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3.
Protein synthesis rates in Xenopus laevis oocytes from stage 1 through stage 6 were measured. In addition, the translational efficiencies, total RNA contents, and percentages of ribosomes in polysomes in growing oocytes at several stages were determined. Stage 1 oocytes synthesize protein at a mean rate of 0.18 ng hr −1 while stage 6 oocytes make protein at a rate of 22.8 ng hr −1. Polysomes from growing and full-grown oocytes sedimented in a sucrose gradient with a peak value of 300 S, corresponding to a weight-average packing density of 10 ribosomes per mRNA. Ribosome transit times of endogenous mRNAs were essentially unchanged at all stages examined. While the oocyte's total ribosomal RNA content was observed to increase about 115-fold during oogenesis, the percentage of ribosomes in polysomes remained constant at approximately 2%. Taken together, the data suggest that the 127-fold increase in protein synthesis which occurs during Xenopus oogenesis involves the progressive recruitment onto polysomes of mRNA from the maternal stockpile. 相似文献
4.
Experiments are described in which the Stoichiometry of the ribosomal proteins before and after ribosome release from mRNA is compared. Polysomes labeled with 3H (or 14C) and run-off 70 S particles (Subramanian el al., 1969) labeled with 14C (or 3H) were separately isolated, mixed, and the ribosomal proteins extracted and fractionated by two-dimensional gel electrophoresis. The measurement of the isotopic ratios shows that 47 proteins out of the 53 investigated are present in the same amount in polysomes as in run-off ribosomes indicating that they remain with the ribosome during the release step. Proteins S 1, S 2, S 6, S 21, (Wittmann et al., 1971), however, show higher amounts in polysomes than in run-off ribosomes. The significance of these results is discussed. 相似文献
5.
Guanine nucleotides and Na + are known to regulate ligand binding to cardiac muscarinic receptors, which are netagively couple to the adenylate cyclase system. In the present study, we found that NH 4+ was more potent than Na + or other monovalent cations in regulating the affinity of the muscarinic receptor for agonists and antagonists. The effect of NH 4+ (or Na +) on the binding of the antagonist [ 3H]quinuclidinyl benzilate (QNB) to muscarinic receptors in homogenates of embryonic chick hearts depended on the assay buffer used. NH 4+ increased Kd in phosphate buffer or histidine and increased Bmax in Tris. NH f4+ (0.1 M) increased the IC 50 value for actylcholine inhibition of [ 3H]QNB binding 20-fold compared to 3–4-fold with 0.1 M Na + or K +. Furthermore, NH 4+ could substitute for and was more potent than Na + in producing synergistic effects with Gpp[NH]p to reduce the affinity of the receptor of acetylcholine. Tris depressed these effects. Gpp[NH]p plus 0.4 M NH 4Cl totally converted the receptor population to a low affinity agonist state and increased the IC 50 for acetylcholine by more than 2000-fold. Two conclusions can be made from the present results. First, NH 4+ appears to be the most potent effector yet studied of the monovalent cation site of the muscarinic receptor system. Second, the use of Tris in muscarinic receptor ligand binding assays will produce anomalous results concerning the properties of both agonist antagonist binding to the receptor. 相似文献
6.
