Diseño de un espacio de acogida como estrategia de cuidados avanzados en enfermería en un servicio de pediatría / Designing a hosting area as an advanced nursing care strategy in a pediatric nursing ward

Antecedentes: las necesidades de confort más relevantes para los cuidadores tienen relación con mejorar las condiciones de permanencia, a través de salas de espera más cómodas, un lugar donde dormir, un baño con ducha, alimentación para el acompañante y un lugar seguro para guardar sus pertenencias. Objetivo: implementar un modelo de cuidado a través de un espacio de acogida, para satisfacer las necesidades de confort de los padres que no cuentan con alojamiento durante la hospitalización de sus hijos. Metodología: estrategia innovadora en la entrega de cuidados avanzados en enfermería, basados en el modelo teórico de Kolcaba el que nos permite llevar la disciplina de enfermería a la práctica asistencial en directo beneficio de los pacientes. Resultados: con la implementación de un espacio de acogida, se espera un aumento en la percepción de confort de los padres durante hospitalización, lo que conllevará a un incremento en el estado de alerta de los padres, participación efectiva en los cuidados de su hijo, mejoría en la respuesta frente a las intervenciones educativas y vinculación efectiva con el equipo de salud. Conclusiones: un espacio de acogida para padres de niños hospitalizados, diseñada para satisfacer las necesidades de confort más relevantes identificadas en la literatura y contextualizadas en la teoría de Kolcaba, contribuiría a implementar el cuidado avanzado en enfermería, rescatando la esencia de la enfermería en el Servicio de Pediatría del Hospital Clínico de la Universidad Católica.

Background: The most important comfort needs for caregivers are related to improving their hospital stay conditions, with more comfortable waiting rooms, a place to sleep, a toilette with shower, food and a safe place where to leave their belongings. Objective: To implement a hosting area that meets the comfort needs of parents who do not have their own accommodations during the hospitalization of their children. Methodology: Innovative strategies in providing advanced nursing care based on the theoretical model of Kolcaba, which allows us to bring the discipline of nursing care into practice, in direct benefit of patients. Results: We expect an increase in comfort perception of parents during hospitalization, which will lead to increased alertness of parents, effective participation in child care, improved response to educational interventions and effective linkages with the caring team. Conclusions: A hosting area for parents of hospitalized children, designed to meet the most important comfort needs reported in the literature and contextualized according to Kolcaba’s theory, can contribute to implement advanced nursing care, recovering the essence of nursing at the Pediatrics Service of the UC Hospital.

Antibacterial and antitumorigenic properties of microcin E492, a pore-forming bacteriocin.

Microcins are a family of low-molecular weight bacteriocins produced and secreted by Gram-negative bacteria. This review is focused on microcin E492, a pore-forming bacteriocin produced by Klebsiella pneumoniae RYC492 that exerts its antibacterial action on related strains. The steps necessary for the production of active microcin E492 involve post-translational modification with a catechol-type siderophore at the C-terminal and proteolytic processing during export to the extracellular space. This bacteriocin has a modular structure, with a toxic domain at the N-terminal and an uptake domain at the C-terminal of the mature protein. The mechanism by which the C-terminal of microcin E492 is recognized by catecholate siderophore receptors is called the "Trojan horse" strategy, because the C-terminal structure mimics essential bacterial elements, which are recognized by the respective receptors and translocated across the outer membrane to exert antibacterial action. The C-terminal uptake module can be exchanged and used with other toxic domains. Microcin E492 also has a cytotoxic effect on malignant human cell lines. The cytotoxic mechanism is through apoptosis, a desired mechanism for cancer therapy. The ability of microcin E492 to form amyloid-like fibrils constitutes a property that can be exploited in the formulation of this bacteriocin as an antitumoral agent, because these fibrils can behave as stable depots to ensure the sustained release of a biologically active molecule. Alternatively, live bacteria can be used as a continuous source of microcin E492 production in specific tumors.

Structure, organization and characterization of the gene cluster involved in the production of microcin E492, a channel-forming bacteriocin.

