Coordination of Eukaryotic Translation Elongation Factor 1A (eEF1A) Function in Actin Organization and Translation Elongation by the Guanine Nucleotide Exchange Factor eEF1B��

Eukaryotic translation elongation factor 1A (eEF1A) both shuttles aminoacyl-tRNA (aa-tRNA) to the ribosome and binds and bundles actin. A single domain of eEF1A is proposed to bind actin, aa-tRNA and the guanine nucleotide exchange factor eEF1Bα. We show that eEF1Bα has the ability to disrupt eEF1A-induced actin organization. Mutational analysis of eEF1Bα F163, which binds in this domain, demonstrates effects on growth, eEF1A binding, nucleotide exchange activity, and cell morphology. These phenotypes can be partially restored by an intragenic W130A mutation. Furthermore, the combination of F163A with the lethal K205A mutation restores viability by drastically reducing eEF1Bα affinity for eEF1A. This also results in a consistent increase in actin bundling and partially corrected morphology. The consequences of the overlapping functions in this eEF1A domain and its unique differences from the bacterial homologs provide a novel function for eEF1Bα to balance the dual roles in actin bundling and protein synthesis.The final step of gene expression takes place at the ribosome as mRNA is translated into protein. In the yeast Saccharomyces cerevisiae, elongation of the polypeptide chain requires the orchestrated action of three soluble factors. The eukaryotic elongation factor 1 (eEF1)² complex delivers aminoacyl-tRNA (aa-tRNA) to the empty A-site of the elongating ribosome (1). The eEF1A subunit is a classic G-protein that acts as a “molecular switch” for the active and inactive states based on whether GTP or GDP is bound, respectively (2). Once an anticodon-codon match occurs, the ribosome acts as a GTPase-activating factor to stimulate GTP hydrolysis resulting in the release of inactive GDP-bound eEF1A from the ribosome. Because the intrinsic rate of GDP release from eEF1A is extremely slow (3, 4), a guanine nucleotide exchange factor (GEF) complex, eEF1B, is required (5, 6). The yeast S. cerevisiae eEF1B complex contains two subunits, the essential catalytic subunit eEF1Bα (5) and the non-essential subunit eEF1Bγ (7).The co-crystal structures of eEF1A:eEF1Bα C terminus:GDP: Mg²⁺ and eEF1A:eEF1Bα C terminus:GDPNP (8, 9) demonstrated a surprising structural divergence from the bacterial EF-Tu-EF-Ts (10) and mammalian mitochondrial EF-Tu_mt-EF-Ts_mt (11). While the G-proteins have a similar topology and consist of three well-defined domains, a striking difference was observed in binding sites for their GEFs. The C terminus of eEF1Bα interacts with domain I and a distinct pocket of domain II eEF1A, creating two binding interfaces. In contrast, the bacterial counterpart EF-Ts and mammalian mitochondrial EF-Ts_mt, make extensive contacts with domain I and III of EF-Tu and EF-Tu_mt, respectively. The altered binding interface of eEF1Bα to domain II of eEF1A is particularly unexpected given the functions associated with domain II of eEF1A and EF-Tu. The crystal structure of the EF-Tu:GDPNP:Phe-tRNA^Phe complex reveals aa-tRNA binding to EF-Tu requires only minor parts of both domain II and tRNA to sustain stable contacts (12). That eEF1A employs the same aa-tRNA binding site is supported by genetic and biochemical data (13-15). Interestingly, eEF1Bα contacts many domain II eEF1A residues in the region hypothesized to be involved in the binding of the aa-tRNA CCA end (8). Because, the shared binding site of eEF1Bα and aa-tRNA on domain II of eEF1A is significantly different between the eukaryotic and bacterial/mitochondrial systems, eEF1Bα may play a unique function aside from guanine nucleotide release in eukaryotes.In eukaroytes, eEF1A is also an actin-binding and -bundling protein. This noncanonical function of eEF1A was initially observed in Dictyostelium amoebae (16). It is estimated that greater than 60% of Dictyostelium eEF1A is associated with the actin cytoskeleton (17). The eEF1A-actin interaction is conserved among species from yeast to mammals, suggesting the importance of eEF1A for cytoskeleton integrity. Using a unique genetic approach, multiple eEF1A mutations were identified that altered cell growth and morphology, and are deficient in bundling actin in vitro (18, 19). Intriguingly, most mutations localized to domain II, the shared aa-tRNA and eEF1Bα binding site. Previous studies have demonstrated that actin bundling by eEF1A is significantly reduced in the presence of aa-tRNA while eEF1A bound to actin filaments is not in complex with aa-tRNA (20). Therefore, actin and aa-tRNA binding to eEF1A is mutually exclusive. In addition, overexpression of yeast eEF1A or actin-bundling deficient mutants do not affect translation elongation (18, 19, 21), suggesting eEF1A-dependent cytoskeletal organization is independent of its translation elongation function (18, 20). Thus, while aa-tRNA binding to domain II is conserved between EF-Tu and eEF1A, this actin bundling function associated with eEF1A domain II places greater importance on its relationship with the “novel” binding interface between eEF1A domain II and eEF1Bα.Based on this support for an overlapping actin bundling and eEF1Bα binding site in eEF1A domain II, we hypothesize that eEF1Bα modulates the equilibrium between actin and translation functions of eEF1A and is perhaps the result of evolutionary selective pressure to balance the eukaryotic-specific role of eEF1A in actin organization. Here, we present kinetic and biochemical evidence using a F163A mutant of eEF1Bα for the importance of the interactions between domain II of eEF1A and eEF1Bα to prevent eEF1A-dependent actin bundling as well as promoting guanine nucleotide exchange. Furthermore, altered affinities of eEF1Bα mutants for eEF1A support that this complex formation is a determining factor for eEF1A-induced actin organization. Interestingly, the F163A that reduces eEF1A affinity is an intragenic suppressor of the lethal K205A eEF1Bα mutant that displays increased affinity for eEF1A. This, along with a consistent change in the actin bundling correlated with the affinity of eEF1Bα for eEF1A, indicates that eEF1Bα is a balancer, directing eEF1A to translation elongation and away from actin, and alterations in this balance result in detrimental effects on cell growth and eEF1A function.     