The effect of NO 3 ?:NH 4 + ratio (14:1, 9:6, 7.5:7.5, 1:14, total 15 mmol/L N) in the nutrient solution on biomass, root morphology, and C and N metabolism parameter in hydroponically grown oilseed rape ( Brassica napus L.) was evaluated. The dry weights of leaves and roots were significantly largest at the equal NO 3 ?:NH 4 + ratio (7.5:7.5) compared with those of high NO 3 ?:NH 4 + ratio (14:1) or low NO 3 ?:NH 4 + ratio (1:14). Additionally, low NO 3 ?:NH 4 + ratio (1:14) reduced total root length and root surface area compared with the equal NO 3 ?:NH 4 + ratio (7.5:7.5), while high NO 3 ?:NH 4 + ratio (14:1) did not show any significant effect on root morphology except average diameter. The maximum of chlorophyll a, chlorophyll b and carotenoid were obtained under 7.5:7.5 treatment, whereas the maximum of the leaf net photosynthetic ( P n), stomatal conductance ( G s) and transpiration rate ( T r) were increased with increase in NH 4 + concentration in the nutrient solution. The activity of nitrate reductase (NR) showed a significant difference at different NO 3 ?:NH 4 + ratios and ranged 9:6 > 7.5:7.5 > 14:1 > 1:14, whereas the range of soluble sugar and soluble protein was 7.5:7.5 > 1:14 > 9:6 > 14:1. Our study reveals that oilseed rape growth is greater under 7.5:7.5 treatment than that under three other treatments. Oilseed rape growth at high or low NO 3 ?:NH 4 + ratios was inhibited by decreased pigments, NR activity, soluble sugar, and soluble protein, whereas subdued root growth should be apprehended considerate under high NH 4 + condition. 相似文献
8.
An improved method for the isolation of Euglena chloroplast ribosomes is described which presents a number of advantages over past procedures. First, ribosomes are prepared from whole cell extracts, thus bypassing the need to isolate intact chloroplasts and resulting in a 10-fold improvement in yield. Second, the inclusion of 40 mm Mg 2+ in the preparation buffers, while stabilizing the chloroplast ribosomes, precipitates and, thereby, virtually eliminates the cytoplasmic 89 S ribosomes. Third, greater than 95% of the chloroplast ribosomes sediment at 68 S rather than as the damaged 53 S particle frequently generated in other preparation procedures. Fourth, even with a high-salt wash to remove endogenous factors, the chloroplast ribosomes still sediment at 68 S and are just as active in in vitro protein synthesis as are E. coli ribosomes. These ribosomes have been tested for activity with elongation factors from prokaryotes, eukaryotes, and the chloroplast itself, and the results have been compared to those obtained with E. coli and wheat germ ribosomes. The data may be summarized as follows: (a) Chloroplast ribosomes use E. coli and EF-G with the same efficiency as do E. coli ribosomes in protein synthesis, (b) E. coli and chloroplast ribosomes can use Euglena chloroplast EF-G to catalyze translocation, but wheat germ ribosomes cannot, (c) Wheat germ EF-1 H and EF-2 are highly active in polymerization with wheat germ ribosomes, but ribosomes from neither E. coli nor the chloroplast are able to recognize these factors, (d) All three types of ribosomes accept Phe-tRNA from E. coli EF-Tu although to differing degrees. However, neither chloroplast nor E. coli ribosomes recognize wheat germ EF-1 H for the binding of Phe-tRNA. 相似文献
9.
When EF G 2 from Escherichia coli or Pseudomonas fluorescens is pre-bound to ribosomes in the presence of GMD, or GTP and fusidic acid, a differential effect is observed on the subsequent EF Tu-catalyzed binding of aminoacyl-tRNA to ribosomes. The EF G from E. coli nearly completely prevents the binding reaction, whereas the corresponding factor from P. fluorescens displays a significantly lower inhibitory effect. Both EF G factors form stable complexes with ribosomes and are equally efficient in the polymerization reaction. The difference in inhibitory properties between the two factors persists over a wide range of NH 4Cl concentration. 相似文献
10.
LYOPHILIZATION has been used to preserve viable microorganisms for extended periods of time 1. Recently, it has been reported that ribosomes functional in in vitro protein synthesis can be isolated from lyophilized fungi 2 and that lyophilized Escherichia coliribosomes retain fully their capacity for poly U-directed phenylalanine incorporation after 5 months storage at room temperature over P 2O 5 (ref. 3). We have now compared the sedimentation profiles and poly U-directed phenylalanine incorporating activity of three types of rabbit reticulocyte ribosome preparations immediately after isolation and after freezing or lyophilization and storage for various times. 相似文献
11.
