Presence Of Guanosine Triphosphate Research Articles

The Impact of Terminal Peptide Extensions of Retinal Inosine 5´Monophosphate Dehydrogenase 1 Isoforms on their DNA-binding Activities.

The main structural difference between the mutation-susceptible retinal isoforms of inosine 5´-monophosphate dehydrogenase-1 (IMPDH-1) with the canonical form resides in the C- and N-terminal peptide extensions with unknown structural/functional impacts. In this report, we aimed to experimentally evaluate the functional impact of these extensions on the specific/non-specific single-stranded DNA (ssDNA)-binding activities relative to those of the canonical form. Our in silico findings indicated the possible contribution of the C-terminal segment to the reduced flexibility of the Bateman domain of the enzyme. In addition, the in silico data indicated that the N-terminal tail acts by altering the distance between the tetramers in the concave octamer complex (the native form) of the enzyme. The overall impact of these predicted structural variations became evident, first, through higher Km values with respect to either of the substrates relative to the canonical isoform, as reported previously (Andashti et al. in Mol Cell Biochem 465(1):155-164, 2020). Secondary, the binding of the recombinant mouse retinal isoform IMPDH1 (603) to its specific Rhodopsin target gene was significantly augmented while its binding to non-specific ssDNA was lower than that of the canonical isoform. The DNA-binding activity of the other mouse retinal isoform, IMPDH1(546), to specific and non-specific ssDNA was lower than that of the canonical form most probably due to the in silico predicted rigidity created in the Bateman domain by the C-terminal peptide extension. Furthermore, the DNA binding to the Rhodopsin target gene by each of the IMPDH isoforms influenced in the presence of GTP (Guanosine triphosphate) and ATP (Adenosine triphosphate).

Thiouronium Salt Derivatives Based on Vicinal Diamines as Potential Neuroprotectors

Most of the medicinal products that are currently approved and used in clinical practice for neurodegenerative diseases, in particular Alzheimer’s disease, have a compensatory mechanism of action that enhances neurotransmitter signalling. It is an urgent need to develop new medicinal products combining cognitive-enhancing, neuroprotective, and disease-specific effects resulting from a multi-target mechanism of action including, in particular, prevention of glutamate-induced neuronal calcium uptake and stabilisation of microtubules.The aim of this study was to search for potentially neuroprotective and tauopathy-alleviating medicines amongst new thiouronium salt derivatives based on vicinal diamines.Materials and methods. The study investigated the ability of thiouronium salts to block glutamate-induced 45Ca2+ uptake by synaptosomes prepared from the brain of Wistar rats. The authors evaluated effects of these new compounds on polymerisation of a preparation of C57bl mouse brain tubulin and microtubule-associated proteins. The evaluation was carried out in the presence of guanosine triphosphate (GTP) and based on specific absorbance changes at 355 nm due to formation of microtubules. The authors analysed the structure of these microtubules, using negative staining followed by transmission electron microscopy. The IC50 determination and the statistical analysis were performed using standard software (Excel and PRISM 6.02).Results. The authors developed a screening algorithm for a number of new thiouronium salt derivatives based on vicinal diamines and studied biological activity of these derivatives by the effects on glutamate-induced calcium uptake by synaptosomes and on microtubule assembly processes. The authors identified compounds suppressing glutamate-induced calcium uptake by synaptosomes, i.e. compounds with neuroprotective potential. In addition, a number of new compounds were able to stimulate GTP-dependent microtubule assembly processes. The authors observed formation of microtubules with a normal structure in the presence of isopropyl-N’-[2-(benzoylamino)-1,2-diphenylethyl]-N-ethylimidothiocarbamate hydrobromide and considered the compound a promising scaffold for further optimisation.Conclusions. Chemical modification of thiouronium salts is a promising direction for developing effective neuroprotectors and microtubule stabilisers.

