Inhibition Of Amino Acid Incorporation Research Articles

Protein Dysfunction and Inhibition of Amino Acid Incorporation in Root Segments from Wheat and Mung Bean Seedlings caused by some Amino Acid Analogues

The effects of para-fluorophenylalanine (p-FPA) and azetidine-2-carboxylic acid (AZ) on acid phosphatase and peroxidase activity, and on L-leucine incorporation, in root segments of Triticum aestivum L. cv. Fenman and Vigna radial a (L.) Wilczek, were studied. Incubation with 50 mmol−3 AZ significantly reduced phosphatase and peroxidase activities in wheat roots, but with 20 mol m−3p-FPA, only the peroxidase activity was reduced. In mung bean roots, phosphatase activity was inhibited by both AZ and p-FPA. Effects of the ortho- and meta- isomers of FPA on wheat root phosphatase and peroxidase, and on mung bean phosphatase, were also examined. Leucine uptake and incorporation were not inhibited by 5 h pre-incubation with either p-FPA or AZ, but were inhibited after 24 h of pre-incubation. The results support the view that, in the shorter term, the analogues inhibit enzyme activity by becoming incorporated to produce non-functional protein and, in the longer term, metabolism is further affected by inhibition of protein synthesis.

Effect of incubation conditions, inhibitors and seminal plasma on protein synthesis in ram spermatozoa.

The effects of incubation time (15 min-4 h), rate of semen to buffer dilution (1/10-1/40), and concentration of glucose (5.5-22 mM) on the rate of protein synthesis by ejaculated washed ram spermatozoa were determined. The rate of protein synthesis increased linearly as incubation time, dilution rate, and the glucose concentration increased. Denaturation of sperm protein with 1% HgCl2 caused an almost complete inhibition of amino acid incorporation. Protein synthesis over a period of 4 h was also inhibited by chloramphenicol but was not affected by cycloheximide. Protein synthesis and uptake of [14C]cAMP by washed ram spermatozoa was also significantly inhibited by the inclusion of 2-8% seminal plasma in the buffer. The present results indicate that the authentic protein synthesis by mature ram spermatozoa is mainly of mitochondrial origin. The data also suggest a role for intracellular cAMP in the regulation of sperm protein synthetic activity.

Synthesis of 2′,5′-oligoadenylate by rat liver nuclear matrix protein

Nuclear matrix was prepared from unstimulated rat liver by treatment of nuclei with DNAse and 0.4 M NaCl and was further extracted with 2.0 M NaCl. Proteins were bound to poly(rI):(rC)-agarose, incubated with (alpha-32P) adenosine 5'-triphosphate and 2',5'-linked oligoadenylate was isolated from the supernatant. The substance inhibited amino acid incorporation in a reticulocyte translation system and was identified after enzymatic treatment followed by thin-layer chromatography on PEI-cellulose. The possible function of 2',5'-oligo(A) synthetase in the maturation of pre-mRNA associated with nuclear matrix is discussed.

Regional impairment of protein synthesis in the rat brain during bicuculline-induced seizures

Protein synthesis was investigated in rats subjected to 30 min of bicuculline-induced seizures using biochemical and autoradiographic techniques. Incorporation studies were performed on freely convulsive animals following systemic administration of either a tracer dose of l-[1- 14C]tyrosine or a flooding dose (7.5 mmol/kg) of l-[1- 14C]valine. Using a tracer dose, amino acid incorporation was only moderately reduced (forebrain) or slightly enhanced (cerebellum/brainstem and spinal cord) but the specific radioactivity of [ 14C]tyrosine in the acid-soluble pool was increased 3- to 5-fold in experimental animals. After a flooding dose of [ 14C]valine the specific activity of the precursor amino acid was similar in control and convulsed animals. Under these conditions incorporation rates in forebrain and cerebellum/brainstem were reduced to 54 and 75%, respectively. Reduction of amino acid incorporation was even more pronounced in extraneural tissues, e.g. liver (6%), intestine (14%) and kidney (15%). Inhibition of protein synthesis in forebrain and cerebellum/brainstem was paralleled by a similar extent of polyribosome disaggregation in these regions (53 and 78% of controls). In anaesthetized, mechanically ventilated rats, 30 min of seizure activity reduced forebrain polyribosomes to a similar extent (57%). Extraneural (hepatic) protein synthesis was also affected in physiologically controlled rats, but cerebellar polysomes were completely preserved. Autoradiographic studies using 3H-labelled amino acids were carried out to identify nerve cell populations most heavily affected. In freely convulsive rats both tracer dose and pool overloading revealed a similar regional pattern with preferential inhibition of amino acid incorporation in forebrain cortex, thalamus and the pyramidal cell layer of the hippocampus. These sites were also affected in the physiologically controlled animal, but the focal distribution of hippocampal and thalamic neurones with reduced protein synthesis differed from that in freely convulsive rats.

