Débora González | Universidad de Buenos Aires (original) (raw)

Papers by Débora González

Research paper thumbnail of the Transient States of the Reaction

distributed under the Creative Commons Attribution License, which permits unrestricted use, distr... more distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The step-by-step free energy (ΔG) profile of the sarcoplasmic reticulum Ca-ATPase cycle during the transient state of simulated reactions was analyzed previously by Alonso and Hecht (1990). ΔG was negative at all the steps. Here we calculated the step-by-step activation energies (ΔG) in the same model according the transition state theory. Transient species were introduced as chemical intermediates at each step; they react with enzyme species accordingly with the mass law, decaying at 6.56x10

Research paper thumbnail of Salivary extracellular vesicles can modulate purinergic signalling in oral tissues by combined ectonucleoside triphosphate diphosphohydrolases and ecto-5′-nucleotidase activities

Molecular and Cellular Biochemistry

Research paper thumbnail of Histamine stimulates secretion of extracellular vesicles with nucleotidase activity in rat submandibular gland

Archives of oral biology, 2018

Extracellular vesicles released by different cells have been isolated from diverse fluids includi... more Extracellular vesicles released by different cells have been isolated from diverse fluids including saliva. We previously reported that rat submandibular glands secrete nanovesicles that catalyze hydrolysis of ATP, ADP and AMP, which are actors of the purinergic signaling system along with adenosine. Extracellular nucleotides like ATP and adenosine are involved in the regulation of inflammatory processes and apoptosis. Histamine, a widely distributed biogenic amine, is involved in inflammatory response. To test if activation of histamine receptors in rat submandibular gland promotes changes in the release of vesicles with nucleotidase activity that could modulate purinergic signaling. Rat submandibular glands were incubated in the absence or presence of histamine and JNJ7777120, an antagonist for H4 receptors. Extracellular vesicles were isolated from incubation media by differential centrifugation. Vesicular nucleotidase activity was measured following Pi release by 3mM MgATP, MgAD...

Research paper thumbnail of Characterization of a functional NTPDase in the endoplasmic reticulum of rat submandibular salivary gland

Physiological research / Academia Scientiarum Bohemoslovaca, 2009

Nucleotidase activity and Ca-uptake were characterized in endoplasmic reticulum (ER) enriched rat... more Nucleotidase activity and Ca-uptake were characterized in endoplasmic reticulum (ER) enriched rat submandibular gland (SMG) microsomal preparations. (i) Ca-uptake had characteristics of an ER Ca-ATPase. (ii) Nucleotidase activity was equally stimulated by calcium, magnesium and manganese, but with different Km values. (iii) Specific inhibitors of P-type Ca-ATPases were ineffective on nucleotidase activity, demonstrating that this activity was not related to calcium uptake and did not correspond to classical Ca(2+) pumps. (iv) ATP and UTP were more efficient substrates, whereas ADP and UDP were hydrolyzed at significantly slower rate. (v) Nucleotidase activity was sensitive to mild detergent solubilization and insensitive to ionophore addition. (vi) Nucleotidase activity was strongly inhibited by suramin, a nucleoside triphosphate diphosphohydrolase (NTPDase) inhibitor. (vii) Nucleotidase activity exponentially diminished as function of time. All these observations are consistent wit...

Research paper thumbnail of Activation free energies in partial steps of the sarcoplasmic reticulum Ca-ATPase cycle during the transient states of the reaction

