The inhibition of elongation factor 1 activity by heparin

The effect of the mucopolysaccharide heparin on elongation factor 1 (EF-1) from embryos of the brine shrimp Artemia salina was investigated. Heparin was found to be a potent inhibitor of the purified enzyme in binding aminoacyl-tRNA to ribosomes and had a comparable effect on polyuridylic acid dependent polyphenylanine synthesis. However, no effect on the binding of GTP to EF-1 or the ability of the factor to form a ternary complex with GTP and aminoacyl-tRNA was observed, suggesting that heparin interferes with the ribosome-attachment site on the ternary complex. In addition EF-1 bound to heparin-Sepharose gels and such gels could be used to partially purify the factor from post-ribosomal supernatant fractions.     

Cooperative binding of adenosine by polyuridylic acid: a further analysis

The dialysis data of Huang and Ts'o for the cooperative binding of adensine to polyuridylic acid are analyzed here using a grand-partition function Ising model method similar to that originally employed for polyelectrolytes by Rice and Nagasawa. An appropriate modification permitting the treatment of the sliding degeneracy of the two polyuridylic acid strands is also included. In addition to the previously estimated stacking energy of about ?6 kcal/mole one also obtains the free energy change F? for the transfer of a single adenosine molecule from a fixed site in solution to a fixed site on the polyuridylic acid. This binding energy falls in the range F? = ?140 to +620 cal/mole, indicating that binding in the 1:2 (purine: pyrimidine) complex is either very weak or repulsive. The absence of any comparable cooperative stacking of adenosines in solution at the same concentration together with the likely repulsive character of the binding implies that the stacking energy must contain a significant contribution from other processes than simple stacking of adenosines. A generalization of the theory to treat multicomponent binding and longer-range interactions is effected, and the form applicable to simultaneous binding of both adenosine and guanosine by polyuridylic acid is presented.     

Studies on the macroconidia of Microsporum canis

The characteristics of an in vitro polyuridylic acid dependent amino acid incorporating system prepared from germinating macroconidia of Microsporum canis are described. The incorporation of ¹⁴C-phenylalanine into polyphenylalanine is dependent on S-30 extract, adenosine triphosphate, magnesium ions and polyuridylic acid. Incorporation is slightly enhanced by yeast transfer ribonucleic acid and pyruvate kinase. The system is highly sensitive to ribonuclease, puromycin and miconazole (an antifungal agent), moderately sensitive to sodium fluoride and much less sensitive to phenethylalcohol, cycloheximide, chloramphenicol and deoxyribonuclease. Cell-free extract from ungerminated conidia has less capacity to synthesize the protein and during germination a marked increase in the protein synthetic activity is observed. The results from experiments wherein ribosomes and S-100 fraction from germinated and ungerminated spores are interchanged, revealed that the defect in the extract from the ungerminated spore is in the ribosomes.Abbreviations Poly(U) polyuridylic acid - tRNA transfer ribonucleic acid - ATP adenosine triphosphate - GTP guanosine triphosphate - BSA bovine serum albumin - RNase ribonuclease - DNase deoxyribonuclease - POPOP 1,4-bis-2(5-phenyl oxazolyl)benzene - PPO 2,5-diphenyl oxazole - TCA trichloracetic acid     

Investigation of non-CB1, non-CB2 WIN55212-2-sensitive G-protein-coupled receptors in the brains of mammals,birds, and amphibians

Abstract

Purpose: Previous studies have found non-CB₁ non-CB₂ G-protein-coupled receptors in rodents that are activated by the aminoalkylindole cannabinoid agonist WIN55212-2. This work obtained evidence for the presence or absence of similar receptors in the brains of other mammals, birds and amphibians.