The cytoplasmic and vacuolar pH and changes thereof in the presence of ammonia (NH 4Cl) and methylamine (CH 3NH 3Cl) have been measured in rhizoid cells of Riccia fluitans by means of a pH-sensitive microelectrode. On addition of 1 micromolar NH4Cl, the cytoplasmic pH of 7.2 to 7.4 drops by 0.1 to 0.2 pH units, but shifts to pH 7.8 in the presence of 50 micromolar NH4Cl or 500 micromolar CH3NH3Cl. The pH of the vacuole increases drastically from 4.5 to 5.7 with these latter concentrations. Since a NH4+/CH3NH3+ uniporter has been demonstrated in the plasmalemma of R. fluitans previously (Felle 1983 Biochim Biophys Acta 602:181-195), the concentration-dependent shifts of cytoplasmic pH are interpreted as results of two processes: first, acidification through deprotonation of the actively transported NH4+; and second, alkalinization through protonation of NH3 which is taken up to a significant extent from high external concentrations. Furthermore, it is concluded that the determination of intracellular pH by means of methylamine distribution is not a reliable method for eucaryotic systems. 相似文献
12.
The interaction of the antibiotic vernamycin Bα with Escherichia coli ribosomes has been studied. The antibiotic is bound to 70S ribosomes and 50S subunits but not to the 30S subunit or to polysomes. The binding of the antibiotic requires K + or NH +4 and Mg 2+. At saturation approximately 0.5 mole of antibiotic is bound per mole of ribosomes. The vernamycin Bα-ribosome complex is unstable. The bound antibiotic is readily displaced by nonradioactive vernamycin Bα and by a number of other antibiotics which are known to interact with the 50S subunit. The dissociation of the vernamycin Bα-ribosome complex is prevented by the simultaneous binding of vernamycin A. The binding sites for A and Bα are distinguishable since both drugs are able to bind simultaneously and neither prevents binding of the other, Ribosomes isolated from an erythromycin-resistant mutant are incapable of binding vernamycin A and Bα, indicating that the mutated protein responsible for resistance to erythromycin distorts the ribosome making it also unreceptive for the vernamycins. 相似文献
13.
The 2:1 reaction of [Ru(H 2O) 2(NH 3) 5] 2+ with 1,2,4,5-tetrazine (tz) gives rise to the formation of the dinuclear complex ion [{Ru(NH 3) 5} 2(μ-tz- N1: N4)] 4+. Its tetraphenylborate and hexafluoro-phosphate salts have been fully characterized; the X-ray structure of the former has also been determined. 相似文献
14.
Deoxycholate-KCl washed reticulocyte ribosomes are purified by affinity chromatography using Sepharose columns to which polyuridylic acid has been covalently bound. Upon passage through the column under nonenzymic conditions (high Mg 2+:K + ratio) approximately 10% of the ribosomes are retained. These ribosomes are then eluted with a buffer containing a high K +:Mg 2+ ratio and are assayed for activity in various steps of the elongation process of protein synthesis. Approximately a 3- to 4-fold increase in the various activities compared with control ribosomes is obtained. 相似文献
15.
Summary The synthesis of E. coli ribosomal proteins ceases after infection with bacteriophages T4 or T7 as does the synthesis of most other host proteins. The shut-off does not affect all ribosomal proteins to the same extent. After T7 infection no new proteins were detected in NH 4Cl-washed ribosomal particles. Bacteriophage T4, however, induces 3–4 new protein bands demonstrated by one-dimensional gel electrophoresis. The appearance of these bands is prevented by the addition of rifampicin at the time of infection but not when rifampicin is added one minute after infection. The NH 4Cl-washed ribosomal particles present at the time of T7 or T4 infection do not show any structural changes by sedimentation, subunit dissociation, or protein analysis on two-dimensional polyacrylamide gels. However, by labeling the T7 infected cells with 32P-phosphate, it is seen that the ribosomes become phosphorylated. The 32P-label comigrates with ribosomal proteins. This phosphorylating activity depends on a T7 gene. The T7 protein phosphokinase utilizes ribosomes as phosphate acceptor in vitro. The T7 ribosomes (NH 4Cl-washed) still function in vitro as do ribosomal particles from uninfected cells.Paper No. 83 on Ribosomal Proteins. Preceding paper is by Isono et al., Mol. gen. Genet. 127, 191–195 (1973). 相似文献
16.