Cyclic Tau-derived peptides for stabilization of microtubules

The cyclization of peptides is a valuable strategy for the development of binding motifs to target proteins with improved affinity. Microtubules (MTs) are important targets for therapeutics, and a variety of MT-targeted drugs and peptides have recently been developed. We have previously designed a Tau-derived peptide (TP) that binds to the interior of MTs. In the present study, the development of a cyclic TP (TCP) for enhanced binding to tubulin and the stabilization of MTs are described. The fluorescently labeled cyclic peptide containing three glycine linkers (TCP3-TMR) exhibited a remarkably enhanced binding affinity to tubulin. The cyclic peptide was also demonstrated to stabilize MTs by enhancing polymerization and reducing depolymerization. Moreover, MTs were effectively formed by the treatment of cyclic peptides in the presence of guanosine triphosphate (GTP), while the linear peptide showed no such effect. These findings indicate that TCP is a useful binding motif that can stabilize MTs and is valuable for various therapeutic and material applications. Microtubules (MTs) consisting of tubulins are important targets of drugs for MT-related diseases. We have previously designed a linear Tau-derived peptide (TP) that binds to the interior of MTs. In this article, a cyclic TP (TCP) was developed for enhanced binding to tubulin and stabilization of MTs. The fluorescently labeled TCP exhibited a remarkably enhanced binding affinity to tubulin compared to the linear TP. The stabilization of MTs by binding of TCP was demonstrated, such as formation of typically unstable MTs in the presence of guanosine triphosphate.

Surface Orientation and Binding Strength Modulate Shape of FtsZ on Lipid Surfaces.

We have used a simple model system to test the prediction that surface attachment strength of filaments presenting a torsion would affect their shape and properties. FtsZ from E. coli containing one cysteine in position 2 was covalently attached to a lipid bilayer containing maleimide lipids either in their head group (to simulate tight attachment) or at the end of a polyethylene glycol molecule attached to the head group (to simulate loose binding). We found that filaments tightly attached grew straight, growing from both ends, until they formed a two-dimensional lattice. Further monomer additions to their sides generated a dense layer of oriented filaments that fully covered the lipid membrane. After this point the surface became unstable and the bilayer detached from the surface. Filaments with a loose binding were initially curved and later evolved into straight thicker bundles that destabilized the membrane after reaching a certain surface density. Previously described theoretical models of FtsZ filament assembly on surfaces that include lateral interactions, spontaneous curvature, torsion, anchoring to the membrane, relative geometry of the surface and the filament ‘living-polymer’ condition in the presence of guanosine triphosphate (GTP) can offer some clues about the driving forces inducing these filament rearrangements.

K-Ras Populates Conformational States Differently from Its Isoform H-Ras and Oncogenic Mutant K-RasG12D

Structures of wild-type K-Ras from crystals obtained in the presence of guanosine triphosphate (GTP) or its analogs have remained elusive. Of the K-Ras mutants, only K-RasG12D and K-RasQ61H are available in the PDB representing the activated form of the GTPase not in complex with other proteins. We present the crystal structure of wild-type K-Ras bound to the GTP analog GppCH2p, with K-Ras in the state1 conformation. Signatures of conformational states obtained by one-dimensional proton NMR confirm that K-Ras has a more substantial population of state1 in solution than H-Ras, which predominantly favors state 2. The oncogenic mutant K-RasG12D favors state 2, changing the balance of conformational states in favor of interactions with effector proteins. Differences in the population of conformational states between K-Ras and H-Ras, as well as between K-Ras and its mutants, can provide a structural basis for focused targeting of the K-Ras isoform in cancer-specific strategies.

Evaluation of Cytotoxicity and Hypoxic Effect of Nitroimidazole Embedded Nanoparticles.

Adenylate cyclase is a key intracellular enzyme involved in energy imbalance leading to tumor hypoxia and cytotoxicity. In this study, adenylate cyclase activities in isolated hepatocytes and Kupffer cells were compared in the presence of several metabolic stimulators. In cultured hepatocyte cells, adenylate cyclase was stimulated by guanylyl imidotriphosphate (GITP), guanosine triphosphate (GTP), progesterone and nitroimidazole embedded nanoparticle (NNP) effectors, while prostaglandin E2 and F2α were used as effectors in cultured Kupffer cells. The results showed that NNPs decreased adenylate cyclase specific activity in a dose-dependent manner after preincubation of hepatocytes with NNPs. The NNPs stimulated adenylate cyclase activities in hepatocytes were evaluated based on measurement of cyclic adenosine monophosphate (cAMP). The stimulatory effects of NNPs on adenylate cyclase were independent of the presence of GTP and may have been due to a direct effect on the catalytic subunit of adenylate cyclase. In addition, basal cAMP generation in hepatocyte cells was efficiently suppressed by the NNPs. In conclusion, NNPs exerted direct effects on the catalytic subunit of the adenylate cyclase system, and adenylate cyclase was hormone sensitive in liver cells.

