Conditions affecting protein synthesis in amphibian oocytes.

The endogenous protein synthesis of Xenopus laevis and Calyptocephalella caudiverbera oocytes was studied by measuring the incorporation into acid-precipitable material of radioactive amino acids placed in the extracellular medium. Large differences of incorporation into protein were observed by using different labeled amino acids. For example, it was found that radioactive aspartic acid or glutamic acid was very poorly incorporated at concentrations under 0.1 mm. These differences are due to differences in uptake constants and in the internal pools of free amino acids which are very large for the acidic amino acids. Both types of oocytes behaved similarly with respect to magnesium ion concentration, temperature optimum and inhibitors of protein synthesis. They differed however in sensitivity to pH since Xenopus laevis oocyte protein synthesis was twofold higher at pH 8.5 than at pH 7 while Calyptocephalella caudiverbera oocytes showed no difference. Isolation of oocyte germinal vesicles allowed a study of the entrance of newly synthesized protein into the cell nuclei.     

Amino acid uptake in Xenopus laevis oocytes.

The isolated oocytes from Xenopus laevis are able to take up radioactive amino acids from the exogenous medium. Most amino acids tested are taken up to reach concentrations higher than the extracellular medium. The initial uptake velocities vary with the external amino acid concentration in a Michaelis-Menten fashion. Aspartic acid requires concentrations an order of magnitude higher than the five other amino acids tested to reach half the maximal uptake velocity. The uptake mechanism seems to be specific for groups of analogous amino acids, as can be determined by competition studies. The amino acid groups for which there is some evidence of uptake specificity would be aromatic, aliphatic, acidic and basic. Amino acid pools of oocytes show that these cells can concentrate amino acids from Xenopus blood, as well as from artificial media.     

Kinetic analysis of amino acid pools and protein synthesis in amphibian oocytes and embryos.

Rates of protein synthesis have been measured in Rana pipiens oocytes and embryos and in Xenopus oocytes from the incorporation kinetics of two different concentrations of amino acid. This method does not require an independent measurement of the amino acid pools, since the pool size can be calculated directly from incorporation data. The effects of the concentration and diffusion of injected amino acid on the calculated values for amino acid pool size and flow rate are discussed. When the endogenous amino acid pool is appreciably expanded by the injected amino acid, the total amino acid pool in the oocytes or embryos may be considered as the precursor pool for protein synthesis. Under these circumstances, compartmentation of amino acids does not affect the results, except when lysine is used as tracer. The rates of protein synthesis in ovarian oocytes of Rana pipiens and Xenopus laevis are 18 and 50–54 ng/hr, respectively. In Rana pipiens, the rate increases 70% during maturation and another 50% before the two-cell stage. Finally, the rate approximately doubles between the two-cell and blastula stages.     

Biochemical research on oogenesis: protein synthesis in whole cells and in cell-free extracts of Xenopus laevis immature ovaries

Nearly all tRNA molecules in previtellogenic oocytes of Xenopus laevis are included in nucleoprotein particles sedimenting at 42S. The tRNA-binding sites of these particles have several properties in common with those of the ribosomes. This suggests that the 42S particles might behave like unprogrammed ribosomes and be the site of a template-independent polymerization of amino acids. We expected this reaction to be insensitive to protein synthesis inhibitors, such as cycloheximide and puromycin. We found that these antibiotics almost completely inhibit the incorporation of labeled amino acids into protein, when added to the incubation medium of whole ovaries or free oocytes. In cell-free extracts of ovaries, the incorporation of amino acids is partially insensitive to cycloheximide and puromycin. When such extracts are fractionated by sucrose density centrifugation and incubated with ATP, a major peak of amino acid incorporation can be detected, which nearly coincides with the 42S particle peak.     

