One of the non-exchangeable nucleotides of the mitochondrial F1-ATPase is bound at a β-subunit: evidence for a non-rotatory two-site catalytic mechanism

AbstractIn active MF1, one of the two non-exchangeable tightly bound adenine nucleotides is an ATP, while the other is an ADP. The respective sites are called the T-site and the D-site. The activity of the enzyme correlates linearly with the amount of bound ATP, ADP at the T-site being inhibitory. When MF1 is stored at room temperature in 50% glycerol and 100 mM Tris-HCl (pH 7.3) after slow passage through a Sephadex column, the tightly bound ATP is slowly dephosphorylated to ADP which is subsequently released, without effect on activity. When enzyme with about one residual ADP left (at the D-site) was incubated at pH 7.3, after dilution of the glycerol, with 400 μM [14C]ATP under varying conditions, the amount of tightly bound nucleotide triphosphate again correlated well with activity, the residual ADP being bound at the D-site. Optimal results were obtained when the incubation was performed in the presence of a regenerating system. Binding of 2-azido-ATP instead of ATP to the T-site as a triphosphate, as indicated by the specific activity of the enzyme, appeared to be optimal when the binding was performed at pH 6.4 in the absence of Mg2+ and with high concentrations of the nucleotide. Under such conditions, 3 mol 2-azido-AXP per mol F1 remained tightly bound after ammonium sulfate precipitation and column centrifugation, in addition to about one residual ADP at the D-site. After a 2-min period of turnover with ATP/Mg2+ as substrate two mol 2-azido-AXP were left on the enzyme, of which one was bound at a β-site. These results show that one of the non-catalytic nucleotide binding sites that contain tightly bound nucleotides, is a β-site, in conflict with the requirements for a rotatory tri-site mechanism for ATP hydrolysis. This β-site can further be identified with the T-site. The validity of these conclusions for F1 from other sources and for catalysis by membrane-bound enzyme is discussed

Analysis of the inhibitory non-catalytic ADP binding site on mitochondrial F1, using NAP3-2N3ADP as probe. Effects of the modification on ATPase and ITPase activity

AbstractThe ADP analogue NAP3-2N3ADP is able to bind to one or two high-affinity sites on mitochondrial F1-ATPase, depending on the nucleotide content of the F1 preparation. In both cases studied (enzyme with three bound nucleotides and enzyme with four bound nucleotides), the binding is accompanied by the exchange of one tightly-bound adenine nucleotide and nearly complete inhibition of the ATPase activity upon UV illumination. In both cases the ADP-analogue binds at a high-affinity catalytic site, replacing a bound nucleotide. The apparent KD value for the exchange equals 25–30 μM, but the newly bound ligand does not dissociate. With F1 containing 3 bound nucleotides NAP3-2N3ADP is able to bind to a second high-affinity site as well. This binding induces already in the absence of illumination 45% inhibition of the ATPase activity. The additionally bound molecule does not exchange within a short period of turnover with Mg-ATP. Therefore it has to be bound at a slowly exchangeable non-catalytic site, with a regulatory influence on the activity of the enzyme. Binding of NAP3-2N3ADP to this non-catalytic site is influenced by the presence of Mg2+ or EDTA: tight binding requires Mg2+ and in the absence of Mg2+ and presence of EDTA the ligand is removed from this site relatively easily, just like ADP. The presence of EDTA instead of Mg2+ lowers the measured affinity of this site for NAP3-2N3ADP with a factor 5. Kinetic measurements after an incubation of F1 with NAP3-2N3ADP show a decrease of the Vmax with ATP as substrate, without effect on the two measured Km values. With ITP as substrate, however, incubation of F1 with NAP3-2N3ADP results in an increase of the Km values, without effect on the Vmax. Comparison of our data with the literature shows that this non-catalytic site is not the site responsible for hysteretic inhibition by ADP. We conclude that this latter form of inhibition is observed when ADP or a suitable analogue is bound at the first (potentially catalytic) β-site, in disagreement with the conclusions of Jault and Allison (J. Biol. Chem. 269 (1994) 319–325)

The aromatic nature of residue 66 of the 11-kDa subunit of ubiquinol-cytochrome <i>c</i> oxidoreductase of the yeast <i>Saccharomyces cerevisiae</i> is important for the assembly of a functional enzyme

AbstractTransformation of multi- and single-copy plasmids carrying a mutated version (LTN2, region 66-YWYWW-70 replaced by SASAA) of QCR8, the gene encoding the 11-kDa subunit of ubiquinol-cytochrome c oxidoreductase of Saccharomyces cerevisiae, to a QCR80 strain indicated the importance of this aromatic region for the assembly of a functional enzyme. Sequencing of plasmids giving spontaneous restoration of growth to some colonies among the single-copy LTN2 transformants showed that changing the sequence SASAA into the sequence FASAA could, to a large extent, overcome the observed assembly defect, indicating the importance of the aromatic nature of residue 66