Microcin E492 is a low-molecular-weight, channel-forming bacteriocin produced and excreted by Klebsiella pneumoniae RYC492. A 13 kb chromosomal DNA fragment from K. pneumoniae RYC492 was sequenced, and it was demonstrated by random Tn5 mutagenesis that most of this segment, which has at least 10 cistrons, is needed for the production of active microcin and its immunity protein. Genes mceG and mceH correspond to an ABC exporter and its accessory protein, respectively, and they are closely related to the colicin V ABC export system. The microcin E492 system also requires the product of gene mceF as an additional factor for export. Despite the fact that this bacteriocin lacks post-translational modifications, genes mceC, mceI and mceJ are needed for the production of active microcin. Genes mceC and mceI are homologous to a glycosyl transferase and acyltransferase, respectively, whereas mceJ has no known homologue. Mutants in these three genes secrete an inactive form of microcin, able to form ion channels in a phospholipidic bilayer, indicating that the mutation of these microcin genes does not alter the process of membrane insertion. On the other hand, microcin isolated from mutants in genes mceC and mceJ has a lethal effect when incubated with spheroplasts of sensitive cells, indicating that the microcin defects in these mutants are likely to alter receptor recognition at the outer membrane. A model for synthesis and export is proposed as well as a novel maturation pathway that would involve conformational changes to explain the production of active microcin E492.

Rate constants determined by nuclear magnetic resonance.

Fast kinetic methods are used to measure reactions that take place in less time than required to mix the reagents manually and to measure the reaction by usual methods, like UV-visible spectrophotometry and fluorescence. The best known of them are rapid-mixing and relaxation methods, which are used for reactions with half-times in the millisecond and microsecond ranges, respectively. The picosecond range is usually measured with electrical field and ultrasonic waves (A. Cornish-Bowden, 1976, Principles of Enzyme Kinetics, pp. 164-167, Butterworths, London). Normally these very fast rates occur when a ligand binds to or dissociates from a protein. When the binding is mediated only by the diffusion, the lower limit of the association rate constant (k(on)) should not exceed the value of a diffusion-controlled reaction (around 10(10) M(-1) s(-1)). Therefore, the values most frequently found for these rate constants, for example, in the association of a substrate with an enzyme, are in the range 10(6) to 10(9) M(-1) s(-1) (M. Eigen and G. G. Hammes, 1963, Adv. Enzymol. 25, 1-38). The values for the dissociation rate constants (k(off)) for these reactions, which depend on the equilibrium constant for the enzyme-substrate complex interaction, are in the range 10(1) to 10(5) s(-1), most often between 10(3) and 10(4) s(-1) (A. Fersht, 1999, Structure and Mechanism in Protein Science, pp. 164-165, Freeman, New York). If the equilibrium constant is known, and the value of koff is determined by nuclear magnetic resonance (NMR), as described in this chapter, the value of k(on) can be calculated; this should not exceed the value of diffusion rate in the media in which the reaction is performed.

High-affinity binding of fatty acyl-CoAs and peroxisome proliferator-CoA esters to glutathione S-transferases effect on enzymatic activity.

Acyl-CoAs are present at high concentrations within the cell, yet are strongly buffered by specific binding proteins in order to maintain a low intracellular unbound acyl-CoA concentration, compatible with their metabolic role, their importance in cell signaling, and as protection from their detergent properties. This intracellular regulation may be disrupted by nonmetabolizables acyl-CoA esters of xenobiotics, such as peroxisome proliferators, which are formed at relatively high concentration within the liver cell. The low molecular mass acyl-CoA binding protein (ACBP) and fatty acyl-CoA binding protein (FABP) have been proposed as the buffering system for fatty acyl-CoAs. Whether these proteins also bind xenobiotic-CoA is not known. Here we have identified new liver cytosolic fatty acyl-CoA and xenobiotic-CoA binding sites as glutathione S-transferase (GST), using fluorescent polarization and a acyl-etheno-CoA derivative of the peroxisome proliferator nafenopin as ligand. Rat liver GST and human liver recombinant GSTA1-1, GSTP1-1 and GSTM1-1 were used. Only class alpha rat liver GST and human GSTA1-1 bind xenobiotic-CoAs and fatty acyl-CoAs, with Kd values ranging from 200 nM to 5 microM. One mol of acyl-CoA is bound per mol of dimeric enzyme, and no metabolization or hydrolysis was observed. Binding results in strong inhibition of rat liver GST and human recombinant GSTA1-1 (IC50 at the nanomolar level for palmitoyl-CoA) but not GSTP1-1 and GSTM1-1. Acyl-CoAs do not interact with the GSTA1-1 substrate binding site, but probably with a different domain. Results suggest that under increased acyl-CoA concentration, as occurs after exposure to peroxisome proliferators, acyl-CoA binding to the abundant class alpha GSTs may result in strong inhibition of xenobiotic detoxification. Analysis of the binding properties of GSTs and other acyl-CoA binding proteins suggest that under increased acyl-CoA concentration GSTs would be responsible for xenobiotic-CoA binding whereas ACBP would preferentially bind fatty acyl-CoAs.