Inhibition by Peptides of Amino Acid Uptake by Bacterial Populations in Natural Waters: Implications for the Regulation of Amino Acid Transport and Incorporation

To investigate the regulatory interactions of amino acid transport and incorporation, we determined the effects of dipeptides on amino acid uptake by bacteria in an estuary and a freshwater lake. Dipeptides noncompetitively inhibited net transport and incorporation of amino acids into macromolecules but had no effect on the ratio of respiration to incorporation. Nearly maximum inhibition occurred at peptide concentrations of <10 nM. In contrast, the initial uptake rate of glycyl-[¹⁴C]phenylalanine was not affected by glycine or phenylalanine. Net amino acid transport appeared to be inhibited by the increased flux into the intracellular pools, whereas the incorporation of labeled monomers into macromolecules was isotopically diluted by the unlabeled amino acids resulting from intracellular hydrolysis of the dipeptide. Chloramphenicol, sodium azide, and dinitrophenol all inhibited the initial uptake rate of leucine and phenylalanine. These results suggest that in aquatic environments amino acids are taken up by active transport which is coupled closely to protein synthesis.     

Phloem Transport of Amino Acids in Relation to their Cytosolic Levels in Barley Leaves

A comparison of barley (Hordeum vulgare L.) leaves was made between the cytosolic content of amino acids and sucrose as determined by subcellular fractionation and the corresponding concentration in phloem sap, which was collected continuously for up to 6 days from severed aphid stylets. Because amino acids were found to be almost absent from the vacuoles, and because the amino acid patterns in the stroma and cytosol are similar, whole leaf contents could be taken as a measure of cytosolic amino acid levels for a comparison of data during a diurnal cycle. The results show that the pattern of amino acids in the phloem sap was very similar to the pattern in the cytosol. Therefore, we concluded that the overall process of transfer of amino acids from the cytosol of the source cells into the sieve tubes, although carrier mediated, may be a passive process and that the translocation of amino acids via the sieve tubes requires the mass flow of sucrose driven by the active sucrose transport involved by the phloem loading.     

Pronase digestion of amino-acid binding components on the surface of Bacillus subtilis cells and minicells

The addition of 250 μg/ml pronase to either cells or minicells of Bacillus subtilis does not interfere with growth, macromolecular synthesis or maintenance of cell integrity. Radioactive amino acids such as proline or isoleucine taken up by cells or minicells are bound to surface components which prevent their release by washing. Pronase treatment causes the release of a significant percentage of these bound amino acids. In cells and minicells exposed to pronase before the addition of labeled amino acids, initial binding capacities are greatly reduced. These data suggest a new approach to the investigation of amino acid binding components on the surface of Bacillus subtilis.     

Gut tract microorganisms supply the precursors for methyl-branched hydrocarbon biosynthesis in the termite,Zootermopsis nevadensis

In vivo and in vitro experiments were performed to examine the role of succinate and other potential precursors of the methylmalonyl-CoA used for methyl-branched hydrocarbon biosynthesis in the termite Zootermopsis nevadensis. The in vivo incorporation of [1,4-¹⁴C]succinate and [2,3-¹⁴C]succinate into hydrocarbon confirmed that succinate is a direct precursor to the methyl branch unit. The other likely precursors, the branched chain amino acids valine and isoleucine, were not efficiently incorporated into hydrocarbon. Carbon-13 NMR showed that one of the labeled carbons of [1,4-¹³C]succinate labeled position 6 of 5-methylalkanes and positions 6 and 18 of 5,17-dimethylalkanes, indicating that succinate, as a methylmalonyl-CoA unit, was incorporated as the third unit to form 5-methylheneicosane and as both the third and ninth units to form 5,17-dimethylheneicosane. Analysis of organic acids after the in vivo metabolism of [2,3-¹⁴C]succinate showed that succinate was converted to propionate and methylmalonate. Labeled succinate injected into the hemolymph was readily taken up by the gut tract. Isolated gut tissue efficiently converted succinate to acetate and propionate, both of which were released into the incubation media. Mitochondria from termite tissue (minus gut tract) converted succinate to methylmalonate and propionate only in the presence of malonic acid, an inhibitor of succinate dehydrogenase. The results of these studies show that while termite mitochondria are able to convert succinate to propionate and methylmalonate, most of the propionate used for methyl-branched hydrocarbon biosynthesis is produced by gut tract microorganisms. The propionate is then presumably transported through the hemolymph to epidermal cells for use in methyl-branched hydrocarbon biosynthesis.     