Single-molecule observations of RNA-RNA kissing interactions in a DNA nanostructure.

RNA molecules uniquely form a complex through specific hairpin loops, called a kissing complex. The kissing complex is widely investigated and used for the construction of RNA nanostructures. Molecular switches have also been created by combining a kissing loop and a ligand-binding aptamer to control the interactions of RNA molecules. In this study, we incorporated two kinds of RNA molecules into a DNA origami structure and used atomic force microscopy to observe their ligand-responsive interactions at the single-molecule level. We used a designed RNA aptamer called GTPswitch, which has a guanosine triphosphate (GTP) responsive domain and can bind to the target RNA hairpin named Aptakiss in the presence of GTP. We observed shape changes of the DNA/RNA strands in the DNA origami, which are induced by the GTPswitch, into two different shapes in the absence and presence of GTP, respectively. We also found that the switching function in the nanospace could be improved by using a cover strand over the kissing loop of the GTPswitch or by deleting one base from this kissing loop. These newly designed ligand-responsive aptamers can be used for the controlled assembly of the various DNA and RNA nanostructures.

Novel effect of 2-aminoethoxydiphenylborate through inhibition of calcium sensitization induced by Rho kinase activation in human detrusor smooth muscle

Since the introduction of 2-aminoethoxydiphenylborate (2-APB) as a membrane permeable modulator of inositol (1,4,5)-trisphosphate receptors, subsequent studies have revealed additional actions of this chemical on multiple Ca2+-permeable ionic channels in the plasma membrane. However, no reports have yet examined 2-APB as a modulator targeting contractile machinery in smooth muscle, independent of Ca2+ mobilization, namely Ca2+ sensitization. Here, we assessed whether or not 2-APB affects intracellular signaling pathways of Ca2+ sensitization for contraction using α-toxin permeabilized human detrusor smooth muscle. Although contractions were induced by application of Ca2+-containing bath solutions, 2-APB had little effect on contractions induced by 1µM Ca2+ alone but significantly reversed the carbachol-induced augmentation of Ca2+-induced contraction in the presence of guanosine triphosphate (carbachol-induced Ca2+ sensitization). The rho kinase inhibitor Y-27632 and protein kinase C inhibitor GF-109203X also reversed the carbachol-mediated Ca2+ sensitization. Additional application of 2-APB caused a small but significant further attenuation of the contraction in the presence of GF-109203X but not in the presence of Y-27632. Like carbachol, the rho kinase activator; sphingosylphosphorylcholine, protein kinase C activator; phorbol 12,13 dibutyrate, and myosin light chain phosphatase inhibitor; calyculin-A all induced Ca2+ sensitization. However, the inhibitory activity of 2-APB was limited with sphingosylphosphorylcholine-induced Ca2+ sensitization. This study revealed a novel inhibitory effect of 2-APB on smooth muscle contractility through inhibition of the rho kinase pathway.

