Selective inhibition of divergent seryl-tRNA synthetases by serine analogues

Seryl-tRNA synthetases (SerRSs) fall into two distinct evolutionary groups of enzymes, bacterial and methanogenic. These two types of SerRSs display only minimal sequence similarity, primarily within the class II conserved motifs, and possess distinct modes of tRNA(Ser) recognition. In order to determine whether the two types of SerRSs also differ in their recognition of the serine substrate, we compared the sensitivity of the representative methanogenic and bacterial-type SerRSs to serine hydroxamate and two previously unidentified inhibitors, serinamide and serine methyl ester. Our kinetic data showed selective inhibition of the methanogenic SerRS by serinamide, suggesting a lack of mechanistic uniformity in serine recognition between the evolutionarily distinct SerRSs.     

Crystal structure of the archaeal asparagine synthetase: interrelation with aspartyl-tRNA and asparaginyl-tRNA synthetases

Asparagine synthetase A (AsnA) catalyzes asparagine synthesis using aspartate, ATP, and ammonia as substrates. Asparagine is formed in two steps: the β-carboxylate group of aspartate is first activated by ATP to form an aminoacyl-AMP before its amidation by a nucleophilic attack with an ammonium ion. Interestingly, this mechanism of amino acid activation resembles that used by aminoacyl-tRNA synthetases, which first activate the α-carboxylate group of the amino acid to form also an aminoacyl-AMP before they transfer the activated amino acid onto the cognate tRNA. In a previous investigation, we have shown that the open reading frame of Pyrococcus abyssi annotated as asparaginyl-tRNA synthetase (AsnRS) 2 is, in fact, an archaeal asparagine synthetase A (AS-AR) that evolved from an ancestral aspartyl-tRNA synthetase (AspRS). We present here the crystal structure of this AS-AR. The fold of this protein is similar to that of bacterial AsnA and resembles the catalytic cores of AspRS and AsnRS. The high-resolution structures of AS-AR associated with its substrates and end-products help to understand the reaction mechanism of asparagine formation and release. A comparison of the catalytic core of AS-AR with those of archaeal AspRS and AsnRS and with that of bacterial AsnA reveals a strong conservation. This study uncovers how the active site of the ancestral AspRS rearranged throughout evolution to transform an enzyme activating the α-carboxylate group into an enzyme that is able to activate the β-carboxylate group of aspartate, which can react with ammonia instead of tRNA.     

Correlations between baltic amber and Pinus resins

The composition of the acid fractions of four pine resins has been studied before and after an ‘ageing’ process. Correlations are established between Baltic amber and ‘aged’ Pinus halepensis resin.     

Evolutionary relationships of bacterial and archaeal glutamine synthetase genes

Glutamine synthetase (GS), an essential enzyme in ammonia assimilation and glutamine biosynthesis, has three distinctive types: GSI, GSII and GSIII. Genes for GSI have been found only in bacteria (eubacteria) and archaea (archaebacteria), while GSII genes only occur in eukaryotes and a few soil-dwelling bacteria. GSIII genes have been found in only a few bacterial species. Recently, it has been suggested that several lateral gene transfers of archaeal GSI genes to bacteria may have occurred. In order to study the evolution of GS, we cloned and sequenced GSI genes from two divergent archaeal species: the extreme thermophile Pyrococcus furiosus and the extreme halophile Haloferax volcanii. Our phylogenetic analysis, which included most available GS sequences, revealed two significant prokaryotic GSI subdivisions: GSI-a and GSI-. GSIa-genes are found in the thermophilic bacterium, Thermotoga maritima, the low G+C Gram-positive bacteria, and the Euryarchaeota (includes methanogens, halophiles, and some thermophiles). GSI--type genes occur in all other bacteria. GSI-- and GSI--type genes also differ with respect to a specific 25-amino-acid insertion and adenylylation control of GS enzyme activity, both absent in the former but present in the latter. Cyanobacterial genes lack adenylylation regulation of GS and may have secondarily lost it. The GSI gene of Sulfolobus solfataricus, a member of the Crenarchaeota (extreme thermophiles), is exceptional and could not be definitely placed in either subdivision. The S. solfataricus GSI gene has a shorter GSI--type insertion, but like GSI-a-type genes, lacks conserved sequences about the adenylylation site. We suspect that the similarity of GSI- genes from Euryarchaeota and several bacterial species does not reflect a common phylogeny but rather lateral transmission between archaea and bacteria.Correspondence to: J.R. Brown 1073     