Presence of nucleoside triphosphates and calcium associated with mycobacteriophage I3

The association of nucleoside triphosphate molecules and calcium ions with purified particles of mycobacteriophage I3 has been documented. The content of nucleoside triphosphate has been determined to be 118 molecules per phage particle by equilibrium dialysis against labelled ATP or 148 molecules per phage particle by the direct determination of labelled nucleoside triphosphate. The concentration of bound Ca²⁺ exhibited a high degree of variation between different batches, which may be due to the nonspecific binding of Ca²⁺ by the virus particles. However, the tightly bound Ca²⁺ not removable by dialysis against calciumspecific chelating agent, showed a constant value of 2985 atoms/phage particle.Abbreviations EGTA Ethylene glycol-bis (-aminoethylether)-N,N¹ tetraacetic acid - PFU plaque forming unit - NTP nucleoside triphosphate     

Aminoacyl-tRNA-charged eukaryotic elongation factor 1A is the bona fide substrate for Legionella pneumophila effector glucosyltransferases

Legionella pneumophila, which is the causative organism of Legionnaireś disease, translocates numerous effector proteins into the host cell cytosol by a type IV secretion system during infection. Among the most potent effector proteins of Legionella are glucosyltransferases (lgt''s), which selectively modify eukaryotic elongation factor (eEF) 1A at Ser-53 in the GTP binding domain. Glucosylation results in inhibition of protein synthesis. Here we show that in vitro glucosylation of yeast and mouse eEF1A by Lgt3 in the presence of the factors Phe-tRNA^Phe and GTP was enhanced 150 and 590-fold, respectively. The glucosylation of eEF1A catalyzed by Lgt1 and 2 was increased about 70-fold. By comparison of uncharged tRNA with two distinct aminoacyl-tRNAs (His-tRNA^His and Phe-tRNA^Phe) we could show that aminoacylation is crucial for Lgt-catalyzed glucosylation. Aminoacyl-tRNA had no effect on the enzymatic properties of lgt''s and did not enhance the glucosylation rate of eEF1A truncation mutants, consisting of the GTPase domain only or of a 5 kDa peptide covering Ser-53 of eEF1A. Furthermore, binding of aminoacyl-tRNA to eEF1A was not altered by glucosylation. Taken together, our data suggest that the ternary complex, consisting of eEF1A, aminoacyl-tRNA and GTP, is the bona fide substrate for lgt''s.     