Luciferase-Based Assay for Adenosine: Application to S-Adenosyl-l-homocysteine Hydrolase

S-Adenosyl-L-homocysteine hydrolase (SAHH) catalyzes the reversible conversion of S-adenosyl-L-homocysteine (SAH) to adenosine (ADO) and L-homocysteine, promoting methyltransferase activity by relief of SAH inhibition. SAH catabolism is linked to S-adenosylmethionine metabolism, and the development of SAHH inhibitors is of interest for new therapeutics with anticancer or cholesterol-lowering effects. We have developed a continuous enzymatic assay for adenosine that facilitates high-throughput analysis of SAHH. This luciferase-based assay is 4000-fold more sensitive than former detection methods and is well suited for continuous monitoring of ADO formation in a 96-well-plate format. The high-affinity adenosine kinase from Anopheles gambiae efficiently converts adenosine to adenosine monophosphate (AMP) in the presence of guanosine triphosphate. AMP is converted to adenosine triphosphate and coupled to firefly luciferase. With this procedure, kinetic parameters (K(m), k(cat)) for SAHH were obtained, in good agreement with literature values. Assay characteristics include sustained light output combined with ultrasensitive detection (10(-7) unit of SAHH). The assay is documented with the characterization of slow-onset inhibition for inhibitors of the hydrolase. Application of this assay may facilitate the development of SAHH inhibitors and provide an ultrasensitive detection for the formation of adenosine from other biological reactions.

Fluorescence detection of adenosine triphosphate through an aptamer–molecular beacon multiple probe

An aptamer–molecular beacon (MB) multiple fluorescent probe for adenosine triphosphate (ATP) assay is proposed in this article. The ATP aptamer was used as a molecular recognition part, and an oligonucleotide (short strand, SS) partially complementary with the aptamer and an MB was used as the other part. In the presence of ATP, the aptamer bound with it, accompanied by the hybridization of MB and SS and the fluorescence recovering. Wherever there is only very weak fluorescence can be measured in the absence of ATP. Based on the relationship of recovering fluorescence and the concentration of ATP, a method for quantifying ATP has been developed. The fluorescence intensity was proportional to the concentration of ATP in the range of 10 to 500nM with a detection limit of 0.1nM. Moreover, this method was able to detect ATP with high selectivity in the presence of guanosine triphosphate (GTP), cytidine triphosphate (CTP), and uridine triphosphate (UTP). This method is proved to be simple with high sensitivity, selectivity, and specificity.

The pH Dependence of Polymerization and Bundling by the Essential Bacterial Cytoskeltal Protein FtsZ

There is a growing body of evidence that bacterial cell division is an intricate coordinated process of comparable complexity to that seen in eukaryotic cells. The dynamic assembly of Escherichia coli FtsZ in the presence of GTP is fundamental to its activity. FtsZ polymerization is a very attractive target for novel antibiotics given its fundamental and universal function. In this study our aim was to understand further the GTP-dependent FtsZ polymerization mechanism and our main focus is on the pH dependence of its behaviour. A key feature of this work is the use of linear dichroism (LD) to follow the polymerization of FtsZ monomers into polymeric structures. LD is the differential absorption of light polarized parallel and perpendicular to an orientation direction (in this case that provided by shear flow). It thus readily distinguishes between FtsZ polymers and monomers. It also distinguishes FtsZ polymers and less well-defined aggregates, which light scattering methodologies do not. The polymerization of FtsZ over a range of pHs was studied by right-angled light scattering to probe mass of FtsZ structures, LD to probe real-time formation of linear polymeric fibres, a specially developed phosphate release assay to relate guanosine triphosphate (GTP) hydrolysis to polymer formation, and electron microscopy (EM) imaging of reaction products as a function of time and pH. We have found that lowering the pH from neutral to 6.5 does not change the nature of the FtsZ polymers in solution—it simply facilitates the polymerization so the fibres present are longer and more abundant. Conversely, lowering the pH to 6.0 has much the same effect as introducing divalent cations or the FtsZ-associated protein YgfE (a putative ZapA orthologue in E. coli)—it stablizes associations of protofilaments.

An Allosteric Self-Splicing Ribozyme Triggered by a Bacterial Second Messenger

Group I self-splicing ribozymes commonly function as components of selfish mobile genetic elements. We identified an allosteric group I ribozyme, wherein self-splicing is regulated by a distinct riboswitch class that senses the bacterial second messenger c-di-GMP. The tandem RNA sensory system resides in the 5' untranslated region of the messenger RNA for a putative virulence gene in the pathogenic bacterium Clostridium difficile. c-di-GMP binding by the riboswitch induces folding changes at atypical splice site junctions to modulate alternative RNA processing. Our findings indicate that some self-splicing ribozymes are not selfish elements but are harnessed by cells as metabolite sensors and genetic regulators.