The widespread use of NO 3− fertilization has had a major ecological impact. NH 4+ nutrition may help to reduce this impact, although high NH 4+ concentrations are toxic for most plants. The underlying tolerance mechanisms are not yet fully understood, although they are thought to include the limitation of C, the disruption of ion homeostasis, and a wasteful NH 4+ influx/efflux cycle that carries an extra energetic cost for root cells.In this study, high irradiance (HI) was found to induce a notable tolerance to NH 4+ in the range 2.5-10 mM in pea plants by inducing higher C availability, as shown by carbohydrate content. This capacity was accompanied by a general lower relative N content, indicating that tolerance is not achieved through higher net N assimilation on C-skeletons, and it was also not attributable to increased GS content or activity in roots or leaves. Moreover, HI plants showed higher ATP content and respiration rates. This extra energy availability is related to the internal NH 4+ content regulation (probably NH 4+ influx/efflux) and to an improvement of the cell ionic balance.The limited C availability at lower irradiance (LI) and high NH 4+ resulted in a series of metabolic imbalances, as reflected in a much higher organic acid content, thereby suggesting that the origin of the toxicity in plants cultured at high NH 4+ and LI is related to their inability to avoid large-scale accumulation of the NH 4+ ion. 相似文献
17.
Phragmites australis and Glyceria maxima are fast-growing littoral grasses often competing for similar wetland habitats. Eutrophication affects their competitiveness, but the outcome is not generally predictable due to the complexity of interrelated factors. We hypotheses that pore water N:P and NH 4 +:NO 3 ? modify their growth and metabolic responses to the trophic status of the habitat. The hypothesis was tested under standardized conditions of long-term sand cultures. Application of N?+?P up to extreme levels in combination with N:P?<?10 and NH 4 +:NO 3 ??<?1 triggered positive growth response in both species. In contrast, similar N levels applied in N:P?>?90 and NH 4 +:NO 3 ??=?4 caused lower productivity, changes in resource allocation, morphology and metabolic relations (e.g. high shoot density, low shoot diameters and heights, reduced root and rhizome growth). Observed signs of stress resembled the factors associated with the reed retreat at the die-back sites. Unbalanced N levels obviously alter plant susceptibility to stresses (altering, e.g. ventilation efficiency, plant anchorage or below-ground storage capacity). The positive effect of sufficient P supply was pronounced in Glyceria. It might therefore favour Glyceria in competition with Phragmites at highly fertile habitats rich in P. 相似文献
18.
Studies on the quantitative binding of [ 3H]anisomycin are useful in determining conformational and/or structural changes on eukaryotic ribosomes. We have shown that yeast ribosomes have different structures depending on their functional states during the ribosome cycle as defined by their affinity for [ 3H]anisomycin.Free ribosomes, either in vivo run-off ribosomes (1 mm-sodium azide treatment or 8 °C incubation of spheroplasts) or puromycin-dependent released ribosomes, have an affinity defined by Kd = 3.3 to 3.6 μm.Ribosomes forming polysomes engaged in protein synthesis have at least two new different conformations (defined by Kd,H = 0.81 μm and Kd,L = 12 μm). These conformations have been ascribed to the pre and post-translocated steps of the elongation cycle in protein synthesis by blocking the polysomes with specific inhibitors of translation. Pre-translocated polysomes (polysomes blocked with cycloheximide) have an affinity of KdCHX = 12 μm and post-translocated polysomes (polysomes blocked with doxycycline) have an affinity of KdDC = 0.82 μm. These dissociation constants are identical to Kd,L and Kd,H obtained with control untreated polysomes, respectively.Moreover, a new ribosome conformation defined by KdDT = 1.5 μm and KdFA = 1.8 μm was found, by blocking the polysomes with the elongation factor, EF-2, bound by using either diphtheria toxin or fusidic acid.We also present evidence of the previously reported heterogeneity of standard preparations of eukaryotic ribosomes (Barbacid & Vazquez, 1974 a) being a direct consequence of the high-salt washing treatment of ribosomes. 相似文献
19.