Kinetics of the acclimational responses of tench to combined hypoxia and hypercapnia

Summary Exposing tench to environmental hypoxia-hypercapnia reduces routine O₂ consumption, sharply decreases arterial O₂ tension and the difference between the water and the blood, and results in marked swelling of the erythrocytes. These changes are rapidly reversed upon return to normoxia.Hypoxic-hypercapnic conditions lower the blood NTP/Hb ratio to a new steady state level within 24 h, by reducing GTP/Hb but not ATP/Hb. A similar selective reduction of eryhtrocytic GTP content forms the initial response of blood incubated in vitro to anoxic conditions.The swelling as well as the reduced GTP/Hb ratio in the erythrocytes appear to improve O₂ loading in the gills during environmental hypoxia-hypercapnia.Symbols and abbreviations a arterial - GTP guanosine triphosphate - Hct hematocrit - I inspired - NTP nucleoside triphosphate - w water     

G-Proteins mediate inhibition and activation of Ca2+-induced exocytosis from SLO-permeabilized peptidergic nerve endings

In SLO-permeabilized isolated nerve endings from the rat neurohypophysis, GTP, guanosine 5[y-thio]triphosphate (GTPyS) and guanosine 5(ßy-imido]triphosphate (GMPPNP) inhibit the Ca²⁺-evoked vasopressin release. Pretreatment with pertussis toxin enhances the inhibitory effects of both GTP-analogues. Omission of Mg²⁺ overcomes the effect of GMPPNP and reverses the inhibitory effect of GTP and GTPyS. In the absence of Mg²⁺, GTP and GTPyS now potentiate Ca²⁺-evoked secretion.     

Respiratory properties of blood and hemoglobin solutions from the piranha

• 1. Respiratory properties of piranha blood are distinguished from those of other fish primarily by the high CO₂ buffering capacity (ΔHCO₃/⁻ΔpH= 19.6mmol/l for oxygenated blood and 39.1 mmol/l for deoxygenated blood).
• 2. The concentration of nucleoside triphosphates (NTP) and the half-saturation tension (P₅₀) of whole blood were found to be inversely related to body size.
• 3. The higherP₅₀ in smaller fish, analogous to values obtained in previous studies involving interspecies comparisons, could be adaptive to a higher weight-specific metabolic rate.
• 4. Both ATP and guanosine triphosphate (GTP) lowered the oxygen affinity of purified hemoglobin solutions, accounting for the size-dependent correlation ofP₅₀ and NTP concentration in whole blood.
• 5. While similar in concentration in red cells, GTP is more potent than ATP as an allosteric modifier of hemoglobin function.

     

Respiratory adaptations in carp blood influences of hypoxia, red cell organic phosphates, divalent cations and CO2 on hemoglobin-oxygen affinity

Summary This study concerns the adaptation of oxygen transporting function of carp blood to environment hypoxia, tracing the roles played by erythrocytic cofactors, inorganic cations, carbon dioxide and hemoglobin multiplicity.Carp acclimated to hypoxia ( 30 mmHg) display striking increases in blood oxygen affinity compared to normoxic ( =120–150 mm) specimens (P ₅₀'s are 3.0 and 7.0 mm, respectively, at pH 7.9 and 20°C). This correlates with a marked decrease in erythrocytic concentrations of NTP (nucleoside triphosphates) (Figs. 1, 2, Table 1), permitting investigation of the time-course of the response (Fig. 3). That GTP (guanosine triphosphate) plays a greater role than ATP in the allosteric regulation of blood oxygen affinity, follows from greater decreases in its concentration during hypoxia, and its greater effect on oxygen affinity of the hemoglobin (Figs. 1, 5). It is furthermore shown that divalent cations (which complex with NTP) inhibit the regulatory role of GTP on O₂ affinity to a lesser extent than that of ATP (Fig. 7). However, the divalent cation, Mg²⁺, occurs in similarly high concentrations in the erythrocytes of hypoxic and normoxic fish (Table 1). CO₂ specifically depresses the O₂ affinity of carp hemoglobin, but below pH 8.3, its effect is obliterated by ATP and GTP suggesting that the chains are the main sites for CO ₂ ^– binding. Four carp hemoglobin components are isolated and their oxygen-binding properties compared with those of the cofactor-free hemolysate (Figs. 4, 8, 9). The results are discussed comparatively with special reference to hemoglobin function in fish and mammals.     

Incorporating efficient radial basis function networks and significant amino acid pairs for predicting GTP binding sites in transport proteins

Background

Guanonine-protein (G-protein) is known as molecular switches inside cells, and is very important in signals transmission from outside to inside cell. Especially in transport protein, most of G-proteins play an important role in membrane trafficking; necessary for transferring proteins and other molecules to a variety of destinations outside and inside of the cell. The function of membrane trafficking is controlled by G-proteins via Guanosine triphosphate (GTP) binding sites. The GTP binding sites active G-proteins initiated to membrane vesicles by interacting with specific effector proteins. Without the interaction from GTP binding sites, G-proteins could not be active in membrane trafficking and consequently cause many diseases, i.e., cancer, Parkinson… Thus it is very important to identify GTP binding sites in membrane trafficking, in particular, and in transport protein, in general.