Helicity of alpha(404-451) and beta(394-445) tubulin C-terminal recombinant peptides.

We have investigated the solution conformation of the functionally relevant C-terminal extremes of alpha- and beta-tubulin, employing the model recombinant peptides RL52alpha3 and RL33beta6, which correspond to the amino acid sequences 404-451(end) and 394-445(end) of the main vertebrate isotypes of alpha- and beta-tubulin, respectively, and synthetic peptides with the alpha-tubulin(430-443) and beta-tubulin(412-431) internal sequences. Alpha(404-451) and beta(394-445) are monomeric in neutral aqueous solution (as indicated by sedimentation equilibrium), and have circular dichroism (CD) spectra characteristic of nearly disordered conformation, consistent with low scores in peptide helicity prediction. Limited proteolysis of beta(394-445) with subtilisin, instead of giving extensive degradation, resulted in main cleavages at positions Thr409-Glu410 and Tyr422-Gln423-Gln424, defining the proteolysis resistant segment 410-422, which corresponds to the central part of the predicted beta-tubulin C-terminal helix. Both recombinant peptides inhibited microtubule assembly, probably due to sequestration of the microtubule stabilizing associated proteins. Trifluoroethanol (TFE)-induced markedly helical CD spectra in alpha(404-451) and beta(394-445). A substantial part of the helicity of beta(394-445) was found to be in the CD spectrum of the shorter peptide beta(412-431) with TFE. Two-dimensional 1H-NMR parameters (nonsequential nuclear Overhauser effects (NOE) and conformational C alphaH shifts) in 30% TFE permitted to conclude that about 25% of alpha(404-451) and 40% of beta(394-451) form well-defined helices encompassing residues 418-432 and 408-431, respectively, flanked by disordered N- and C-segments. The side chains of beta(394-451) residues Leu418, Val419, Ser420, Tyr422, Tyr425, and Gln426 are well defined in structure calculations from the NOE distance constraints. The apolar faces of the helix in both alpha and beta chains share a characteristic sequence of conserved residues Ala,Met(+4),Leu(+7),Tyr(+11). The helical segment of alpha(404-451) is the same as that described in the electron crystallographic model structure of alphabeta-tubulin, while in beta(394-451) it extends for nine residues more, supporting the possibility of a functional coil --> helix transition at the C-terminus of beta-tubulin. These peptides may be employed to construct model complexes with microtubule associated protein binding sites.

Identification and properties of the genes encoding microcin E492 and its immunity protein.

The gene coding for the immunity protein (mceB) and the structural gene of microcin E492 (mceA), a low-molecular-weight channel-forming bacteriocin produced by a strain of Klebsiella pneumoniae, have been characterized. The microcin gene codes for a precursor protein of either 99 or 103 amino acids. Protein sequencing of the N-terminal region of microcin E492 unequivocally identified this gene as the microcin structural gene and indicated that this microcin is synthesized as a precursor protein that is cleaved at either amino acid 15 or 19, at a site resembling the double-glycine motif. The gene encoding the 95-amino-acid immunity protein (mceB) was identified by cloning the DNA segment that encodes only this polypeptide into an expression vector and demonstrating the acquisition of immunity to microcin E492. As expected, the immunity protein was found to be associated with the inner membrane. Analysis of the DNA sequence indicates that these genes belong to the same family as microcin 24, and they do not share structural motifs with any other known channel-forming bacteriocin. The organization of the microcin- and immunity protein-encoding genes suggests that they are coordinately expressed.