Recognition of the amber UAG stop codon by release factor RF1

We report the crystal structure of a termination complex containing release factor RF1 bound to the 70S ribosome in response to an amber (UAG) codon at 3.6‐Å resolution. The amber codon is recognized in the 30S subunit‐decoding centre directly by conserved elements of domain 2 of RF1, including T186 of the PVT motif. Together with earlier structures, the mechanisms of recognition of all three stop codons by release factors RF1 and RF2 can now be described. Our structure confirms that the backbone amide of Q230 of the universally conserved GGQ motif is positioned to contribute directly to the catalysis of the peptidyl‐tRNA hydrolysis reaction through stabilization of the leaving group and/or transition state. We also observe synthetic‐negative interactions between mutations in the switch loop of RF1 and in helix 69 of 23S rRNA, revealing that these structural features interact functionally in the termination process. These findings are consistent with our proposal that structural rearrangements of RF1 and RF2 are critical to accurate translation termination.     

Enzymatic biosynthesis of ergotamine and investigation on some aminoacyl-tRNA synthetases in Claviceps

An enzyme system from Claviceps purpurea (Fr.) Tul. catalyzing the incorporation of l-phenylalanine into ergotamine - ergotamine synthetase - was purified 172-fold. This was done by a combination of ammonium sulfate precipitation, gel filtration, ion-exchange chromatography on DEAE-Sepharose CL-6B, and hydroxyapatite chromatography. The activation of ergotamine specific amino acids as well as d-lysergic acid and dihydrolysergic acid via adenylates, as determined by the ATP-³²PP_i exchange, was investigated. Phenylalanyl-tRNA synthetase, catalyzing the same type of activation reaction, could not be separated from ergotamine synthetase by the purification procedure applied. Therefore, at the present stage of enzyme purification, phenylalanine-dependent ATP-³²PP_i exchange cannot be used to measure ergotamine synthetase activity specifically.Phenylalanyl-tRNA synthetase and leucyl-tRNA synthetase were separated into mitochondrial and cytoplasmic isoenzymes by hydroxyapatite chromatography. Their charging activities of procaryotic versus eucaryotic tRNA and their molecular masses were determined.     

Disrupted [Ca2+]i Homeostasis Contributes to the Toxicity of Nitric Oxide in Cultured Hippocampal Neurons

Abstract: Nitric oxide (NO) has been shown to be an important mediator in several forms of neurotoxicity. We previously reported that NO alters intracellular Ca²⁺ concentration ([Ca²⁺]_i) homeostasis in cultured hippocampal neurons during 20-min exposures. In this study, we examine the relationship between late alterations of [Ca²⁺]_i homeostasis and the delayed toxicity produced by NO. The NO-releasing agent S -nitrosocysteine (SNOC; 300 µ M ) reduced survival by about one half 1 day after 20-min exposures, as did other NO-releasing agents. SNOC also was found to produce prolonged elevations of [Ca²⁺]_i, persisting at 2 and 6 h. Hemoglobin, a scavenger of NO, blocked both the late [Ca²⁺]_i elevation and the delayed toxicity of SNOC. Removal of extracellular Ca²⁺ during the 20-min SNOC treatment failed to prevent the late [Ca²⁺]_i elevations and did not prevent the delayed toxicity, but removal of extracellular Ca²⁺ for the 6 h after exposure as well blocked most of the toxicity. Western blots showed that SNOC exposure resulted in an increased proteolytic breakdown of the structural protein spectrin, generating a fragment with immunoreactivity suggesting activity of the Ca²⁺-activated protease calpain. The spectrin breakdown and the toxicity of SNOC were inhibited by treatment with calpain antagonists. We conclude that exposures to toxic levels of NO cause prolonged disruption of [Ca²⁺]_i homeostatic mechanisms, and that the resulting persistent [Ca²⁺]_i elevations contribute to the delayed neurotoxicity of NO.     