Results

We developed the proposed model with a cross-validation and examined with an independent dataset. We achieved an accuracy of 95.6% for evaluating with cross-validation and 98.7% for examining the performance with the independent data set. For newly discovered transport protein sequences, our approach performed remarkably better than similar methods such as GTPBinder, NsitePred and TargetSOS. Moreover, a friendly web server was developed for identifying GTP binding sites in transport proteins available for all users.

Conclusions

We approached a computational technique using PSSM profiles and SAAPs for identifying GTP binding residues in transport proteins. When we included SAAPs into PSSM profiles, the predictive performance achieved a significant improvement in all measurement metrics. Furthermore, the proposed method could be a power tool for determining new proteins that belongs into GTP binding sites in transport proteins and can provide useful information for biologists.

     

Novel N-terminal and Lysine Methyltransferases That Target Translation Elongation Factor 1A in Yeast and Human

Eukaryotic elongation factor 1A (eEF1A) is an essential, highly methylated protein that facilitates translational elongation by delivering aminoacyl-tRNAs to ribosomes. Here, we report a new eukaryotic protein N-terminal methyltransferase, Saccharomyces cerevisiae YLR285W, which methylates eEF1A at a previously undescribed high-stoichiometry N-terminal site and the adjacent lysine. Deletion of YLR285W resulted in the loss of N-terminal and lysine methylation in vivo, whereas overexpression of YLR285W resulted in an increase of methylation at these sites. This was confirmed by in vitro methylation of eEF1A by recombinant YLR285W. Accordingly, we name YLR285W as elongation factor methyltransferase 7 (Efm7). This enzyme is a new type of eukaryotic N-terminal methyltransferase as, unlike the three other known eukaryotic N-terminal methyltransferases, its substrate does not have an N-terminal [A/P/S]-P-K motif. We show that the N-terminal methylation of eEF1A is also present in human; this conservation over a large evolutionary distance suggests it to be of functional importance. This study also reports that the trimethylation of Lys⁷⁹ in eEF1A is conserved from yeast to human. The methyltransferase responsible for Lys⁷⁹ methylation of human eEF1A is shown to be N6AMT2, previously documented as a putative N(6)-adenine-specific DNA methyltransferase. It is the direct ortholog of the recently described yeast Efm5, and we show that Efm5 and N6AMT2 can methylate eEF1A from either species in vitro. We therefore rename N6AMT2 as eEF1A-KMT1. Including the present work, yeast eEF1A is now documented to be methylated by five different methyltransferases, making it one of the few eukaryotic proteins to be extensively methylated by independent enzymes. This implies more extensive regulation of eEF1A by this posttranslational modification than previously appreciated.Protein methylation is emerging as one of the most prominent posttranslational modifications in the eukaryotic cell (1). Often showing high evolutionary conservation, it is increasingly recognized for its role in modulating protein–protein interactions (2). Indeed, it has been documented in protein interaction codes (3), such as those of the histones and p53 (4, 5), where it shows interplay with modifications such as acetylation and phosphorylation. Despite this, there remains a paucity of understanding of the enzymes that catalyze protein methylation. Many of the known methyltransferases target histones. However, many other methyltransferases have been discovered recently that act on nonhistone proteins (6).While protein methylation predominantly occurs on lysine and arginine residues, it is also known to occur on glutamine, asparagine, glutamate, histidine, cysteine, and the N- and C termini of proteins. Although the presence of N-terminal methylation on numerous proteins has been known for decades (7), the first enzymes responsible for this methylation have only recently been discovered (8, 9). The Saccharomyces cerevisiae protein Tae1 and its human ortholog N-terminal methyltransferase 1 (NTMT1) catalyze N-terminal methylation of proteins with an N-terminal [A/P/S]-P-K motif (after methionine removal). Yet there is evidence that these enzymes may recognize a more general N-terminal motif (10). Human NTMT2 is a monomethyltransferase that methylates the same substrates as NTMT1 and may prime substrate proteins with monomethylation to assist subsequent trimethylation by NTMT1 (11).The biological function of N-terminal methylation on some proteins has been recently revealed. For example, N-terminal methylation of regulator of chromatin condensation protein 1 (RCC1) is known to affect its binding to chromatin and thereby the correct chromosomal segregation during mitosis (12, 13), and N-terminal methylation of DNA damage-binding protein 2 (DDB2) is important for its role in UV-damaged DNA repair (14). Interestingly, there is evidence of interplay between N-terminal methylation and other posttranslational modifications (15), suggesting that, like lysine and arginine methylation, it may be incorporated into protein interaction codes (3). N-terminal methylation therefore appears to be a modification of functional importance in the cell.Eukaryotic elongation factor 1A (eEF1A), and its bacterial ortholog EF-Tu, is an essential translation elongation factor that is found in all living organisms. Its canonical function is in facilitating delivery of aminoacyl-tRNAs to the ribosome; however, it is also known to have a role in many other cellular functions, such as actin bundling, nuclear export, and proteasomal degradation (16). A number of methyltransferases have been discovered in both S. cerevisiae and human that target translation elongation factors. In yeast, four of these elongation factor methyltransferases (EFMs) act on eEF1A, namely Efm1, Efm4, Efm5, and Efm6, generating monomethylated Lys³⁰, dimethylated Lys³¹⁶, trimethylated Lys⁷⁹, and monomethylated Lys³⁹⁰, respectively (17–19). Human METTL10 is the ortholog of Efm4 in that it trimethylates eEF1A at Lys³¹⁸, which is equivalent to Lys³¹⁶ in yeast (20). Interestingly, eukaryotic elongation factor 2 (eEF2) is also methylated by a number of lysine methyltransferases. In yeast, Efm2 and Efm3 act on eEF2, generating dimethylated Lys⁶¹³ and trimethylated Lys⁵⁰⁹, respectively (21–24). Human eEF2-KMT is the ortholog of Efm3 in that it trimethylates eEF2 at Lys⁵²⁵, which is equivalent to Lys⁵⁰⁹ in yeast eEF2 (23).Here, we report the N-terminal methylation of eEF1A in S. cerevisiae and the identification of the methyltransferase that catalyzes this event. Using parallel reaction monitoring and MS/MS/MS (MS3), we unambiguously localize the modification to the N-terminal glycine and show it is conserved in the human cell. We also show that YLR285W, which we rename elongation factor methyltransferase 7 (Efm7), is responsible for this modification in yeast, as well as dimethylation at the adjacent lysine. We also characterize the methyltransferases responsible for methylation of lysine 79 in eEF1A. Human N6AMT2 is shown to be the ortholog of yeast Efm5 through its capacity to methylate yeast and human eEF1A at Lys⁷⁹ in vitro. We therefore rename N6AMT2 as eEF1A-KMT1.     