Materials and methods: Antagonism of the stimulation of [³⁵S]GTPγS binding by WIN55212-2 and CP55940 was assessed in multiple CNS regions of rat and canine, and in whole brain membranes from shrew, pigeon, frog and newt. A bioinformatics approach searched for orthologs of GRP3, GPR6, and GPR12 (closely related to cannabinoid receptors) in the genomes of these or related species. Orthologs were examined for amino acid motifs known to impart functionality to receptors.

Results: In mammals and pigeon, but not amphibians, a significant fraction of the stimulation of [³⁵S]GTPγS binding by WIN55212-2 was not blocked by the CB₁ antagonist SR141716A. BLAST searches found that GPR3 was restricted to mammals. GPR12 orthologs existed in all species, and they shared identical amino acid motifs. GPR6 orthologs existed all species, but with significant departures in the identity of some critical amino acids in bird, more so in amphibian.

Conclusions: The portion of WIN55212-2-stimulated [³⁵S]GTPγS binding that was antagonized by SR141716A was consistent with stimulation via CB₁ receptors, indicating that antagonist-insensitive activity was via a different G-protein coupled receptor. Pharmacological evidence of this receptor was found in the brains of mammals and pigeon, but not frog or newt. Bioinfomatics results implicate GPR6 as a possible candidate for the additional WIN55212-2-sensitive receptor.     

Further observations on the polynucleotide-induced stimulation of protein synthesis by cell-free preparations from rabbit reticulocytes.

1. The effect of high-molecular-weight RNA from reticulocyte polyribosomes (messenger RNA) on protein synthesis by subcellular fractions derived from reticulocytes, reported by Arnstein, Cox & Hunt (1964), has now been studied in detail. Optimum response of the cell-free system requires 30-50mm-K(+) and approx. 5mm-Mg(2+) in the pH range 7.4-7.6. 2. RNA stimulates the incorporation into protein of both free amino acids and of aminoacyl residues from s-RNA. Stimulation by either RNA or polyuridylic acid is dependent on a labile factor or enzyme, which is present in the ;pH5 fraction' and may be concerned with the formation of new polysomes. Quantitatively the response of the cell-free system to RNA is similar to that of polyuridylic acid, and there appears to be competition between messenger RNA and polyuridylic acid or polyadenylic acid.     

Partial reaction of peptide initiation inhibited by the reticulocyte hemin-controlled repressor

The hemin-controlled repressor from rabbit reticulocytes inhibits binding of Met-tRNA_f to reticulocyte 40S ribosomal subunits in a partial reaction containing these components, two initiation factor fractions and GTP. The inhibitor does not interfere with the formation of the Met-tRNA_f· initiation factor IF-E₂·GTP complex.     

IN VlTRO BINDING OF PHENYLALANYL-tRNA TO NEONATAL AND ADULT MOUSE BRAIN RIBOSOMES

The ability of brain ribosomes, isolated from mice of various ages, to bind phenylalanyl-tRNA was measured under various reaction conditions. In the presence of template RNA (polyuridylic acid) the binding could be measured by both enzymic and non-enzymic assays. In general, the binding requirements for the brain system were similar to those previously described for microbial and eukaryotic systems. Although previous studies have shown that ribosomes obtained from increasingly older mow brain tissue were less active in polyphenylalanine synthesis, no significant differences in phenylalanyl-tRNA binding to polysome complexes could be detected. The binding of phenylalanyl-tRNA by ribosomes isolated from both neonatal and mature mouse brain tissue was similar with regard to GTP and polyuridylic acid dependence, magnesium ion concentration and reaction kinetics. Similar binding of phenylalanyl-tRNA by young and mature brain ribosomes was also measured with ribonucleoprotein particles previously stripped with puromycin. The results are discussed in light of the rapid alteration of macromolecular synthesis during postnatal brain development and the possible role of the interaction between ribosomes and tRNA.     