The nitrogen (N) uptake kinetic parameters for Microcystis field assemblages collected from the San Francisco Bay Delta (Delta) in 2012 and non-toxic and toxic laboratory culture strains of M. aeruginosa were assessed. The 15N tracer technique was used to investigate uptake of ammonium (NH 4+), nitrate (NO 3−), urea and glutamic acid over short-term incubations (0.5–1 h), and to study inhibition of NO 3−, NH 4+ and urea uptake by NH 4+, NO 3− and NH 4+, respectively. This study demonstrates that Delta Microcystis can utilize different forms of inorganic and organic N, with the greatest capacity for NH 4+ uptake and the least for glutamic acid uptake, although N uptake did not always follow the classic Michaelis–Menten hyperbolic relationship at substrate concentrations up to 67 μmol N L −1. Current ambient N concentrations in the Delta may be at sub-saturating levels for N uptake, indicating that if N loading (especially NH 4+) were to increase, Delta Microcystis assemblages have the potential for increased N uptake rates. Delta Microcystis had the highest specific affinity, α, for NH 4+ and the lowest for NO 3−. In culture, N uptake by non-toxic and toxic M. aeruginosa strains was much higher than from the field, but followed similar N utilization trends to those in the field. Neither strain showed severe inhibition of NO 3− uptake by NH 4+ or inhibition of NH 4+ uptake on NO 3−, but both strains showed some inhibition of urea uptake by NH 4+. 相似文献
20.
The swamp eel, Monopterus albus, can survive in high concentrations of ammonia (>75 mmol l −1) and accumulate ammonia to high concentrations in its brain (∼4.5 µmol g −1). Na +/K +-ATPase (Nka) is an essential transporter in brain cells, and since NH 4
+ can substitute for K + to activate Nka, we hypothesized that the brain of M. albus expressed multiple forms of Nka α-subunits, some of which might have high K + specificity. Thus, this study aimed to clone and sequence the nka α-subunits from the brain of M. albus, and to determine the effects of ammonia exposure on their mRNA expression and overall protein abundance. The effectiveness of NH 4
+ to activate brain Nka from M. albus and Mus musculus was also examined by comparing their Na +/K +-ATPase and Na +/NH 4
+-ATPase activities over a range of K +/NH 4
+ concentrations. The full length cDNA coding sequences of three nkaα ( nkaα1, nkaα3a and nkaα3b) were identified in the brain of M. albus, but nkaα2 expression was undetectable. Exposure to 50 mmol l −1 NH 4Cl for 1 day or 6 days resulted in significant decreases in the mRNA expression of nkaα1, nkaα3a and nkaα3b. The overall Nka protein abundance also decreased significantly after 6 days of ammonia exposure. For M. albus, brain Na +/NH 4
+-ATPase activities were significantly lower than the Na +/K +-ATPase activities assayed at various NH 4
+/K + concentrations. Furthermore, the effectiveness of NH 4
+ to activate Nka from the brain of M. albus was significantly lower than that from the brain of M. musculus, which is ammonia-sensitive. Hence, the (1) lack of nkaα2 expression, (2) high K + specificity of K + binding sites of Nkaα1, Nkaα3a and Nkaα3b, and (3) down-regulation of mRNA expression of all three nkaα isoforms and the overall Nka protein abundance in response to ammonia exposure might be some of the contributing factors to the high brain ammonia tolerance in M. albus. 相似文献
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