Partial purification and properties of guanosine triphosphate cyclohydrolase from Drosophila melanogaster

The first enzyme (named GTP cyclohydrolase) in the pathway for the biosynthesis of pteridines has been partially purified from extracts of late pupae and young adults of Drosophila melanogaster. This enzyme catalyzes the hydrolytic removal from GTP of carbon 8 as formate and the synthesis of 2-amino-4-hydroxy-6-(d-erythro-1,2,3-trihydroxypropyl)-7,8-dihydropteridine triphosphate (dihydroneopterin triphosphate). Some of the properties of the enzyme are as follows: it functions optimally at pH 7.8 and at 42 C; activity is unaffected by KCl and NaCl, but divalent cations (Mg²⁺, Mn²⁺, Zn²⁺, and Ca²⁺) are inhibitory; the K _m for GTP is 22 m; and the molecular weight is estimated at 345,000 from gel filtration experiments. Of a number of nucleotides tested, only GDP and dGTP were used to any extent as substrate in place of GTP, and these respective compounds were used only 1.8% and 1.5% as well as GTP.This work was supported by research grants from the National Institutes of Health (AM03442) and the National Science Foundation (GB33929).     

Acclimation to hypoxic water in facultative air-breathing fish: Blood oxygen affinity and allosteric effectors

• 1. Blood oxygen affinities, erythrocytic nucleoside triphosphate concentrations (NTP) and other hematological parameters were measured in facultative air-breathing fish from the Amazon after acclimation to well-aerated (“normoxic”) and hypoxic water (PO₂ = 125–135 and 20–25 mm, respectively).
• 2. In the armored catfishHypostomus sp. andPterygoplichthys sp., hypoxia induces intermittent surfacing to gulp air and results in lower NTP levels, chiefly through significant decreases in guanosine triphosphate (GTP). The subsequent increases in blood O₂ affinity appear adaptive to lowered time average internal O₂ tensions. No similar changes were seen in the ellSynbranchus which breathes air almost continuously when kept in hypoxic water.
• 3. The results are discussed in terms of their adaptive significance, and compared with data on temperate fish.