Inhibition of mammalian microsomal protein synthesis by aminoglycoside antibiotics.

The aminoglycoside antibiotics gentamicin, kanamycin and netilmicin produce a dose-dependent inhibition of amino acid incorporation in microsomes isolated from human liver and rat brain, kidney and liver. Inhibitory effects on microsomal protein synthesis occur at concentrations that have been shown to accumulate in rodent and human renal cortex and perilymph following therapeutic administration. Inhibition of translation in those tissues which specifically accumulate aminoglycoside antibiotics may, in part, explain toxicities observed following exposure to aminoglycosides.

Effect of swainsonine, an inhibitor of glycoprotein processing, on cultured mammalian cells.

AbstractSwainsonine is an indolizidine alkaloid that inhibits glycoprotein processing by inhibiting mannosidase II. Thus, cells grown in the presence of this alkaloid exhibit a decreased amount of complex types of oligosaccharides at their cell surface, and instead have hybrid types of structures. Since this compound could be useful for studying functional roles of glycoproteins, it was important to determine whether it affected the growth of mammalian cells in culture, and whether it was cytotoxic to these cells. At levels of up to 1 μg/ml, swainsonine did not affect the growth rate of Madin‐Darby canine kidney (MDCK) cells, Chinese hamster ovary (CHO), simian virus‐181 (SV‐101), B‐16 melanoma, or intestine 407 cells, as measured by the increase in cell numbers over a 5‐day period. There was also no apparent change in cell size or cell shape in cells grown in the presence of this inhibitor. Swainsonine also did not appear to be cytotoxic, nor to cause alterations in cell morphology, as evidenced by comparison of thin sections of normal and swainsonine‐grown cells in the electron microscope. Since alterations in the oligosaccharide chains of cell surface glycoproteins could greatly affect cell surface properties, we examined the binding of various lectins and bacteria to cells grown in swainsonine as a measure of changes in their cell surface carbohydrates. Thus, when MDCK cells, CHO cells, or B‐16 melanoma cells were grown for several days in the presence of swainsonine (100–500 ng/ml), these cells showed a 50–100% increase in their ability to bind [3H]concanavalin A, and a substantial decrease in the binding of [3H]wheat germ agglutinin. These alterations suggested an increase in high‐mannose (or hybrid) types of receptors and a decrease in the complex types. The adhesion of E. coli B‐886, a bacterium that binds to high‐mannose glycoproteins, was also increased 1.5‐to twofold, in cells grown in swainsonine. However, the binding of E. coli SS‐142, another bacterial strain that does not bind to high‐mannose receptors, was not altered by growth in swainsonine. In addition to the decrease in wheat germ agglutinin binding, another indication of a decrease in complex chains was the finding that CHO cells grown in swainsonine were more resistant to the toxic effects of the lectin, ricin. This increased resistance could be measured microscopically by the decrease in the number of cells remaining attached to the plates, or by the inhibition of amino acid incorporation, at various ricin concentrations. The effect of swainsonine on the incorporation of amino acids and sugars into protein was also examined. When MDCK cells were grown overnight in swainsonine (1 μg/ml), or were incubated in the alkaloid for several hours before the start of the experiment, there was no alteration in the incorporation of [3H]leucine or [3H]proline into protein. There was, however, a significant inhibition in the incorporation of [3H]fucose, [3H]glucosamine, and [3H]galactose caused by this alkaloid. Fucose incorporation was decreased by about 40%, glucosamine by about 40 or 50%, and galactose by about 50%. In many cases (but not all), the incorporation of mannose was enhanced about 20–30% in cells grown in swainsonine.

Ethanol potentiates the inhibitory effect of D-galactosamine on protein synthesis in isolated hepatocytes

The effect of the combined addition of D-galactosamine and ethanol on hepatic protein synthesis was studied in isolated mouse hepatocytes. 2.5 mM D-galactosamine or 40 mM ethanol alone caused slight or no inhibition of amino acid incorporation into proteins. However, a profound inhibition (about 80%) was observed if D-galactosamine of the same dose was added after a preincubation of the cells with 40 mM ethanol and vice versa. It shows that there is a strong mutual potentiation between D-galactosamine and ethanol in the inhibition of protein synthesis.