Fatty acid synthesis by isolated chromoplasts from the daffodil. Energy sources and distribution patterns of the acids

1. Fatty acid synthesis in isolated intact chromoplasts from [1-¹⁴C]acetate was made possible by using ATP, ADP (via adenylate kinase), and, with decreasing efficiency, UTP, CTP, and GTP as energy sources. 2. The glycolytic path from dihydroxyacetone phosphate to acetyl-CoA operates within the chromoplasts. The glycolytic intermediates, especially 2-phosphoglycerate and phosphoenolpyruvate, served as very effective energy donors for fatty acid synthesis by phosphorylating the endogenous adenine nucleotide pool. 3. In the presence of exogenous ATP or ADP, appreciable amounts of in vitro formed fatty acids were found as acyl-CoA and subsequent products, mainly phosphatidylcholine. When other energy sources were used most of the acids formed were in the free form, and to a minor extent, in the phosphatidic acid and diacylglycerol fractions. Similar results have recently been reported for spinach chloroplasts (Kleinig and Liedvogel 1979, FEBS Lett.101, 339–342).Abbreviations ATP adenosine triphosphate - ADP adenosine diphosphate - UTP uridine triphosphate - CTP cytidine triphosphate - GTP gnanosine triphosphate     

GTP binding by Arabidopsis extra-large G protein 2 is not essential for its functions

The extra-large guanosine-5′-triphosphate (GTP)-binding protein 2, XLG2, is an unconventional Gα subunit of the Arabidopsis (Arabidopsis thaliana) heterotrimeric GTP-binding protein complex with a major role in plant defense. In vitro biochemical analyses and molecular dynamic simulations show that affinity of XLG2 for GTP is two orders of magnitude lower than that of the conventional Gα, AtGPA1. Here we tested the physiological relevance of GTP binding by XLG2. We generated an XLG2(T476N) variant with abolished GTP binding, as confirmed by in vitro GTPγS binding assay. Yeast three-hybrid, bimolecular fluorescence complementation, and split firefly-luciferase complementation assays revealed that the nucleotide-depleted XLG2(T476N) retained wild-type XLG2-like interactions with the Gβγ dimer and defense-related receptor-like kinases. Both wild-type and nucleotide-depleted XLG2(T476N) restored the defense responses against Fusarium oxysporum and Pseudomonas syringae compromised in the xlg2 xlg3 double mutant. Additionally, XLG2(T476N) was fully functional restoring stomatal density, root growth, and sensitivity to NaCl, but failed to complement impaired germination and vernalization-induced flowering. We conclude that XLG2 is able to function in a GTP-independent manner and discuss its possible mechanisms of action.

Arabidopsis extra-large GTP-binding proteins have nucleotide-independent functions.     

Evaluation of the Role of GTP Hydrolysis in the Interaction Between Mg2+ and GTP in Regulating Agonist Binding to the Adenosine A1 Receptor

Abstract

Binding of agonists to adenosine receptors is reduced by GTP, whereas it is enhanced by Mg²⁺. The effect of GTP can be completely reversed by divalent cations, in contrast to the effect of the nonhydrolyzable analogue 5′-guanylylimidodiphosphate (GPPNHP). The present study addresses the role of divalent cation-stimulated specific and nonspecific GTP-ases in this reversal process. Under the conditions commonly employed in binding assays, almost all GTP is rapidly converted to GMP and P_i, indicating that maintenance of GTP levels is essential for the proper interpretation of results. A combination of a GTP-generating system and a competing substrate for high K_m GTP-ases minimizes GTP breakdown. In the presence of these additions, the reversal of GTP effects is almost eliminated, and the inhibitory effects of both GTP and GPPNHP on agonist binding are reduced by divalent cations to a similar extent. Besides enhancing nonspecific GTP hydrolysis, Mg²⁺, but not Mn²⁺ or Ca²⁺, also stimulates specific agonist-dependent GTP-ase activity. Thus, it is evident that specific regulatory effects of Mg²⁺ and other divalent cations can only be identified when other, nonspecific, effects have been evaluated and controlled.     