     

Structural Models of Human eEF1A1 and eEF1A2 Reveal Two Distinct Surface Clusters of Sequence Variation and Potential Differences in Phosphorylation

Background

Despite sharing 92% sequence identity, paralogous human translation elongation factor 1 alpha-1 (eEF1A1) and elongation factor 1 alpha-2 (eEF1A2) have different but overlapping functional profiles. This may reflect the differential requirements of the cell-types in which they are expressed and is consistent with complex roles for these proteins that extend beyond delivery of tRNA to the ribosome.

Methodology/Principal Findings

To investigate the structural basis of these functional differences, we created and validated comparative three-dimensional (3-D) models of eEF1A1 and eEF1A2 on the basis of the crystal structure of homologous eEF1A from yeast. The spatial location of amino acid residues that vary between the two proteins was thereby pinpointed, and their surface electrostatic and lipophilic properties were compared. None of the variations amongst buried amino acid residues are judged likely to have a major structural effect on the protein fold, or to affect domain-domain interactions. Nearly all the variant surface-exposed amino acid residues lie on one face of the protein, in two proximal but distinct sub-clusters. The result of previously performed mutagenesis in yeast may be interpreted as confirming the importance of one of these clusters in actin-bundling and filament disorganization. Interestingly, some variant residues lie in close proximity to, and in a few cases show differences in interactions with, residues previously inferred to be directly involved in binding GTP/GDP, eEF1Bα and aminoacyl-tRNA. Additional sequence-based predictions, in conjunction with the 3-D models, reveal likely differences in phosphorylation sites that could reconcile some of the functional differences between the two proteins.

Conclusions

The revelation and putative functional assignment of two distinct sub-clusters on the surface of the protein models should enable rational site-directed mutagenesis, including homologous reverse-substitution experiments, to map surface binding patches onto these proteins. The predicted variant-specific phosphorylation sites also provide a basis for experimental verification by mutagenesis. The models provide a structural framework for interpretation of the resulting functional analysis.     

Region of Elongation Factor 1A1 Involved in Substrate Recognition by Legionella pneumophila Glucosyltransferase Lgt1: IDENTIFICATION OF Lgt1 AS A RETAINING GLUCOSYLTRANSFERASE*

Lgt1 is one of the glucosyltransferases produced by the Gram-negative bacterium Legionella pneumophila. This enzyme modifies eukaryotic elongation factor 1A (eEF1A) at serine 53, which leads to inhibition of protein synthesis and death of target cells. Here we studied the region of eEF1A, which is essential for substrate recognition by Lgt1. We report that the decapeptide ⁵⁰GKGSFKYAWV⁵⁹ of eEF1A is efficiently modified by Lgt1. This peptide covers the loop of the helix-loop-helix region formed by helices A* and A′ of eEF1A and is part of the first turn of helix A′. Substitution of either serine 53, phenylalanine 54, tyrosine 56, or tryptophan 58 by alanine abolished or severely decreased glucosylation. Lgt1 modified the decapeptide ⁵⁰GKGSFKYAWV⁵⁹ with a higher glucosylation rate than full-length eEF1A purified from yeast, suggesting that a specific conformation of eEF1A is the preferred substrate of Lgt1. A GenBank^TM search on the basis of the substrate decapeptide for similar peptide sequences retrieved heat shock protein 70 subfamily B suppressor 1 (Hbs1) as a target for glucosylation by Lgt1. Recombinant Hbs1 and the corresponding fragment (³⁰³GKASFAYAWV³¹²) were gluco syl a ted by Lgt1. NMR studies with the gluco syl a ted eEF1A-derived decapeptide identified an α-anomeric structure of the glucose-serine 53 bond and characterize Lgt1 as a retaining glucosyltransferase.Legionella pneumophila is a Gram-negative bacterium, causing pulmonary infectious disease in humans. This microorganism is able to infect various free-living protozoa in natural environment as well as macrophages, monocytes, and lung epithelial cells during human disease (1, 2). A plethora of virulence factors, which are important for intracellular proliferation of the bacteria in target eukaryotic cells, and a type IVB secretion system for intracytoplasmic delivery of these effectors have been identified (3). Among the best studied Legionella products are RalF (4) and DrrA (5), which act as exchange factors for Arf1 and Rab1 small GTPases, respectively. Additionally, DrrA has been shown to possess activity of a guanine nucleotide dissociation inhibitor-displacement factor (6). These two proteins were suggested to participate in recruitment of endosomal vesicles and construction of a replicative phagosome, which is a characteristic intracellular niche of Legionella and prerequisite for subsequent proliferation of the bacteria in host cells (7). However, despite considerable progress, many aspects of intracellular biology of L. pneumophila, in particular those apart from processes associated with alterations in vesicular trafficking, remain poorly understood.In our previous investigations we identified three proteins in L. pneumophila (Lgt1, Lgt2, and Lgt3), which possess enzymatic activity and modify eukaryotic elongation factor eEF1A³ at serine 53 by mono-O-glucosylation (8–10). This modification inhibits protein synthesis and is eventually lethal to target cells. Expression of Lgt1 is strongly increased during late phase of bacterial growth in broth medium and in Acanthamoeba castellanii (10). Because bacteria taken at the stationary phase of growth are known to possess maximal pathogenic potential (11, 12), up-regulation of the glucosyltransferase has been suggested to be involved in virulence of L. pneumophila. Here we studied the recognition of eEF1A1 by Lgt1 and identified the type of glucosylation catalyzed by the enzyme.     