Calcium and gadolinium ions stimulate the GTPase activity of purified chicken brain tubulin through a conformational change.

Ca2+ and Gd3+ stimulated the GTPase activity of chicken brain tubulin 13- and 26-fold, respectively. Mg2+, Tb3+, and Na+ had no effect. This GTPase activity showed a saturation behavior with Ca2+ and Gd3+ with a maximal activity of 0.26 +/- 0.026 and 1.15 +/- 0.78 nmol min-1 per mg of tubulin and semisaturation constants, expressed as the concentration of the cation needed for 50% of saturation, of 0.32 +/- 0.18 and 0.011 +/- 0.007 mM, respectively. In the presence of Ca2+, the GTPase activity was proportional to tubulin concentration in the range 0.9-31.8 microM. The semisaturation constants for the inhibition of tubulin polymerization and for the depolymerization of microtubules by Ca2+ were 0.71 +/- 0.1 and 0.049 +/- 0.043 mM, respectively. The similarity of the Ca2+ semisaturation constants for inhibition of tubulin assembly and stimulation of the GTPase activity suggests that these processes are correlated. These results support the hypothesis that the GTPase activity is related to but not directly involved in the mechanism of inhibition of Ca2+ -dependent tubulin assembly. This inhibition could be better explained by the formation of a nonfunctional conformational state of tubulin induced by Ca2+ that is responsible for the GTPase activity. Quenching of the intrinsic fluorescence of tryptophan induced by Ca2+ showed an apparent dissociation constant of 0.14 +/- 0.005 mM, in the range of values determined through tubulin polymerization inhibition or through the induction of GTPase activity by Ca2+. Acrylamide-induced quenching of the intrinsic fluorescence showed values of the Stern-Volmer constants of 5.4 +/- 0.12 and 5.0 +/- 0.15 M-1 in the absence and presence of Ca2+, respectively. These results support the hypothesis that the inhibition of tubulin polymerization and the induction of the GTPase activity by Ca2+ is mediated by a conformational change. Ca2+ failed to induce depolymerization of GDP-AIF4-microtubules; this could be explained by a model in which Ca-tubulin is unable to assemble into microtubules and the rate of dissociation of GDP-Pi-tubulin from the microtubule ends is extremely slow compared with the rate of GDP-subunit dissociation, supporting the concept that the GTP- and GDP-Pi-tubulin cap at the ends of microtubules regulates their dynamic instability.

Applications of nuclear magnetic resonance to determine the structure and interactions of ligands, peptides and enzymes.

Nuclear magnetic resonance (NMR) spectroscopy is emerging as a powerful tool for the study of enzyme structure and function. This article discusses the general principles of NMR and the potential information this technique can provide in the study of enzymes along with its limitations.

Recovery of soluble protein after expression in Escherichia coli depends on cellular disruption conditions.

Proteins overproduced in Escherichia coli are frequently recovered from bacterial extracts as insoluble material. The influence of different cell disruption procedures on the recovery of soluble protein, after recombinant protein expression in E. coli, was assessed using two beta-tubulin derivatives. Nonionic detergents such as Triton X-100 and Nonidet P-40 promote aggregation when present in the lysis buffer. The effect of Triton X-100 is reversed by the addition of 1 M NaCl in the lysis buffer indicating that the recombinant protein aggregation is probably caused by interactions with membrane proteins. The importance of the cellular disruption method on the recovery of potentially soluble recombinant proteins is discussed.

Applications of nuclear magnetic resonance to determine the structure and interactions of ligands, peptides and enzymes

Nuclear magnetic resonance (NMR) spectroscopy is emerging as a powerful tool for the study of enzyme structure and function. This article discusses the general principles of NMR and the potential information this technique can provide in the study of enzymes along with its limitations

Tubulin-tyrosine ligase catalyzes covalent binding of 3-fluoro-tyrosine to tubulin: kinetic and [19F]NMR studies.