Effects of GTP on 3H-Domperidone Binding and its Displacement by Dopamine in Rat Striatal Homogenates

Abstract

The potency of dopamine in displacing ³H-domperidone from rat striatal membranes was reduced by GTP and enhanced by Mg++. GTP also increased specific binding, and this effect was larger in the presence of Mg++, which itself inhibited binding. In differently washed and incubated preparations the magnitude of both GTP and Mg++ effects correlated well with the endogenous dopamine content of the membranes. Na+ reduced the potency of dopamine, the GTP effect and specific binding. Saturation experiments revealed that these effects of ions and GTP reflected changes in both the apparent ^Bmax and K_D of ³H-domperidone. It was possible to eliminate the GTP effect on specific binding by extensive washing and incubation, but this treatment may also alter other receptor binding characteristics. It remains unclear whether the enhancing effect of GTP reflects contamination of endogenous dopamine or a genuine property of the D₂ receptor.     

Small GTP‐binding proteins in the nuclei of human placenta

Mitochondria and crude nuclei containing fractions from human placenta have been shown to contain proteins which bind [α³²P]‐GTP. Prior to this study the number of GTP‐binding proteins in placental nuclei and their nucleotide specificity was not known. Also unknown was the identity of any of the GTP‐binding proteins in mitochondria of human placenta. Nuclei and mitochondria were purified from human placental extracts by sedimentation. Proteins were separated by electrophoresis and transferred to nitrocellulose membranes. Overlay blot with [α³²P]‐GTP identified two nuclei proteins with approximate molecular weights of 24 and 27 kDa. Binding of [α³²P]‐GTP to the 27 and 24 kDa proteins was significantly displaced by guanine nucleotides but not by adenine, thymine or cytosine nucleotides or deoxy (d) GTP. Western blot with a specific antibody to Ran identified a band at 27 kDa in nuclei and in mitochondrial fractions. These data indicate that both nuclei and mitochondria contain 24 and 27 kDa GTP‐binding proteins. The GTP‐binding proteins in nuclei display binding specificity for guanine nucleotides and the hydroxylated carbon 2 on the ribose ring of GTP appears essential for binding. It will be important in future studies to determine the functions of these small GTP‐binding proteins in the development and physiology of the placenta. J. Cell. Biochem. 84: 100–107, 2002. © 2001 Wiley‐Liss, Inc.     

Thermodynamics of GTP and GDP Binding to Bacterial Initiation Factor 2 Suggests Two Types of Structural Transitions

During initiation of messenger RNA translation in bacteria, the GTPase initiation factor (IF) 2 plays major roles in the assembly of the preinitiation 30S complex and its docking to the 50S ribosomal subunit leading to the 70S initiation complex, ready to form the first peptide bond in a nascent protein. Rapid and accurate initiation of bacterial protein synthesis is driven by conformational changes in IF2, induced by GDP-GTP exchange and GTP hydrolysis. We have used isothermal titration calorimetry and linear extrapolation to characterize the thermodynamics of the binding of GDP and GTP to free IF2 in the temperature interval 4-37 °C. IF2 binds with about 20-fold and 2-fold higher affinity for GDP than for GTP at 4 and 37 °C, respectively. The binding of IF2 to both GTP and GDP is characterized by a large heat capacity change (− 868 ± 25 and − 577 ± 23 cal mol^− 1 K^− 1, respectively), associated with compensatory changes in binding entropy and enthalpy. From our data, we propose that GTP binding to IF2 leads to protection of hydrophobic amino acid residues from solvent by the locking of switch I and switch II loops to the γ-phosphate of GTP, as in the case of elongation factor G. From the large heat capacity change (also upon GDP binding) not seen in the case of elongation factor G, we propose the existence of yet another type of conformational change in IF2, which is induced by GDP and GTP alike. Also, this transition is likely to protect hydrophobic groups from solvent, and its functional relevance is discussed.     