Analysis of teleost hemoglobin by Adair and Monod-Wyman-Changeux models

Summary The allosteric effects of the erythrocytic nucleoside triphosphates (NTP) and of proton concentrations were investigated by precise measurement of Hb–O₂ equilibria of tench hemoglobin (including extreme, high and low saturation ranges) and analysed in terms of the MWC two state model and the Adair four step oxygenation theory.At low concentrations (NTP/Hb ratio=1.0, and pH>7.3) ATP, GTP and protons decrease Hb–O₂ affinity by increasing the allosteric constantL and reducingK _T, the association constant¹ of the deoxy, tense state of the Hb, without significantly affecting that (K _R) of the oxy state, increasing the free energy of cooperativity (G). High concentrations of these effectors, however, also reduceK _R. The greater sensitivity of the half-saturation O₂ tension (P ₅₀) of the Hb to GTP than to ATP at the same concentration, correlates with greater effects of GTP on bothK _T andK _R. The pH and NTP dependence of the four Adair association constants and the calculated fractional populations of Hb molecules in different stages of oxygenation show that the autochthonous NTP effectors and protons stabilize the T structure and postpone the TR transition basic to cooperativity in fish Hb.The possible implications of the findings for aquatic respiration are discussed.Abbreviations ATP adenosine triphosphate - DPG 2,3-diphosphoglycerate (glycerate-2,3-bisphosphate) - GTP guanosine triphosphate - IHP inositol hexaphosphate - NTP nucleoside triphosphates In this paperK _T andK _R are defined as theassociation equilibrium constants instead of dissociation constants (as originally defined by Monod et al. 1965) to facilitate comparison with the Adair constants     

A fetal-maternal shift in the oxygen equilibrium of hemoglobin from the viviparous caecilian,Typhlonectes compressicauda

• 1. The equilibria and kinetics of oxygen binding by blood and hemoglobin from adult and fetal caecilians,Typhlonectes compressicauda, have been measured.
• 2. The oxygen affinity of fetal blood is higher than that of adult blood.
• 3. Electrophoresis of adult and fetal hemoglobins suggests that they may be identical: a major and minor component occurs in each.
• 4. Adult and fetal hemoglobins have identical oxygen equilibria. Stripped hemoglobins have a high oxygen affinity and no Bohr effect between pH 6.5 and 10.0. An “acid”, reversed Bohr effect is present below pH 6.5. The addition of 1 mM ATP reduces the oxygen affinity markedly and produces a moderate, normal Bohr effect.
• 5. The major nucleoside triphosphate in fetal and adult erythrocytes is adenosine triphosphate: about 10% of the nucleoside triphosphates is guanosine triphosphate. Adult erythrocytes contain 3 times as much ATP as do the fetal erythrocytes.
• 6. The fetal to maternal shift in the oxygen equilibrium is mediated entirely by the difference in ATP content of the maternal and fetal red blood cells.