Modification of membrane-bound F1 by p-fluorosulfonylbenzoyl-5′-adenosine: sites of binding and effect on activity

AbstractBovine heart submitochondrial particles (smp) were incubated with p-fluorosulfonylbenzoyl-5′-adenosine (FSBA) in order to study the binding of this ligand and its effect on ATP synthesis and ATP hydrolysis in smp and to compare the results with those obtained with isolated F1. The binding was measured with the 14C-labeled compound. ATP hydrolysis was in all cases as much inhibited as succinate-driven ATP synthesis and ITP hydrolysis was more inhibited than ATP hydrolysis. The binding experiments show that modification of three nucleotide binding sites results in nearly complete inhibition of ATPase activity. In the presence of pyrophosphate up to 6 mol [14C]SBA/mol F1 can be bound. FSBA preferentially modifies amino acids of the α-subunits but also β-subunits are modified. It is concluded that modification of both subunits results in inhibition of activity. The results are very well comparable with the results obtained with isolated F1, which indicates that our preparation of F1 is a good model for F1 in the intact system. Furthermore it is concluded that each α-subunit of F1 in smp, just like in the isolated form, contains two pockets where adenosine moieties can bind, one located above the P-loop, modifying α-Tyr-244 and α-Tyr-300 and the other one located below the P-loop where also the adenosine moiety of AD(T)P binds, modifying β-Tyr-368

Plurilingüismo: por qué ser personas plurilingües y cuándo iniciar el aprendizaje

Treball Final de Grau en Mestre o Mestra d'Educació Primària. Codi: MP1040. Curs acadèmic: 2016/2017Entendemos como plurilingüismo aquellas variedades lingüísticas que utiliza un mismo locutor, ya sea la lengua materna y todas aquellas adquiridas posteriormente. Tenemos al alcance la opción de enriquecernos con lenguas diferentes a la nuestra y esto se considera un aspecto muy importante para el acceso a las manifestaciones culturales. La lengua regula nuestro pensamiento y todas nuestras ideas apoyadas por bases lingüísticas. Además, como seres sociales contamos con la necesidad de relacionarnos y de comunicarnos, para hacernos hueco en el progreso continuo de la sociedad. Por esta misma razón, se deben tener en cuenta ciertos aspectos culturales, características personales de los individuos de una sociedad y a qué estratificación social pertenecen, para poder llevar a cabo una Competencia Comunicativa adecuada. La conciencia es cada vez mayor en cuanto a la función que desempeñan los idiomas en el desarrollo, la diversidad cultural y en el fortalecimiento de la cooperación y la conservación del patrimonio cultural. Siempre se ha considerado esencial el aprendizaje de idiomas desde edades tempranas para poder exprimir esos conocimientos de cara al futuro, pero, ¿qué impacto tiene el plurilingüismo y el aprendizaje de nuevas lenguas en edades adultas? El derecho a aprender a lo largo de toda la vida es cada vez más ratificado, no obstante, la educación de adultos se encuentra muy rezagada con respecto a los demás sectores de la enseñanza. A lo largo de este trabajo de investigación descubriremos las ventajas cognitivas que presenta el plurilingüismo en la Educación para Adultos así como la eficiencia y los beneficios a los cuales llevan

Continuous wave optical parametric oscillator for quartz-enhanced photoacoustic trace gas sensing

A continuous wave optical parametric oscillator, generating up to 300 mW idler output in the 3–4 μm wavelength region, and pumped by a fiber-amplified DBR diode laser is used for trace gas detection by means of quartz-enhanced photoacoustic spectroscopy (QEPAS). Mode-hop-free tuning of the OPO output over 5.2 cm-1 and continuous spectral coverage exceeding 16.5 cm-1 were achieved via electronic pump source tuning alone. Online monitoring of the idler wavelength, with feedback to the DBR diode laser, provided an automated closed-loop control allowing arbitrary idler wavelength selection within the pump tuning range and locking of the idler wavelength with a stability of 1.7×10-3 cm-1 over at least 30 min.\ud \ud Using this approach, we locked the idler wavelength at an ethane absorption peak and obtained QEPAS data to verify the linear response of the QEPAS signal at different ethane concentrations (100 ppbv-20 ppmv) and different power levels. The detection limit for ethane was determined to be 13 ppbv (20 s averaging), corresponding to a normalized noise equivalent absorption coefficient of 4.4×10-7 cm-1  W/Hz1/2