Role of Amino Acids and Vitamins in Nutrition of Mesophilic Methanococcus spp

In this study we found that autotrophic methanococci similar to Methanococcus maripaludis obtained up to 57% of their cellular carbon from exogenous amino acids. About 85% of the incorporation was into protein. Primarily nonpolar and basic amino acids and glycine were incorporated; only small amounts of acidic and some polar amino acids were taken up. An additional 10% of the incorporation was into the nucleic acid fraction. Because little ¹⁴CO₂ was formed from the ¹⁴C-amino acids, little metabolism of the amino acids occurred. Therefore the growth stimulation by amino acids was probably due to the sparing of anabolic energy requirements. Of the amino acids incorporated, only alanine was also a sole nitrogen source for these methanococci. In contrast, Methanococcus vannielii and “Methanococcus aeolicus” are autotrophic methanococci which did not incorporate amino acids and did not utilize alanine as a sole nitrogen source. Although glutamine served as a sole nitrogen source for the autotrophic methanococci and Methanococcus voltae, a heterotrophic methanococcus, growth was due to chemical deamination in the medium. M. voltae requires leucine and isoleucine for growth. However, these amino acids were not significant nitrogen sources, and alanine was not a sole nitrogen source for the growth of M. voltae. The branched-chain amino acids were not extensively metabolized by M. voltae. Pantoyl lactone and pantoic acid were readily incorporated by M. voltae. The intact vitamin pantothenate was neither stimulatory to growth nor incorporated. In conclusion, although amino acids and vitamins are nutritionally important to both autotrophic and heterotrophic methanococci, generally they are not subject to extensive catabolism.     

Amino acid utilization by Gymnodinium breve

The marine dinoflagellate Gymnodinium breve utilizes erogenous amino acids for the synthesis of proteins in the light. During logarithmic growth, l-valine and l-methionine are incorporated into proteinaceous material which is retained by the cell. Glycine is also incorporated, but the glycine-containing proteins are extruded. When cells are no longer growing exponentially, all proteins that incorporated these supplied amino acids are extruded. The pronase-susceptible extruded material has a MW in excess of 300 000. When chloramphenicol is used to inhibit protein synthesis, glycine is not taken up. l-Methionine is rapidly metabolized intracellularly and is used in the synthesis of other macromolecules. l-Valine accumulates intracellularly and remains unaltered. Glycine and l-methionine appear to be transported via facilitated diffusion systems, while l-valine uptake appears to be active.     

Incorporation of different labelled precursors into chloroplast RNA of Chlorella

Summary Radioactive uridine is incorporated by Chlorella strain 211-8b/p into ribosomal subunits and their rapidly labelled RNA comigrates with chloroplast RNA on polycrylamide gels.Ribosomal particles which can be labelled by short pulses of orotic acid cosediment with the particles labelled by uridine pulses and contain the same RNA species as these when separated either on sucrose gradients or on polycrylamide gels. This incorporation is, like that of uridine, sensitive to rifampin and chloramphenicol, but insensitive to cycloheximide.A comparative study of short-time incorporation of uridine, orotic acid and guanosine into the RNA of Chlorella showed that all three precursors were incorporated mainly into RNA of chloroplastic origin. However, guanosine was also partly incorporated into cytoplasmic rRNA. Nitrogen-deficient cells always incorporated part of all three precursors into cytoplasmic rRNA, but the proportions of these were different among the different precursors.These results are consistent with the hypothesis that the described differences in the incorporation of the above mentioned precursors into RNA of different cellular compartments are largely attributable to effects of pool sizes.     

The stimulatory effect of human chorionic gonadotropin on amino acid uptake by amphibian follicles.