Involvement of β-adrenergic receptor in synaptic vesicle swelling and implication in neurotransmitter release

Secretory vesicle swelling is required for vesicular discharge during cell secretion. The Gαo-mediated water channel aquaporin-6 (AQP-6) involvement in synaptic vesicle (SV) swelling in neurons has previously been reported. Studies demonstrate that in the presence of guanosine triphosphate (GTP), mastoparan, an amphiphilic tetradecapeptide from wasp venom, activates Go protein GTPase, and stimulates SV swelling. Stimulation of G proteins is believed to occur via insertion of mastoparan into the phospholipid membrane to form a highly structured α-helix that resembles the intracellular loops of G protein-coupled adrenergic receptors. Consequently, the presence of adrenoceptors and the presence of an endogenous β-adrenergic agonist at the SV membrane is suggested. Immunoblot analysis of SV using β-adrenergic receptor antibody, and vesicle swelling experiments using β-adrenergic agonists and antagonists, demonstrate the presence of functional β-adrenergic receptors at the SV membrane. Since a recent study shows vH+-ATPase to be upstream of AQP-6 in the pathway leading from Gαo-mediated swelling of SV, participation of an endogenous β-adrenergic agonist, in the binding and stimulation of its receptor to initiate the swelling cascade is demonstrated.

Hydroxyl radical oxidation of guanosine 5′-triphosphate (GTP): requirement for a GTP–Cu(II) complex

Levels of oxidized guanosine base in DNA have become a hallmark biomarker in assessing oxidative stress implicated in a variety of disease and toxin-induced states. However, there is evidence that the guanosine in the nucleotide triphosphate pool (GTP) is more susceptible to oxidation than guanosine residues incorporated into nucleic acids and this causes a substantial amount of the oxidized product, 8-oxoguanosine 5′-triphosphate (oxo8GTP), to accumulate in cell-free and in cell-culture preparations. Electron paramagnetic resonance (EPR) spectroscopy and direct EPR analysis of free radical production by copper sulfate and L-ascorbic acid demonstrates that the hydroxyl radical (HO•) is produced via oxidation of Cu+ to Cu2+ while in a complex with GTP. This HO• production is dependent on the availability of oxygen and the presence of GTP in the reaction milieu. Verification of free radical-mediated production of oxo8GTP is presented using HPLC with electrochemical detection and matrix-assisted laser desorption/ionization linear time-of-flight mass spectrometry (MALDI-LTOF-MS). The sum of these results is presented in a novel mechanism of GTP oxidation by Cu2+ and L-ascorbic acid. A better understanding of the chemistry involved in this oxidative modification of GTP facilitates a more comprehensive understanding of its potential physiological consequences.

Semisynthesis of H‐Ras with a glutamic acid methylester at position 61

The mechanism of the guanosine triphosphate (GTP) hydrolysis reaction of small G-proteins such as Ras is generally understood; however, some important molecular details are still missing. One example concerns the role of Gln61 in the catalysis of the GTP hydrolysis reaction. This amino acid is frequently mutated in oncogenic Ras leading to constitutively active variants of the protein. To elucidate the role of Gln61, subtle structural changes were introduced at this position by exchanging the natural occurring glutamine against a glutamic acid methyl ester (GluOme). Thereby the H-bond donor properties of this residue are changed and analysis of the GTP hydrolysis reaction can provide information on the function of the native carboxamide moiety. Using a semisynthetic approach, Ras(1-166)Gln61GluOMe was synthesized by sequential native chemical ligation of three unprotected peptide segments. Peptides Ras(1-50) and Ras(51-79)Gln61GluOMe were synthesized using Boc chemistry. The C-terminal peptide Ras(80-166) was expressed in E. coli. Initial tests of this semisynthetic strategy were performed by synthesis of the N- and C-terminally truncated protein variant Ras(39-101)Gln61GluOMe. The identified optimal reaction conditions were then applied to the synthesis of Ras(1-166)Gln61GluOMe. Refolding of the semisynthetic product in the presence of GTP was successful and revealed intrinsic GTPase activity of Ras(1-166)Gln61GluOMe.