     

Use of scanning cytometry in studying bradykinin binding in MRC-5 cells

Ligand-receptor affinity is classically demonstrated by measuring ligand binding density to a specific site on membrane preparations, and receptor function is studied by measuring calcium flux, cell by cell, using microspectrofluorimetry. In order to study these phenomena in a larger cell population, calcium flux was measured in MRC-5 cell line expressing the B₂ receptor for bradykinin using an ACAS 570 scanning cytometer. Following incorporation of fluo3/AM, different ligands were studied, singly or in association with bradykinin. This study confirmed that only the B₂ receptor is present on the plasma membrane of MRC-5 cells. Bradykinin binding to the B₂ receptor was not modified by a B₁ agonist (Des-Arg⁹-bradykinin) or by a B₁ antagonist (Des-Arg⁹-[Leu⁸]-bradykinin) but was inhibited by a B₂ agonist ([Hyp³]-bradykinin) and a B₂ antagonist (HOE 140). The source of free calcium was also studied in comparison with ionomycin. The intensity of the calcium peak after binding of bradykinin is independent of the concentration of extracellular calcium. Preincubation with diltiazem or TMB-8 did not modify calcium flux indicating that transduction of the signal after bradykinin binding in this cell line is independent of voltage-dependent channels and does not require mobilization of intracellular calcium blocked by TMB-8. In conclusion, scanning cytometry can be used to study ligand-receptor binding and to obtain results rapidly from multiple cells. Recording of individual cell variations and kinetics enables identification of active agonists or antagonists and consequently the selection of new compounds.Abbreviations 9AA 9 amino acids - CCD charged-coupled device - DMEM Dulbecco's Modified Eagle's Medium - EDTA ethylenediamine tetraacetic acid - EGTA ethylene glycol-bis (-amino-ethyl ether)N,N,N,N-tetraacetic acid - FCS Fetal Calf Serum - GTP guanosine triphosphate - HBSS Hank's Buffer Salt Solution - IP3 inositol triphosphate     

Guanine nucleotides do not inhibit tritiated dopamine agonist binding to bovine striatal membranes

The addition of GTP (50 M), MnCl₂ (1 mM) or EDTA (2 mM) had no effect on the affinity or capacity of bovine striatal plasma membranes for [³H]spiperone. However, GTP caused a decrease in the potency of dopamine as an inhibitor of [³H]spiperone binding under all conditions tested. Manganese enhanced the potency of dopamine both in the presence and absence of GTP, but NaCl (100 mM) had no effect. Neither manganese nor GTP caused any change in the affinity or capacity of bovine striatal membranes for the tritiated agonists dopamine, apomorphine or ADTN. GPPNHP, a nonhydrolyzable analog of GTP, was also ineffective. However, in identical experiments using rat striatal membranes, 50 M GTP caused a decrease in affinity for all three tritiated agonists and this effect was observed both in the presence and absence of manganese (1 mM). In addition, binding capacities for [³H]dopamine and [³H]ADTN were doubled when manganese was present. In light of this and other reports that GTP inhibits tritiated agonist binding in rat striatum, it is suggested that the absence of such inhibition in bovine striatal membranes may reflect a fundamental difference between the two species with regard to their receptors for dopamine agonists.     

Cannabinoid receptor stimulation of guanosine-5′-O-(3-[35S]thio)triphosphate binding in rat brain membranes

Cannabinoid receptors belong to the class of G-protein-coupled receptors which inhibit adenylyl cyclase. Coupling of receptors to G-proteins can be assessed by the ability of agonists to stimulate guanosine-5′-O-(3-[³⁵S]thio)triphosphate ([³⁵S]GTPγS) binding in the presence of excess GDP. The present study examined the effect of cannabinoid agonists on [³⁵S]GTPγS binding in rat brain membranes. Assays were conducted with 0.05 nM [³⁵S]GTPγS, incubated with rat cerebellar membranes, 1–30 μM GDP and the cannabinoid agonist WIN 55212-2. Results showed that the ability of WIN 55212-2 to stimulate [³⁵S]GTPγS binding increased with increasing concentrations of GDP, with 10–30 μM GDP providing approximately 150–200% stimulation by the cannabinoid agonist. The pharmacology of cannabinoid agonist stimulation of [³⁵S]GTPγS binding paralleled that of previously reported receptor binding and adenylyl cyclase assays, and agonist stimulation of [³⁵S]GTPγS binding was blocked by the cannabinoid antagonist SR141716A. Brain regional studies revealed widespread stimulation of [³⁵S]GTPγS binding by WIN 55212-2 in a number of brain areas, consistent with in vitro [³⁵S]GTPγS autoradiography. These results demonstrate that [³⁵S]GTPγS binding in the presence of excess GDP is an effective measure of cannabinoid receptor coupling to G-proteins in brain membranes.