Genotyping of bovine k-casein (k-CNA, k-CNB, k-CNC, k-CNE) following DNA sequence amplification and direct sequencing of k-CNE PCR product

Genomic DNA isolated from blood and semen of dairy cattle with known kappa-casein (kappa-CN) genotypes was subjected to Southern blot hybridization and polymerase chain reaction (PCR) using up to 14 restriction endonucleases. kappa-casein genotypes AA, AB and BB were identified using Hin dIII and Hin fI while genotypes with kappa-CNC and kappa-CNE were misidentified. Direct sequencing of the PCR product (kappa-CN EE) showed a substitution of guanine (kappa-CNA,B) by adenine (kappa-CNE) which creates a HaeIII restriction site. Therefore using PCR followed by Hin dIII or HinfI and Hae III digest allows discrimination between kappa-casein A, B and E directly at the DNA level.     

Excretion of ions (Cd2+, Li+, Na+ and Cl−) by Tamarix aphylla

Excretion of minerals by the NaCl-resistant and comparatively cadmium-resistant tree Tamarix aphylla (L.) Karst, was investigated. Cd²⁺ was excreted by plants exposed for 1–10 days to 9 or 45 μ M Cd²⁺ solutions. Excretion of this toxic ion increased considerably with time but was less than 5% of the quantities that had been accumulated in the shoots. Excretion of Na⁺ and Cl⁻ was positively correlated with NaCl concentration (1.5, 10, 50 m M ) of the medium. The Na⁺/Cl⁻ ratios of the excrete were positively correlated with the concentration of the treatment solution. Ca²⁺ excretion decreased with increasing NaCl concentrations of the solution. Excretion of K⁺ and Mg²⁺ was only little affected by NaCl. Excretion of Li⁺ occurred whenever this element was supplied in the uptake solution; daily excretion rates of Li⁺ increased with time. The ecological significance of excretion is discussed in relation to the low selectivity of the mechanism in T. aphylla .     

Kinetics of NH4+ and K+ uptake by ectomycorrhizal fungi. Effect of NH4+ on K+ uptake

NH₄⁺ and K⁺ uptake experiments have been conducted with 3 ectomycorrhizal fungi, originating from Douglas fir (Pseudotsuga menziesii (Mirb.] Franco) stands. At concentrations up to 250 μM, uptake of both NH₄⁺ and K⁺ follow Michaelis-Menten kinetics. Laccaria bicolor (Maire) P. D. Orton, Lactarius rufus (Scop.) Fr. and Lactarius hepaticus Plowr. ap. Boud. exhibit K_m values for NH₄⁺ uptake of 6, 35, and 55 μM, respectively, and K_m values for K⁺ uptake of 24, 18, and 96 μM, respectively. Addition of 100 μM NH₄⁺ raises the K_m of K⁺ uptake by L. bicolor to 35 μM, while the V_max remains unchanged. It is argued that the increase of K_m is possibly caused by depolarization of the plasma membrane. It is not due to a competitive inhibition of K⁺ by NH₄⁺ since the apparent inhibitor constant is much higher than the K_m, for NH₄⁺ uptake. The possibility that NH₄⁺ and K⁺ are taken up by the same carrier can be excluded. The K_m, values for K⁺ uptake in the two other fungi are not significantly affected by 100 μM NH₄⁺. Except for a direct effect of NH₄⁺ on influx of K⁺ into the cells, there may also be an indirect effect after prolonged incubation of the cells in the presence of 100 μM NH₄⁺.     

Involvement of Ca2+ and cGMP in bacterial taxis

Abstract The level of cGMP in a suspension of Escherichia coli cells increased transiently upon the addition of chemoattractants. Ca²⁺ (1 mM), but not Mg²⁺, produced constant tumbling of cells in the presence of the ionophore A23187. The effect was observed either in stationary-state cells, or in a logarithmic culture treated with EDTA to increase permeability by A23187. Under the same conditions, Ca²⁺ decreased the cytoplasmic level of cGMP. In Phormidium uncinatum , rapid ⁴⁵Ca²⁺ accumulation followed a light-dark stimulus, or the addition of tetramethylquinone (TMQ), a chemorepellent. La³⁺, which increases the reversal rate, also enhanced the level of cytoplasmic Ca²⁺, presumably by blocking the outward Ca²⁺ flux. In both E. coli and P. uncinatum Ca²⁺ inhibited methylaccepting chemotaxis protein (MCP) methylation. It is concluded that cGMP and Ca²⁺ are secondary messengers in taxis information-processing.     