Altering the GTP binding site of the DNA/RNA-binding protein, Translin/TB-RBP, decreases RNA binding and may create a dominant negative phenotype

The DNA/RNA-binding protein, Translin/Testis Brain RNA-binding protein (Translin/TB-RBP), contains a putative GTP binding site in its C-terminus which is highly conserved. To determine if guanine nucleotide binding to this site functionally alters nucleic acid binding, electrophoretic mobility shift assays were performed with RNA and DNA binding probes. GTP, but not GDP, reduces RNA binding by ~50% and the poorly hydrolyzed GTP analog, GTPγS, reduces binding by >90% in gel shift and immunoprecipitation assays. No similar reduction of DNA binding is seen. When the putative GTP binding site of TB-RBP, amino acid sequence VTAGD, is altered to VTNSD by site directed mutagenesis, GTP will no longer bind to TB-RBP_GTP and TB-RBP_GTP no longer binds to RNA, although DNA binding is not affected. Yeast two-hybrid assays reveal that like wild-type TB-RBP, TB-RBP_GTP will interact with itself, with wild-type TB-RBP and with Translin associated factor X (Trax). Transfection of TB-RBP_GTP into NIH 3T3 cells leads to a marked increase in cell death suggesting a dominant negative function for TB-RBP_GTP in cells. These data suggest TB-RBP is an RNA-binding protein whose activity is allosterically controlled by nucleotide binding.     

Guanine nucleotides in protein synthesis. Utilization of pppGpp and dGTP by initiation factor 2 and elongation factor Tu

Guanosine 5′-triphosphate, 3′-diphosphate (pppGpp), and dGTP support the initiation factor 2 (IF-2) and elongation factor Tu (EF-Tu) partial reactions of Escherichia coli protein synthesis. These natural analogs of GTP were as effective as GTP in supporting (1) IF-2-dependent binding of f-Met-tRNA to ribosomes, (2) IF-2-dependent formation of N-formylmethionylpuromycin, (3) EF-Tu-dependent binding of Phe-tRNA to a ribosome-polyuridylic acid-N-acetyl-Phe-tRNA complex, and (4) EF-Tu-dependent formation of N-acetyl-Phe-Phe-tRNA. GTP, pppGpp, and dGTP behaved similarly in time-course studies and across a broad concentration range with both enzymes. In addition, both GDP and guanosine 5′-diphosphate, 3′-diphosphate were found to be competitive inhibitors of both GTP and pppGpp in the IF-2- and EF-Tu-dependent reactions.     

Alanine scan mutagenesis of the switch I domain of the Caulobacter crescentus CgtA protein reveals critical amino acids required for in vivo function

The Caulobacter crescentus CgtA protein is a member of the Obg/GTP1 subfamily of monomeric GTP-binding proteins. In vitro, CgtA displays moderate affinity for both GDP and GTP and displays rapid exchange rate constants for either nucleotide, indicating that the guanine nucleotide-binding and exchange properties of CgtA are different from those of the well-characterized Ras-like GTP-binding proteins. The Obg/GTP1 proteins share sequence similarity along the putative effector-binding domain. In this study, we examined the functional consequences of altering amino acid residues within this conserved domain, and identified that T193 was critical for CgtA function. The in vitro binding, exchange and GTP hydrolysis of the T192A, T193A and T192AT193A mutant proteins was examined using fluorescent guanine nucleotide analogues (mant-GDP and mant-GTP). Substitution of either T192 and/or T193 for alanine modestly reduced binding to GDP and significantly reduced the binding affinity for GTP. Furthermore, the T193A mutant protein was more severely impaired for binding GTP than the T192A mutant. The T193A mutation appeared to account solely for the impaired GTP binding of the T192AT193A double mutation. This is the first report that demonstrates that a confirmed defect in guanine nucleotide binding and GTP hydrolysis of an Obg-like protein results in the lack of function in vivo.     