The use of 3-fluoro-tyrosine as an alternative substrate for the enzyme tubulin:tyrosine ligase which catalyzes the incorporation of tyrosine into the alpha-tubulin subunit was investigated. The incorporation of tyrosine into tubulin was inhibited competitively by 3-fluoro-tyrosine with an apparent Ki of approximately 25 microM. The affinity for this analog was similar to that of tyrosine, confirming that the hydrogen at position 3 of the aromatic ring is not essential for the reaction catalyzed by TTLase. The incorporation of 3-fluoro-tyrosine into the C-terminus of the alpha-tubulin subunit was demonstrated through [19F]NMR spectroscopy. The 3-fluoro-tyrosine signal at -58.6 ppm (trifluoroacetic acid as external standard), with a bandwidth of 24.7 Hz presented a chemical shift of 0.75 ppm upfield and an enlargement in the bandwidth (30.5 Hz) when incorporated into tubulin. These results strongly suggest that this amino acid is exposed to the solvent in tubulin. Tubulin covalently labeled with 3-fluoro-tyrosine was competent to polymerize into microtubules. The use of fluorinated tubulin in [19F]NMR spectroscopy for studying questions concerning protein conformation and interactions will be discussed.

Interaction between the C-terminal peptides of tubulin and tubulin S detected with the fluorescent probe 4',6-diamidino-2-phenylindole.

The digestion of tubulin with subtilisin and the reassociation of the digestion products was followed by means of the fluorescent probe 4',6-diamidino-2-phenylindole (DAPI). The fluorescence spectra of DAPI bound to chicken brain tubulin and to the main products of tubulin digested with subtilisin-agarose (tubulin S and C-terminal peptides) were analyzed. The corrected emission spectrum of DAPI in the presence of tubulin showed an enhancement of fluorescence intensity with a maximum at 452 nm. The digestion reaction was followed by the diminution of the area of DAPI-tubulin emission spectra, which showed biphasic pseudo-first-order kinetics. The values for the rate constants were 1.2 x 10(-2) min-1 and 3.5 x 10(-2) min-1 for the alpha and beta subunits, respectively, and were similar to those determined from the undigested subunits using polyacrylamide gel electrophoresis. Tubulin S and the C-terminal peptides were purified by means of a Bio-Gel P-60 column. The C-terminal peptides obtained from this column were analyzed by urea-sodium dodecyl sulfate polyacrylamide gel electrophoresis, and an apparent molecular weight around 3000 was determined. The corrected emission spectrum of DAPI in the presence of tubulin S showed a maximum shifted to 460 nm and a lower enhancement of fluorescence than the emission spectrum of the DAPI-tubulin complex. Titration of purified tubulin S with the C-terminal peptides of tubulin showed, after the addition of DAPI, an increase in the fluorescence intensity at 460 nm with a saturation function dependent on the concentration of peptides added. On the other hand, the emission spectrum of DAPI in the presence of the C-terminal peptides was unchanged from that of free DAPI in the solution. From these results we propose that the DAPI binding site is located on tubulin S and that the C-terminal peptides interact with tubulin S after digestion with subtilisin.

Microcin E492 forms ion channels in phospholipid bilayer membrane.

Microcin E492, a polypeptide antibiotic, has been shown to have an M(r) of 6,000 by urea-SDS-polyacrylamide gel electrophoresis of the fluorescently labelled compound. It is known that the bactericidal action of microcin involves a loss of the transmembrane potential. In this study we show that microcin forms cation-selective channels in planar phospholipid bilayers. The channels have two main conductance states the current-voltage curves of which rectify. The reversal potentials measured under biionic conditions indicate a permeability sequence of NH4+ > K+ = Rb+ = Cs+ > Na+ = Na+ = Li+ > Tris+. The results suggest that membrane potential dissipation induced by microcin is a consequence of the formation of pores in the bacterial membrane.

The binding of terbium ions to tubulin induces ring formation.

The intrinsic fluorescence excitation and emission spectra of chicken brain tubulin showed the characteristic tryptophan fluorescence. The emission spectrum of Tb3+ in the presence of tubulin and GTP excited at 295 nm, showed four peaks, with the maxima at 490, 545, and 586 nm and a minor peak around 620 nm. Titration of tubulin with Tb3+ was followed by the increment in luminescence at 545 nm and showed a sigmoidal curve where the initial lag interval and the maximal luminescence intensity depended on tubulin concentration. The presence of Mg2+, Co2+, and Zn2+ diminished both the sigmoidicity of the curve and the maximal luminescence intensity. Titration of tubulin with Tb3+ also produced a sigmoidal increase in turbidity, which was shifted to the left with respect to the luminescence curve. The dependence of turbidity on the wavelength of the Tb(3+)-induced polymers revealed that the large structures formed were not microtubules. Electron microscopy of the aggregates induced by Tb3+ showed mainly a lattice of double rings with side-by-side contacts. These results indicate that Tb3+ induces principally double ring formation and that these rings (33 +/- 2 nm external diameter) aggregate in large-ordered arrays. The luminescence of Tb3+ seems to be induced mainly by the aggregation of rings.