A minor role for Ca2+ sensitization in sustained contraction through activation of muscarinic receptor in circular muscle of rat distal colon

We previously demonstrated that Ca(2+) sensitization has an essential role for carbachol-induced contraction in the longitudinal muscle of the rat distal colon. In the present study, we extended these studies to clarify the role of Ca(2+) sensitization in contraction induced by the activation of muscarinic receptors in the circular muscle of the rat distal colon. Carbachol induced a rapid phasic contraction followed by a sustained contraction that was significantly lower than the phasic and was superimposed with the rhythmic contractions. The extent of increase in intracellular Ca(2+) concentration that was measured simultaneously with tension recording was dissociated from the phasic contraction, whereas it exhibited to a similar extent as sustained contraction. In alpha-toxin-permeabilized preparations, Ca(2+) induced contraction comprising a rapid phasic and a subsequent low sustained component. After Ca(2+)-induced sustained contraction reached a constant level, guanosine triphosphate (GTP) addition resulted in the enhancement of contractile force in a concentration-dependent manner. Carbachol in the presence of GTP caused a further minimal increase in tension (Ca(2+) sensitization). Chelerythrine, a protein kinase C (PKC) inhibitor, inhibited carbachol-induced Ca(2+) sensitization but not GTP-induced Ca(2+) sensitization. In contrast, Y-27632, a Rho kinase inhibitor, inhibited GTP-induced Ca(2+) sensitization but not that induced by carbachol. Phorbol 12,13-dibutyrate, a PKC activator, increased the sustained contraction. These results suggest that the activation of muscarinic receptor with carbachol induces Ca(2+) sensitization via activation of PKC, but this action is minor in the circular muscle of the rat distal colon as a result of limited coupling between muscarinic receptors and Ca(2+) sensitization via the PKC pathway.

The roles of initiation factor 2 and guanosine triphosphate in initiation of protein synthesis.

The role of IF2 from Escherichia coli was studied in vitro using a system for protein synthesis with purified components. Stopped flow experiments with light scattering show that IF2 in complex with guanosine triphosphate (GTP) or a non-cleavable GTP analogue (GDPNP), but not with guanosine diphosphate (GDP), promotes fast association of ribosomal subunits during initiation. Biochemical experiments show that IF2 promotes fast formation of the first peptide bond in the presence of GTP, but not GDPNP or GDP, and that IF2-GDPNP binds strongly to post-initiation ribosomes. We conclude that the GTP form of IF2 accelerates formation of the 70S ribosome from subunits and that GTP hydrolysis accelerates release of IF2 from the 70S ribosome. The results of a recent report, suggesting that GTP and GDP promote initiation equally fast, have been addressed. Our data, indicating that eIF5B and IF2 have similar functions, are used to rationalize the phenotypes of GTPase-deficient mutants of eIF5B and IF2.

Activation of NADPH Oxidase without GTP Exchange

The NADPH oxidase complex [gp91 phox , p22 phox , p47 phox , p40 phox , and the guanosine triphosphatase (GTPase) Rac] is responsible for the production of superoxide radicals in phagocytes during killing of engulfed microorganisms. The cytochrome b 559 part of the complex consisting of gp91 phox and p22 phox is activated by the cytosolic factors p47 phox , p40 phox , and Rac. This activation can be achieved in vitro with phagocyte membranes supplemented only with these cytosolic factors in the presence of an anioinic amphiphile. Prenylated Rac can substitute for the amphiphile and for p47 phox in the in vitro system. Sigal et al. reconstituted the NADPH complex in vitro and determined that prenylated Rac1 bound to guanosine diphosphate (GDP) was able to stimulate oxidase activity in the presence of the functional fragment of the guanine exchange factor (GEF) Trio. However, the stimulation did not require the presence of guanosine triphosphate (GTP) or guanine exchange and the presence of nonhydrolyzable GTP analogs prevented Trio stimulation of Rac-mediated oxidase activation. Using mutants of Rac, the authors determined that Trio binding was required and that a conformational change associated with guanine exchange was required for stimulation of oxidase activity by prenylated Rac. Thus, the authors propose that Trio stimulates a conformational change, but not guanine nucleotide exchange, in Rac that alters Rac's affinity for the NADPH oxidase complex components allowing Rac to recruit the cytosolic factors to the catalytic subunits of the NADPH complex. N. Sigal, Y. Gorzalczany, R. Sarfstein, C. Weinbaum, Y. Zheng, E. Pick, The guanine nucleotide exchange factor Trio activates the phagocyte NADPH oxidase in the absence of GDP to GTP exchange on Rac. J. Biol. Chem. 278 , 4854-4861 (2003). [Abstract] [Full Text]