Inhibition of amino acid incorporation in a cell-free system and inhibition of protein synthesis in cultured cells by reaction products of selenite and thiols

Reaction products of selenite with thiols were tested for an inhibitory effect on amino acid incorporation in a cell-free system derived from rat liver and on protein synthesis in intact P815 and L1210 cells. In the cell-free system maximum inhibition, up to 96%, was reached at about 10 μM selenium. In intact cells inhibitory effect varied depending on which reaction product or cell line was used. Maximum inhibition was obtained after 30 min of incubation with selenium concentrations ranging from 0.25 μM to over 7 μM. Selenite itself also inhibited protein synthesis of L1210 cells, but only after 90 min of incubation and starting at selenium concentrations of 2 μM. Inhibition of protein synthesis in intact cells was followed by cell death. Pre-incubation of the reaction products of a monothiol (2-propanethiol) and of a vicinal dithiol (2,3-dimercapto-1-propanol) in culture medium showed a rapid decrease of the inhibitory capability of the product from the monothiol, but not of the product from the dithiol. The results indicate that selenite and a thiol react to form products which have differential toxic effects to cells in vitro.

Glucocorticoids inhibit precursor incorporation into protein in splenic lymphocytes by stimulating protein degradation and expanding intracellular amino acid pools

In the presence of tracer concentrations of extracellular leucine (5 μM), treatment of rat splenic lymphocyte suspensions in vitro with 1 μM dexamethasone for 2.5–4 h caused a 30–35% inhibition of [ 3H]leucine incorporation into protein. As the extracellular leucine concentration was raised to 5 mM, this inhibition was progressively reduced to 0–12%. This phenomenon correlated with a marked dependence on extracellular leucine concentration of the dexamethasone-dependent enlargement of free intracellular leucine pools in splenic lymphocytes: a 123% increase in pool size with tracer extracellular leucine; a 10% increase with 5 mM leucine. Varying extracellular leucine had no effect on: (1) nuclear [ 3H]dexamethasone binding by the cells; (2) the concentration of dexamethasone needed for half-maximal inhibition of [ 3H]leucine incorporation; (3) the time course of onset and maximal expression of the hormonal inhibition of [ 3H]leucine incorporation; or (4) the magnitude of dexamethasone-dependent inhibition of [ 3H]uridine incorporation into RNA by these cells. There was no detectable effect of dexamethasone on uptake and retention of [ 3H]leucine by the cells, regardless of the extracellular leucine concentration. Treatment of splenic lymphocytes for 4 h in vitro with 1 μM dexamethasone caused a small shift of ribosomes from larger aggregate polysomes to smaller forms. Thus, glucocorticoid-induced inhibition of amino acid incorporation in splenic lymphocytes is a multicomponent response, of which an actual decrease in protein synthesis is only a small part. Enlargement of free intracellular amino acid pools, probably resulting from increased protein degradation, is the major contributing factor to the hormonal inhibition of amino acid incorporation.

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.

Streptococcus mutans dextransucrase: effect of cerulenin on lipid synthesis and enzyme production.

The effect of the fatty acid synthesis inhibitor cerulenin on growth and dextransucrase (EC 2.4.1.5) production by Streptococcus mutans 6715 was analyzed. Growth was markedly inhibited by less than 1 microgram of the antibiotic per ml. Under conditions where cerulenin did not inhibit amino acid incorporation into protein but did block acetate metabolism into lipid, the production of extracellular dextransucrase was suppressed. Inhibition was not due to a direct effect of the antibiotic on the enzyme or the lack of release of enzyme from the bacterial cell surface. Gel column chromatography demonstrated that enzyme produced in the presence of cerulenin was highly aggregated, similar to the control enzyme. Although the addition of ysophosphatidylcholine to enzyme which had been synthesized in the presence of cerulenin stimulated glucan formation from sucrose, the increase was not greater than that produced with the control enzyme. The differential inhibition of dextransucrase production by cerulenin indicates that enzyme secretion requires the production of lipid and may reflect the mechanism by whch this enzyme is transported from the bacterial cell.