Properties of auxin binding sites in different subcellular fractions from maize coleoptiles

In-vitro binding of labeled auxins to sedimentable particles was tested in subcellular fractions from homogenates of maize (Zea mays L.) coleoptiles. The material was fractionated by differential centrifugation or on sucrose density gradients. It was confirmed that the major saturable binding activity (site I) for 1-naphthyl[1-¹⁴C]acetic acid is associated with vesicles derived from the endoplasmatic reticulum. A second type of specific auxin binding (site II) could be distinguished by several criteria, e.g. by the low affinity towards phenylacetic acid. The particles carrying site II could be clearly separated from markers of the endoplasmatic reticulum, the plasmalemma, the mitochondria and the nuclei, while their density as well as sedimentation velocity correlated with particle-bound acid phosphatase, indicating a localization at the tonoplast. In contrast to site I, binding at site II was hardly affected by a supernatant factor and by sulfhydryl groups. However, the specificity pattern of site II towards auxins and auxin analogs was very similar to that of site I tested in the presence of supernatant factor. The existence of a third auxin receptor localized in plasma membrane-rich gradient fractions was indicated by a preferential in-vitro binding of 2,4-dichlorophenoxyacetic acid.Abbreviations 1-NAA 1-naphthyl acetic acid - 2-NAA 2-naphthyl acetic acid - IAA 3-indolyl acetic acid - PAA phenyl acetic acid - 2,4-D 2,4-D-dichlorophenoxy acetic acid - D-2,4-DP dichlorophenoxy isopropionic acid - NPA 1-N-naphthyl phthalamic acid - ER endoplasmatic reticulum - SF supernatant factor     

Molecular characterization of novel immunodominant molybdenum cofactor biosynthesis protein C1 (Rv3111) from Mycobacterium tuberculosis H37Rv

BackgroundIn the molybdenum cofactor biosynthesis pathway, MoaA and MoaC catalyze the first step of transformation of GTP to cPMP. In M. tuberculosis H37Rv, three different genes (Rv3111, Rv0864 and Rv3324c) encode for MoaC homologs. Out of these three only MoaC1 (Rv3111) is secretory in nature.MethodsWe have characterized MoaC1 protein through biophysical, in-silico, and immunological techniques.ResultsWe have characterized the conformation and thermodynamic stability of MoaC1, and have established its secretory nature by demonstrating the presence of anti-MoaC1 antibodies in human tuberculosis patients' sera. Further, MoaC1 elicited a dominant Th1 immune response in mice characterized by increased induction of IL-2 and IFN-γ.ConclusionIntegrating these results, we conclude that MoaC1 is a structured secretory protein capable of binding with GTP and eliciting induced immune response.General significanceThis study would be useful for the development of vaccines against tuberculosis and to improve methods used for diagnosis of tuberculosis.     

Initiation of mammalian protein synthesis. II. The assembly of the initiation complex with purified initiation factors

The assembly of initiation complexes is studied in a protein synthesis initiation assay containing ribosomal subunits, globin [¹²⁵I]mRNA, [³H]Met-tRNA_f, seven purified initiation factors, ATP and GTP. By omitting single components from the initiation assay, specific roles of the initiation factors, ATP and GTP are demonstrated. The initiation factor eIF-2 is required for the binding of Met-tRNA_f to the 40 S ribosomal subunit. The initial Met-tRNA_f binding to the small ribosomal subunit is a stringent prerequisite for the subsequent mRNA binding. The initiation factors eIF-3, eIF-4A, eIF-4B and eIF-4C together with ATP promote the binding of mRNA to the 40 S initiation complex. The association of the 40 S initiation complex with the 60 S ribosome subunit to form an 80 S initiation complex is mediated by the initiation factor eIF-5 and requires the hydrolysis of GTP. The factor eIF-1 gives a twofold overall stimulation of initiation complex formation. A model of the sequential steps in the assembly of the 80 S initiation complex in mammalian protein synthesis is presented.