Binding of Cd2+, Ni2+, Cu2+ and Zn2+ by isolated envelopes of Pseudomonas fluorescens

Abstract Cell envelopes of Pseudomonas fluorescens , cytoplasmic membrane, peptidoglycan and outer membrane were obtained from a fractionation procedure and tested for their metal binding capacity. Isolated envelopes (cytoplasmic membrane, peptidoglycan and outer membrane) were chemically modified and functional carboxyl groups transformed to electropositive amine groups, using carbodiimide ethylenediamine. Transformation of carboxyl groups was evaluated by measuring total amine groups in all fractions (modified or not). Using equilibrium dialysis and Scatchard plots for the data, we have established that isolated unmodified cell envelopes (cytoplasmic membrane, peptidoglycan and outer membrane) possess at least two types of metal binding sites with different association constants ( K _a and K '_a). Introduction of positive charges into the bacterial envelopes resulted in the disappearance of one type of metal binding site which had the highest association constant value for Ni²⁺, Cu²⁺ and Zn²⁺. All fractions, modified or not, always presented at least two types of binding sites with different association constants for Cd²⁺.     

Suppression of IIIGlc-defects by Enzymes IINag and IIBgl of the PEP:carbohydrate phosphotransferase system

The Enzymes II of the PEP:carbohydrate phosphotransferase system (PTS) specific for N-acetylglucosamine (IINag) and beta-glucosides (IIBgl) contain C-terminal domains that show homology with Enzyme IIIGlc of the PTS. We investigated whether one or both of the Enzymes II could substitute functionally for IIIGlc. The following results were obtained: (i) Enzyme IINag, synthesized from either a chromosomal or a plasmid-encoded nagE+ gene could replace IIIGlc in glucose, methyl alpha-glucoside and sucrose transport via the corresponding Enzymes II. An Enzyme IINag with a large deletion in the N-terminal domain but with an intact C-terminal domain could also replace IIIGlc in IIGlc-dependent glucose transport. (ii) After decryptification of the Escherichia coli bgl operon, Enzyme IIBgl could substitute for IIIGlc. (iii) Phospho-HPr-dependent phosphorylation of methyl alpha-glucoside via IINag/IIGlc is inhibited by antiserum against IIIGlc as is N-acetylglucosamine phosphorylation via IINag. (iv) In strains that contained the plasmid which coded for IINag, a protein band with a molecular weight of 62,000 D could be detected with antiserum against IIIGlc. We conclude from these results that the IIIGlc-like domain of Enzyme IINag and IIBgl can replace IIIGlc in IIIGlc-dependent carbohydrate transport and phosphorylation.     

Distribution of NO3− reduction between roots and shoots of peachtree seedlings as affected by NO3− uptake rate

The distribution of NO₃^? reduction between roots and shoots was studied in hydro-ponically-grown peach-tree seedlings (Prunus persica L.) during recovery from N starvation. Uptake, translocation and reduction of NO₃^?, together with transport through xylem and phloem of the newly reduced N were estimated, using ¹⁵N labellings, in intact plants supplied for 90 h with 0.5 mM NH₄⁺ and 0.5, 1.5 or 10 mM NO₃^?. Xylem transport of NO₃^? was further investigated by xylem sap analysis in a similar experiment. The roots were the main site of NO₃^? reduction at all 3 levels of NO₃^? nutrition. However, the contribution of the shoots to the whole plant NO₃^? reduction increased with increasing external NO₃^? availability. This contribution was estimated to be 20, 23 and 42% of the total assimilation at 0.5, 1.5 and 10 mM NO₃^?, respectively. Both ¹⁵N results and xylem sap analysis confirmed that this trend was due to an enhancement of NO₃^? translocation from roots to shoots. It is proposed that the lack of NO₃^? export to the shoots at low NO₃^? uptake rate resulted from a competition between NO₃^? reduction in the root epidermis/cortex and NO₃^? diffusion to the stele. On the other hand, net xylem transport of newly reduced N was very efficient since ca 70% of the amino acids synthesized in the roots were translocated to the shoots, regardless of the level of NO₃^? nutrition. This net xylem transport by far exceeded the net downward phloem transport of the reduced N assimilated in shoots. As a consequence, the reduced N resulting from NO₃^? assimilation, principally occurring in the roots, was mainly incorporated in the shoots.     