Ligand-Induced Conformational Changes in Tissue Transglutaminase: Monte Carlo Analysis of Small-Angle Scattering Data

Small-angle neutron and x-ray scattering experiments have been performed on type 2 tissular transglutaminase to characterize the conformational changes that bring about Ca 2+ activation and guanosine triphosphate (GTP) inhibition. The native and a proteolyzed form of the enzyme, in the presence and in the absence of the two effectors, were considered. To describe the shape of transglutaminase in the different conformations, a Monte Carlo method for calculating small-angle neutron scattering profiles was developed by taking into account the computer-designed structure of the native transglutaminase, the results of the Guinier analysis, and the essential role played by the solvent-exposed peptide loop for the conformational changes of the protein after activation. Although the range of the neutron scattering data is rather limited, by using the Monte Carlo analysis, and because the structure of the native protein is available, the distribution of the protein conformations after ligand interaction was obtained. Calcium activation promotes a rotation of the C-terminal with respect to the N-terminal domain around the solvent-exposed peptide loop that connects the two regions. The ψ angle between the longest axes of the two pairs of domains is found to be above 50°, larger than the ψ value of 35° calculated for the native transglutaminase. On the other hand, the addition of GTP makes possible conformations characterized by ψ angles lower than 34°. These results are in good agreement with the proposed enzyme activity regulation: in the presence of GTP, the catalytic site is shielded by the more compact protein structure, while the conformational changes induced by Ca 2+ make the active site accessible to the substrate.

GTP-dependent permeabilized neutrophil secretion requires a freely diffusible cytosolic protein.

Guanosine triphosphate (GTP) has been implicated in the regulation of Ca(2+)-mediated secretion from neutrophils. We further examined the role of GTP in neutrophil secretion using streptolysin O permeabilized cells. We found that, in the presence of GTP, 1.0 microM free Ca(2+) causes maximum secretion-equivalent to that achieved with 100 microM free Ca(2+)-whereas GTPgammaS inhibits Ca(2+)-stimulated secretion. Interestingly, GTP by itself stimulates secretion. These results indicate the existence of a GTP-regulated mechanism of secretion in neutrophils that requires GTP hydrolysis to stimulate secretion in the presence and absence of Ca(2+). The stimulatory effect of GTP is only observed when GTP is present during permeabilization. Addition of GTP after permeabilization, when the cytosolic contents have leaked out from cells, gives no stimulatory response, implying that the GTP-dependent secretory apparatus requires at least one cytosolic protein. GTP-dependent secretion can be reconstituted with crude HL-60 and bovine liver cytosol. The reconstituting activity binds to GTP-agarose, suggesting that the cytosolic factor is a GTP-binding protein or forms a complex with a GTP-binding protein. However, it is not a member of the rho or rac families of GTPases. By gel filtration chromatography, the secretion-reconstituting activity eluted at 870 and 200 kDa, but in the presence of GTP, eluted at 120 kDa, indicating that it is part of a high-molecular-weight complex that dissociates in the presence of GTP. Retention of adenosine diphosphate-ribosylation factor (ARF) in permeabilized cells and insensitivity of the cytosolic reconstituting activity to brefeldin A led to our speculation that ARF6 may be the GTPase involved in GTP-dependent secretion, and that activity from a BFA-insensitive ARF6 guanine nucleotide exchange factor reconstitutes secretion.