exponential phase of growth, the dosesurvival curve was of the simple exponential type, without any shoulder. If the bacteria were subjected to amino acid starvation prior to irradiation, an shoulder corresponding to a quasi-treshold dose (D_q) of about 780 ergs/mm² appeared in the curve. The administration of protein or RNA-synthesia inhibitors prior to irradiation had the same effect. The effect of pre-irradiation amino acid starvation was abolished by simultaneous thymidine starvation. It was likewise abolished if amino acid starvation was followed by incubation in the presence of amino acids (without thymidine) and then by irradiation of the cells. Post-irradiation amino acid starvation did not lead to the formation of an shoulder but if combined with thymidine starvation it did. It can be concluded from the results that post-irradiation repair processes are facilitated or promoted if, during the post-irradiation interval DNA synthesis is delayed. This delay represents a compensation of the pre-irradiation increase of cellular DNA-content, taking place during inhibition of proteosynthesis. The postirradiation administration of caffeine did not abolish the formation of the shoulder induced by pre-irradiation amino acid starvation; on the contrary, it induced its formation even in exponentially growing, irradiated control bacteria.     

PROTEIN BIOSYNTHESIS IN SALT-SHOCKED CELLS OF STICHOCOCCUS BACILLARIS (CHLOROPHYCEAE)1

We have developed an in vivo¹⁴C-amino acid labelling procedure for monitoring protein synthesis in salt-shocked cells of Stichococcus bacillaris Naeg. This alga possesses an efficient transport system for the uptake of leucine, methionine, and phenylalanine and rapidly incorporates these amino acids into proteins. Of the three amino acids tested, ¹⁴C-phenylalanine is ideally suited for labelling proteins in S. bacillaris, as it establishes an early equilibrium between uptake and incorporation of the amino acid into proteins. The uptake of phenylalanine shows little inhibition following transfer of cells to higher salinities and is also not affected in short-term experiments by the presence of the protein inhibitors cycloheximide and chloramphenicol. While Stichococcus bacillaris grows slowly at salinities equal to, or higher than, 150% artificial seawater (ASW), it shows surprising rates of recovery of major physiological functions following considerable salt shocks. Cells transferred from 33 to 150% ASW show complete recovery of photosynthetic activity and protein synthesis within 10–15 min, and cell transferred from 33 to 300% ASW recover 50% of their capacity to synthesize proteins within. 1 h. Cytoplasmic and organellar protein synthesis appears to be equally sensitive to the effects of salt shocks according to studies with protein synthesis inhibitors.     

The Effects of Canavanine on Protein and Nucleic Acid Synthesis in Canavanine Resistant and Sensitive Species of Higher Plants

The effectiveness of the inhibitor, canavanine, was evaluated by examining its action in Canavalia ensiformis and Glycine max. Isolated roots were grown in culture tubes containing White's medium plus canavanine or arginine. A differential effect of canavanine on the incorporation of precursors of DNA, RNA, and protein was found, which is assumed to be related to the ability of the plant to utilize canavanine in reactions typically involving arginine. Canavanine was not found to affect DNA, RNA, or protein synthesis in Canavalia ensiformis, a plant in which this amino acid is synthesized naturally. In the canavanine sensitive species, Glycine max, of the same subfamily Papilionoideae, canavanine was observed to inhibit strongly DNA, RNA, and protein synthesis. A primary inhibition of the RNA synthesizing system is suggested. The data indicate the canavanine inhibitions are more complex than a simple competition with arginine in protein synthesis.