Studies on amino acid incorporation in isolated toad bladder epithelial cells. Seasonal changes in protein synthesis

Amino acid incorporation in isolated toad bladder epithelial cells was investigated by examining the rate of radiolabel incorporation into trichloroacetic acid-insoluble material. The results demonstrated that during the summer months, a large fraction of the radiolabel incorporated was not inhibited by cycloheximide and did not seem to correspond to incorporation of the radioactive amino acid into elongating peptides. A similar effect was not observed in other organs of the toad (e.g. liver, spleen). During the winter months, however, virtually all the acid-insoluble radiolabel appears to correspond to incorporation of the amino acid into elongating peptides. Ouabain, an inhibitor of sodium transport, inhibited amino acid incorporation into acid-insoluble material by affecting the acid-soluble pool and this effect appeared to be independent of the effect of ouabain on sodium transport. The isolated epithelial cells appeared to derive the energy for protein synthesis almost entirely from glycolysis and in this regard they resemble certain tissue culture cells.

Production of a lesion on liver rough endoplasmic reticulum after prolonged ethanol ingestion and its reversal through abstinence

Prolonged ethanol ingestion by adult rats resulted in an inhibition of amino acid incorporation in vitro by liver membrane-bound ribosomes. The same treatment, however, produced a stimulation in the activity of free ribosomes. The addition of endoplasmic reticulum membranes, extracted from the microsomal fraction of control or ethanol-treated rats, had an inhibitory effect on the protein synthetic activities of free ribosomes. However, the membrane preparations from the livers of ethanol-treated animals had relatively greater inhibitory activity if compared with the similar preparation from the control livers. The observed effect of ethanol intake on liver protein synthesis in vitro could be almost completely reversed if the animals abstained from ethanol for a period of 4 weeks.

Cigarette Smoke Components and Alveolar Macrophage Protein Synthesis

A comparison was made of the effects of acrolein and aqueous cigarette smoke extracts on amino acid incorporation into protein by rabbit pulmonary alveolar macrophages. Studies were on cells maintained in vitro as adherent monolayers. Freshly prepared acrolein inhibited amino acid incorporation by significant amounts after approximately 30 min and aqueous smoke extracts after approximately 15 min of incubation. Fifty percent inhibition by acrolein occurred with a dose of 5.5 microgram acrolein/ml, an amount four times that in the amount of aqueous smoke extract required for 50% inhibition according to previously reported findings. Analysis by a dual-isotope technique and sodium dodecyl sulfate polyacrylamide gel electrophoresis showed the inhibitory effect of acrolein to be nonspecific, as had previously been found for aqueous smoke extracts. The presence of the sulfhydryl reagent cysteine, reduced the inhibitory effect of acrolein by 57.5%, but reduced inhibition induced by aqueous smoke extracts by only 12.2%. These results suggest the effects of acrolein are both quantitatively and qualitatively different than those of aqueous smoke extracts.

Radiation-induced fluctuation of the polysome content in adequately oxygenated tetrahymena pyriformis.

The polysome content of the ciliate protozoan Tetrahymena pyriformis is transiently reduced by gamma-irradiation. In order to test whether this is a result of a respiration-produced or radiation-produced hypoxia or some other mechanism, the oxygen content of the culture was determined during and after irradiation, and the polysome contents and rates of amino acid incorporation were measured with and without air bubbling. Irradiation (40 krad at approximately 3 krad/min) produced approximately a 25 per cent loss in dissolved O2 content in the medium. This decrease is not sufficient to affect the polysome level, since (a) the same radiation-induced loss of polysomes and inhibiition of amino acid incorporation was observed whether or not the culture was bubbled with air during the irradiation and (b) bubbling unirradiated cultures with gas mixtures containing as little as 17 per cent of the normal O2 content did not influence the polysome level. As long as the cells are irradiated as a shallow layer in open flasks, replacement of O2 from the gas phase appears adequate, and neither respiration-induced nor radiation-induced hypoxia masks the effects of the radiation.

The physiological effects of restrictive environmental conditions on Dictyostelium discoideum spore germination

Spores may be reversibly activated by the application of heat, dimethyl sulfoxide, urea, or ethylene glucol. Severe changes in four environmental variables (high osmotic pressure, low oxygen tension, low or high pH, and low or high temperature) interfere with the germination process. Spores at the end of the postactivation lag phase of germination were usually deactivated if exposed to severe environmental conditions and thus did not swell; spores in the swelling and oxygen uptake which began during spore activation was primarily attributable to a cyanide-sensitive pathway and secondarily to a salicylhydroxamic acid (SHAM) sensitive pathway. Inhibition of the SHAM-sensitive pathway did not cause spore deactivation while the addition of cyanide resulted in rapid spore deactivation. Treatment of activated spores with azide or environmental shifts also resulted in inhibition of oxygen uptake and spore deactivation. Deactivating spores did not demonstrate the amino acid incorporation, uridine incorporation, and expression of trehalase activity which is found in the later stages of germinating control spores. Protein synthesis inhibitors did not cause spore deactivation or a decrease in oxygen uptake but they inhibited amino acid incorporation and the expression trehalase activity in swollen spores. It is concluded that control of respiratory activity is involved in regulation of reversible activation.