NADP(+)-dependent D-xylose dehydrogenase from pig liver. Purification and properties.

An NADP(+)-dependent D-xylose dehydrogenase from pig liver cytosol was purified about 2000-fold to apparent homogeneity with a yield of 15% and specific activity of 6 units/mg of protein. An Mr value of 62,000 was obtained by gel filtration. PAGE in the presence of SDS gave an Mr value of 32,000, suggesting that the native enzyme is a dimer of similar or identical subunits. D-Xylose, D-ribose, L-arabinose, 2-deoxy-D-glucose, D-glucose and D-mannose were substrates in the presence of NADP+ but the specificity constant (ratio kcat./Km(app.)) is, by far, much higher for D-xylose than for the other sugars. The enzyme is specific for NADP+; NAD+ is not reduced in the presence of D-xylose or other sugars. Initial-velocity studies for the forward direction with xylose or NADP+ concentrations varied at fixed concentrations of the nucleotide or the sugar respectively revealed a pattern of parallel lines in double-reciprocal plots. Km values for D-xylose and NADP+ were 8.8 mM and 0.99 mM respectively. Dead-end inhibition studies to confirm a ping-pong mechanism showed that NAD+ acted as an uncompetitive inhibitor versus NADP+ (Ki 5.8 mM) and as a competitive inhibitor versus xylose. D-Lyxose was a competitive inhibitor versus xylose and uncompetitive versus NADP+. These results fit better to a sequential compulsory ordered mechanism with NADP+ as the first substrate, but a ping-pong mechanism with xylose as the first substrate has not been ruled out. The presence of D-xylose dehydrogenase suggests that in mammalian liver D-xylose is utilized by a pathway other than the pentose phosphate pathway.

Nuclear relaxation rates study of GTP(gamma F)-tubulin interaction using 19F-nuclear magnetic resonance.

To study the relationship between the exchangeable GTP binding site (E-site) and the high affinity metal binding site we synthesized P3-fluoro P1-5'-guanosine tripaosphate (GTP(gamma F), an analog of GTP. Our results show that this analog binds to the exchangeable GTP binding site of calf brain tubulin. The values of the dissociation constant and the stoichiometry of the GTP(gamma F)-Mn(II) complex as determined by EPR spectroscopy were 1.64 x 10(-4) M and one mole of manganese per mole of nucleotide, respectively. The distance separating the high-affinity binding site for the divalent metal ion and the exchangeable nucleotide binding site was evaluated by using high-resolution 19F-NMR. The 31P- and 19F-NMR spectra of GTP(gamma F) were studied, both the fluorine and the gamma-phosphate were split in a doublet with a coupling constant of 936 Hz. Tubulin purified by the method of Weisenberg (Weisenberg, R.C., and Timashef, S.N. (1970) Biochemistry 9, 4110-4116) was treated with colchicine to stabilize it, GTP(gamma F) was added and the 254.1 MHz 19fluorine relaxation rates measured within the first four hours. Longitudinal and transversal relaxation rates were determined in the presence of colchicine-tubulin-Mn(II), (paramagnetic complex), or the ternary complex with magnesium (diamagnetic complex). The analysis of the temperature-dependent relaxation data indicates that the metal and the exchangeable nucleotide binding sites are separated by a maximal distance of 6 at 35 degrees C, to 8.1 A at 12 degrees C.

Inhibition of tubulin self-assembly and tubulin-colchicine GTPase activity by guanosine 5'-(gamma-fluorotriphosphate).