Human chorionic gonadotropin (hCG) stimulates the uptake of eight different amino acids and four nucleosides by Xenopus laevis ovarian follicles. This hormone also stimulates amino acid uptake in the follicles of another amphibian, Callyptocephallela caudiverbera. The stimulation of uptake is due to a reduction in the amino acid concentration required for half-maximal uptake velocity and not to an increment in V_max. The effect of hCG does not require protein synthesis but requires physiological conditions of temperature and pH. Incorporation of radioactive exogenous amino acid into proteins is also stimulated by the hormone, but high-resolution electrophoresis shows that there are no drastic qualitative changes in the pattern of proteins synthesized at early times after hCG treatment. The effect of hCG on the uptake of exogenous amino acids does not appear to be required for oocyte maturation because other hormones such as progesterone and testosterone which induce maturation do not increase amino acid uptake. Also the concentration of hCG required for oocyte maturation is significantly lower than that required for an effect on amino acid transport. Inhibitors of oocyte maturation such as theophylline and cycloheximide do not inhibit the action of hCG on amino acid uptake by the amphibian follicles.     

Ammonia regulation of carbon metabolism in photosynthesizing leaf discs

Alfalfa (Medicago sativa L., var. El Unico) leaf discs, floating on buffer containing NH₄Cl and photosynthesizing with ¹⁴CO₂, produced more labeled amino acid and less sucrose than did control discs (no added NH₄Cl). The level of pyruvate increased and that of phosphoenolpyruvate decreased. These and other changes in levels of labeled compounds led us to conclude that pyruvate kinase was activated by ammonia, resulting in increased transfer of photosynthetically incorporated carbon to synthesis of amino acid skeletons at the expense of sucrose synthesis. Carbon flow through enzymes catalyzing the anaplerotic reactions was apparently stimulated.     

Molecular Insights into Variable Electron Transfer in Amphibian Cryptochrome

Cryptochrome proteins are activated by the absorption of blue light, leading to the formation of radical pairs through electron transfer in the active site. Recent experimental studies have shown that once some of the amino acid residues in the active site of Xenopus laevis cryptochrome DASH are mutated, radical-pair formation is still observed. In this study, we computationally investigate electron-transfer pathways in the X. laevis cryptochrome DASH by extensively equilibrating a previously established homology model using molecular dynamics simulations and then mutating key amino acids involved in the electron transfer. The electron-transfer pathways are then probed by using tight-binding density-functional theory. We report the alternative electron-transfer pathways resolved at the molecular level and, through comparison of amino acid sequences for cryptochromes from different species, we demonstrate that one of these alternative electron-transfer pathways could be general for all cryptochrome DASH proteins.     

Amino acid contents and transport in oilseed rape (<Emphasis Type="Italic">Brassica napus</Emphasis> L.) under different nitrogen conditions

Oilseed rape (Brassica napus L.) needs very high nitrogen fertilizer inputs. Significant amounts of this nitrogen are lost during early leaf shedding and are a source of environmental and economic concern. The objective of this study was to investigate whether the remobilization of leaf amino acids could be limiting for nitrogen use efficiency. Therefore, amino acid concentrations were analyzed in subcellular compartments of leaf mesophyll cells of plants grown under low (0.5 mM NO₃^–) and high (4 mM NO₃^–) nitrogen supply. With high nitrogen supply, young leaves showed an elevated amino acid content, mainly in vacuoles. In old leaves, however, subcellular concentrations were similar under high and low nitrogen conditions, showing that the excess nitrogen had been exported during leaf development. The phloem sap contained up to 650 mM amino acids, more than four times as much than the cytosol of mesophyll cells, indicating a very efficient phloem-loading process. Three amino acid permeases, BnAAP1, BnAAP2, and BnAAP6, were identified and characterized. BnAAP1 and BnAAP6 mediated uptake of neutral and acidic amino acids into Xenopus laevis oocytes at the actual apoplastic substrate concentrations. All three transporters were expressed in leaves and the expression was still detectable during leaf senescence, with BnAAP1 and BnAAP2 mRNA levels increasing from mature to old leaves. We conclude that phloem loading of amino acids is not limiting for nitrogen remobilization from senescing leaves in oilseed rape.     

Inhibitor of ribosomal RNA synthesis in Xenopus laevis embryos: VI. Occurrence in mature oocytes and unfertilized eggs

Occurrence of a factor(s) which can selectively inhibit ribosomal RNA synthesis in isolated neurula cells of Xenopus laevis was examined in oocytes, unfertilized eggs, and embryos of Xenopus laevis. It was found that acid-soluble materials from full-sized oocytes, white-banded mature oocytes, unfertilized eggs, and pregastrular embryos were all active in significantly reducing the relative ratio of the [³H]uridine incorporation into 18S and 28S ribosomal RNA to that into 4S RNA from the control value. These results suggest that the inhibitor appears in the terminal step of oogenesis and, hence, may be assumed as a maternal regulator.     