Cell suspensions for kinetic studies of inhibitor action

Because of uniformity and small distances for transport, cell suspensions offer a system for rapid measurements of initial reactions of phytotoxic compounds. We had previously shown that a growth regulator, dikegulac (2,3:4,6 di-o-isopropylidine-2-keto-L-gulonate) inhibits amino acid incorporation into proteins. Using Solanum nigrum suspension cultures, it was found that dikegulac rapidly inhibits amino acid uptake into cells, before inhibiting incorporation, with time points starting at a few minutes, and kinetics that can be extrapolated back to time zero. With more rapid kinetics this compound induces leakage of a preloaded dye. The rate of leakage was less with stationary cells in suspension, reiterating that they are more resistant to the effects of this compound. It was thus concluded that at the concentrations used, the first effect of dikegulac (or one very close to the first effect) is on the cell membrane.

Effect of calcium on brain metabolism in vitro

In attempts to distinguish between direct and indirect effects of Ca on brain cell metabolism, respiration, glycolysis, ATP, phosphocreatine, incorporation of [14C] leucine into protein, and accumulation of 45Ca was determined in brain slices. Incubation was carried out in normal salt-balanced medium, in high-potassium-or ouabain-containing medium under aerobic and anaerobic conditions. Calcium ions inhibited slightly glycolysis and respiration in normal medium and activated amino acid incorporation into proteins. Levels of ATP and phosphocreatine remained normal. The effects were interpreted as due to a stabilization of plasma membranes by Ca ions to prevent their spontaneous depolarization. Incubation of slices in high-potassium and ouabain media in aerobic conditions in the presence of Ca resulted in activation of respiration and glycolysis, decrease of ATP and phosphocreatine levels, and inhibition of amino acid incorporation into proteins. The disturbances in energy metabolism, caused by the respiration-linked Ca uptake in brain mitochondria and concomitant inhibition of oxidative phosphorylation, may lead to the inhibition of amino acid incorporation into proteins. An increase in Ca levels in the cytoplasm may only be expected in anaerobic conditions during the incubation in high-potassium and ouabain media. This is manifested by a direct inhibition of glycolysis by Ca ions and a drastic decrease of ATP and phosphocreatine in slices. The results suggest that stimulation of aerobic glycolysis and inhibition of anaerobic glycolysis by Ca may explain the unknown mechanism of the so-called "reversed Pasteur effect" of brain slices incubated in high-potassium media.

Bacteriophage P2-lambda interference: III. Essential role of an early step in the initiation of lambda replication

Induction of bacteriophage λ in the presence of a P2 prophage results in inactivation of cellular transfer RNA, inhibition of amino acid and uridine incorporation in the host, as well as inhibition of phage replication. A red gam double mutation allows λ to escape from interference, and a mutation in gene O or P abolishes the effects on the host. It is shown here that phage and plasmid DNA extracted from cells undergoing P2-λ interference are still active in a transfection assay. Mutations in bacterial gene dna B or in phage site ori suppress the inhibition of amino acid incorporation, whereas genes dnaE and dna G have no such effect. Derepression of bacterial exonuclease VIII totally suppresses the interference, and mutations in genes recA and lexA, which control the SOS functions, suppress it partially if the λ phage is red +. Our results suggest that P2-λ interference is due to the action of old at an early step of the initiation of λ replication.

Cordycepin. An inhibitor of newly synthesized globin messenger RNA.

The effect of cordycepin (3'-deoxyadenosine) on newly synthesized globin mRNA in cultured mouse fetal liver erythroid cells is investigated. At cordycepin concentrations that do not inhibit amino acid incorporation into acid-precipitable material, the quantity of pulse-labeled (radioactive) globin mRNA nucleotide sequences is reduced by 90%, as compared to adenosine-treated controls. The reduction of radioactivity in globin-specific RNA sequences is greater than the inhibition of total RNA synthesis in experiments in which the labeling times range from 6 to 60 min. Control experiments demonstrate that cordycepin does not reduce the recovery of total cell RNA or steady state (unlabeled) globin mRNA. The hybridization assay used to detect radioactive globin mRNA sequences is independent of the cellular location or the number of 3'-terminal adenylate residues in the mRNA-containing molecules. These data thus indicate that cordycepin inhibits newly synthesized mRNA as effectively as it inhibits ribosomal and transfer RNA synthesis.