The inhibitory effects of guanosine 5'-(gamma-fluorotriphosphate) [GTP(gamma F)] on both the polymerization and the colchicine-dependent GTPase activity of calf brain tubulin have been studied. The results demonstrate that this analogue of GTP, with a fluorine atom on the gamma-phosphate, is a reversible competitive dead-end inhibitor of the colchicine-induced GTPase activity with a K1 value of (1.8 +/- 0.6) X 10(-4) M. GTP(gamma F) did not promote assembly of tubulin from which the E-site guanine nucleotide had been removed. It binds to the exchangeable nucleotide site competitively with respect to GTP, diminishing both the rate and extent of tubulin polymerization. Treatment in terms of the Oosawa-Kasai model of the inhibitory effect of GTP(gamma F) on the assembly led to a value of Kdis = 1.1 X 10(-6) M for the complex GTP(gamma F)-tubulin. This analogue does not bind to the postulated third site. The growing of tubulin polymers at 37 degrees C was arrested by GTP(gamma F), and only limited depolymerization was induced by the addition of this analogue after assembly in the presence of GTP. This result confirms that the E-site is blocked in the polymer and that this analogue can bind only to the ends of the polymers. Sedimentation velocity and circular dichroism studies showed that the conformation of the tubulin-GTP(gamma F) complex is not identical with that of tubulin-GTP. This is caused by the replacement of the hydroxyl group in the gamma-phosphate by the fluorine group, which have 2.20- and 1.35-A van der Waals radii, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

19F nuclear magnetic resonance measurement of the distance between the E-site GTP and the high-affinity Mg2+ in tubulin.

The distance separating the divalent metal ion high-affinity binding site and the exchangeable nucleotide binding site on tubulin was evaluated by using high-resolution 19F NMR. The 31P and 19F NMR spectra of guanosine 5'-(gamma-fluorotriphosphate) [GTP (gamma F)] were studied. Both the fluorine and the gamma-phosphate were split into a doublet with a coupling constant of 936 Hz. Tubulin purified according to the method of Weisenberg [Weisenberg, R.C., & Timasheff, S.N. (1970) Biochemistry 9, 4110-4116] was incubated with 1 mM Mn2+. After one cycle of assembly, Mn2+ replaced Mg2+ only partially, i.e., 60% at the high-affinity binding site. After colchicine treatment of tubulin to stabilize it, GTP(gamma F) was added, and the 254-MHz fluorine-19 relaxation rates were measured within the first 4 h. Longitudinal and transversal relaxation rates were determined at two concentrations of GTP(gamma F) and variable concentrations of colchicine-tubulin-Mn(II) (paramagnetic complex) or the ternary complex with magnesium (diamagnetic complex). The analysis of the relaxation data indicates that the rate of exchange of GTP(gamma F) from the exchangeable nucleotide site has a lower limit of 8.7 X 10(4) s-1 and the metal and exchangeable nucleotide binding sites are separated by an upper distance between 6 and 8 A. These data confirm that the high-affinity divalent cation site is situated in the same locus as that of the exchangeable nucleotide, forming a metal-nucleotide complex.

Role of the dianionic form of the GTP gamma-phosphate in the polymerization process of tubulin.

To determine whether tubulin polymerization requires the bivalent metal-GTP complex with the gamma-phosphate in the dianionic form, the effect of GTP(gamma F) on the polymerization process was studied, in the presence of either magnesium or manganese. P3-fluoro P1-5'-guanosine triphosphate (GTP(gamma F)) was a competitive inhibitor (Ki = 1.8 X 10(-4) M) of the GTPase activity of tubulin-colchicine complex, stopped the polymerization process during the course of reaction and no depolymerization occurred. This indicates that GTP(gamma F) has access only to the nucleotide exchangeable site of the free tubulin dimer. Tubulin has one mole of magnesium tightly bound per mole of dimer. In order to know whether the inhibitory effect of GTP(gamma F) was due to the release of the metal, magnesium was replaced for manganese (a paramagnetic ion) and the paramagnetic effect of manganese on the fluorine NMR signal from the GTP(gamma F)-tubulin-metal complex was followed. Longitudinal and transversal relaxation rates measurements of the 1 degree F-NMR signal allowed to determine that the upper distance from the manganese site to the fluorine atom was between 6 and 8 A. These studies demonstrate that the dianionic form of the terminal phosphate of the metal-GTP complex, at the nucleotide exchangeable site, is essential to stimulate tubulin polymerization.