Alteration of Ca2+ Fluxes in Brain Microsomes by K+ and Na+: Modulation by Sulfated Polysaccharides and Trifluoperazine

Abstract: Rat brain microsomes accumulate Ca²⁺ at the expense of ATP hydrolysis. The rate of transport is not modulated by the monovalent cations K⁺, Na⁺, or Li⁺. Both the Ca²⁺ uptake and the Ca²⁺-dependent ATPase activity of microsomes are inhibited by the sulfated polysaccharides heparin, fucosylated chondroitin sulfate, and dextran sulfate. Half-maximal inhibition is observed with sulfated polysaccharide concentrations ranging from 0.5 to 8.0 µg/ml. The inhibition is antagonized by KCl and NaCl but not by LiCl. As a result, Ca²⁺ transport by the native vesicles, which in the absence of polysaccharides is not modulated by monovalent cations, becomes highly sensitive to these ions. Trifluoperazine has a dual effect on the Ca²⁺ pump of brain microsomes. At low concentrations (20–80 µM) it stimulates the rate of Ca²⁺ influx, and at concentrations >100 µM it inhibits both the Ca²⁺ uptake and the ATPase activity. The activation observed at low trifluoperazine concentrations is specific for the brain Ca²⁺-ATPase; for the Ca²⁺-ATPases found in blood platelets and in the sarcoplasmic reticulum of skeletal muscle, trifluoperazine causes only a concentration-dependent inhibition of Ca²⁺ uptake. Passive Ca²⁺ efflux from brain microsomes preloaded with Ca²⁺ is increased by trifluoperazine (50–150 µM), and this effect is potentiated by heparin (10 µg/ml), even in the presence of KCl. It is proposed that the Ca²⁺-ATPase isoform from brain microsomes is modulated differently by polysaccharides and trifluoperazine when compared with skeletal muscle and platelet isoforms.     

Stimulation of proton extrusion by K+ and divalent cations (Ni2+, Co2+ Zn2+) in maize root segments

Entry of the divalent cations Ni²⁺, Co²⁺ and Zn²⁺ into cells of maize ( Zea mays L. cv. Dekalb XL 85) root tissue is accompanied by an acidification of the incubation medium, a decrease in both the pH of the cell sap and the level of malate in the cells, and by an inhibition of dark fixation of CO₂. K⁺, on the contrary, induces only a very low acidification of the incubation medium, does not change either the pH of the cell sap or the malate level in the cells, and induces an increase in CO₂ dark fixation. Different mechanisms are postulated for the stimulation of proton extrusion by divalent cations and K⁺.     

Blue-light-induced pH changes associated with NO3−, NO2− and Cl− uptake by the green alga Monoraphidium braunii

In M. braunii, the uptake of NO₃⁻ and NO₂⁻ is blue-light-dependent and is associated with alkalinization of the medium. In unbuffered cell suspensions irradiated with red light under a CO₂-free atmosphere, the pH started to rise 10s after the exposure to blue light. When the cellular NO₃⁻ and NO₂⁻ reductases were active, the pH increased to values of around 10, since the NH₄⁺ generated was released to the medium. When the blue light was switched off, the pH stopped increasing within 60 to 90s and remained unchanged under background red illumination. Titration with H₂SO₄ of NO₃⁻ or NO₂⁻ uptake and reduction showed that two protons were consumed for every one NH₄⁺ released. The uptake of Cl⁻ was also triggered by blue light with a similar 10 s time response. However, the Cl⁻ -dependent alkalinization ceased after about 3 min of blue light irradiation. When the blue light was turned off, the pH immediately (15 to 30 s) started to decline to the pre-adjusted value, indicating that the protons (and presumably the Cl⁻) taken up by the cells were released to the medium. When the cells lacked NO₃⁻ and NO₂⁻ reductases, the shape of the alkalinization traces in the presence of NO₃⁻ and NO₂⁻ was similar to that in the presence of Cl⁻, suggesting that NO₃⁻ or NO₂⁻ was also released to the medium. Both the NO₃⁻ and Cl⁻-dependent rates of alkalinization were independent of mono- and divalent cations.