Protein synthesis in interspecies hybrid embryos of the amphibian Xenopus

The newly synthesized abundant proteins of early Xenopus laevis and Xenopus borealis embryos have been examined by two-dimensional electrophoresis after labelling with [³⁵S]methionine. Six prominent polypeptides specific to Xenopus laevis embryos and a further six specific to Xenopus borealis have been identified. Overall, embryos of the two species are estimated to differ by approx. 15% in their protein synthetic patterns from blastula to tailbud stage. Interspecific hybrid embryos (Xenopus laevis (♀)/Xenopus borealis (♂)) synthesise only the maternally specified set of proteins until gastrulation after which they produce the full complement of both Xenopus laevis and Xenopus borealis specific proteins. The possible use of such molecular markers of parental genome activity in facilitating further embryological study is discussed.     

Characteristics of transport of selenoamino acids by epithelial amino acid transporters

Selenoamino acids are the main form of organic selenium derived from the diet. They are efficiently absorbed in the intestine and reabsorbed in kidney, but the transporter proteins that mediate their cellular uptake have not yet been identified. We here describe the transport pathways of selenoamino acids and derivatives, including selenomethionine, methylselenocysteine, selenocystine, selenobetaine and selenocystamine. Transport studies employed the Xenopus laevis oocyte system expressing the amino acid transporters SIT1, b^0,+rBAT, B⁰ or PAT1 and intestinal Caco-2 and renal OK cell lines that possess a multitude of amino acid transporters. Our results suggest that the major route for the uptake of selenomethionine is the system b^0,+ rBAT in Caco-2 cells and B⁰ in OK cells. Affinity of selenomethionine or methionine for these transporters did not differ, but for SIT1 selenomethionine shows a higher affinity than methionine. Methylselenocysteine displayed a higher affinity than cysteine for all transporters tested and in both OK and Caco-2 cells, system B⁰ seems to be the primary uptake route. Selenocystine is taken up well by the b^0,+ rBAT system, while selenobetaine is a low-affinity substrate only for SIT1 and PAT1. Selenocystamine was not transported by any of the transport systems investigated. When cells were exposed to selenoamino acids, intracellular selenium levels in OK cells considerably exceeded those in Caco-2 cells, indicating effective renal reabsorption capacity. In conclusion, selenoamino acids but not the seleno-derivatives selenobetaine and selenocystamine, are effectively transported by various intestinal and renal amino acid transporters and are thus available for selenium metabolism and therapeutic approaches.     

Cloning,sequencing and expression of the two genes encoding the mitochondrial single-stranded DNA-binding protein in Xenopus laevis

In Xenopus laevis the single-stranded DNA binding protein imported into the mitochondria consists of two highly related polypeptides. The establishment of the genomic nucleotide sequences reveals that they are encoded by two different genes, XLSSB1 and XLSSB2. The deduced amino acid sequence is identical to the direct amino acid sequence determined by Edman degradation of the mitochondrial polypeptides [Ghrir, R., Lecaer, J.P., Dufresne, C. and Gueride, M. (1991) Primary structure of the two variants of Xenopus laevis mtSSB, a mitochondrial DNA binding protein. Arch. Biochem. Biophys. 291, 395–400]. Both genes are organized in seven exons and six introns, the sequence of the peptide leader is interrupted by an intervening sequence (intron 2). The exon/intron junctions are in exactly conserved positions, splitting the same codon. A high level of identity is observed between corresponding introns of the two genes over part or most of their lengths. Structural features of intronic sequences reveal multiple rearrangements and exchanges during the evolution of X. laevis species. A CCAAT box and the potential regulatory elements NRF-2 and Sp 1 are observed in the 5′-flanking region of both genes. During oogenesis, XLSSB gene expression is correlated with the replicative activity of the mitochondrial DNA.