Advanced Studies in Biology, 2014

The step-by-step free energy (ΔG) profile of the sarcoplasmic reticulum Ca-ATPase cycle during th... more The step-by-step free energy (ΔG) profile of the sarcoplasmic reticulum Ca-ATPase cycle during the transient state of simulated reactions was analyzed previously by Alonso and Hecht (1990). ΔG was negative at all the steps. Here we calculated the step-by-step activation energies (ΔG) in the same model according the transition state theory. Transient species were introduced as chemical intermediates at each step; they react with enzyme species accordingly with the mass law, decaying at 6.56x10 12 s-1. Other rate constants are as in the referenced paper. The transient evolution of the model was followed with a computer program. The evolution of the ligands concentrations agree with experimental results. Enzymatic species concentrations lie within 10-10-10-6 M, transient species lie within 10-19-10-17 M, at 0.1 s, when the reaction is in a cuasi-steady-state. These differences preclude the use of the standard (ΔG o) or basic (ΔG basic) free energies, to describe ΔG fall along the cycle. Forward and backward ΔG are shown for 10 and 100 ms of simulated reaction; both are negative in forward cycles, at any reaction time, and their sum equals the partial ΔG. The results show the absence of any activation energy hill to be surmounted upon the reaction advance. Similar results were obtained for the successive binding of 2 Ca ions to the enzyme through (+)cooperative reactions.

Research paper thumbnail of Rat submandibular glands secrete nanovesicles with NTPDase and 5′-nucleotidase activities

Purinergic Signalling, 2014

Extracellular nucleotides modulate a wide number of biological processes such as neurotransmissio... more Extracellular nucleotides modulate a wide number of biological processes such as neurotransmission, platelet aggregation, muscle contraction, and epithelial secretion acting by the purinergic pathway. Nucleotidases as NTPDases and ecto-5'-nucleotidase are membrane-anchored proteins that regulate extracellular nucleotide concentrations. In a previous work, we have partially characterized an NTPDase-like activity expressed by rat submandibular gland microsomes, giving rise to the hypothesis that membrane NTPDases could be released into salivary ducts to regulate luminal nucleotide concentrations as was previously proposed for ovarian, prostatic, and pancreatic secretions. Present results show that rat submandibular glands incubated in vitro release membrane-associated NTPDase and ecto-5'-nucleotidase activities. Electron microscopy images show that released membranes presenting nucleotidase activity correspond to exosome-like vesicles which are also present at microsomal fraction. Both exosome release and nucleotidase activities are raised by adrenergic stimulation. Nucleotidase activities present the same kinetic characteristics than microsomal nucleotidase activity, corresponding mainly to the action of NTPDase2 and NTPDase3 isoforms as well as 5'-nucleotidase. This is consistent with Western blot analysis revealing the presence of these enzymes in the microsomal fraction.

Research paper thumbnail of Kinetic Analysis of a Model of the Sarcoplasmic Reticulum Ca-ATPase, with Variable Stoichiometry, which Enhances the Amount and the Rate of Ca Transport

Journal of Theoretical Biology, 2001

The sarcoplasmic reticulum (SR) Ca-dependent adenosinetriphosphatase (Ca-ATPase) actively transpo... more The sarcoplasmic reticulum (SR) Ca-dependent adenosinetriphosphatase (Ca-ATPase) actively transports Ca2+ from the myoplasm to the SR lumen. Under optimal conditions a 2:1 stoichiometry of Ca transport/ATP hydrolysis has been observed, but lower stoichiometries have been reported under several circumstances. A lower stoichiometry under conditions of high Ca2+ load, although thermodynamically less efficient, could in theory increase the rate and the maximal amount of Ca uptake. We analysed, by computing simulation, the transient kinetics of a model of the SR Ca-ATPase with variable stoichiometry. The model is based on current experimental reports and includes the most relevant properties of the system. The results show an acceleration in the rate of Ca uptake, an increase in the net Ca transport, and an increase in the rate of [Ca2+] reduction in the medium, which might be physiologically useful to increase the rate of Ca pumping at high Ca load of the sarcoplasmic reticulum.

Research paper thumbnail of In Silico Kinetic Study of the Glucose Transporter

Journal of Biological Physics, 2007

Glucose transport in plasma membranes is the prototypic example of facilitated diffusion through ... more Glucose transport in plasma membranes is the prototypic example of facilitated diffusion through biological membranes, and transport in erythrocytes is the most widely studied. One of the oldest and simplest models describing the kinetics of the transport reaction is that of alternating conformers, schematized in a cycle of four partial reactions where glucose binds and dissociates at two opposite steps, and the transporter undergoes transconformations at the other two opposite steps. The transport kinetics is entirely defined by the forward and backward rate constants of the partial reactions and the glucose and transporter concentrations at each side of the membrane, related by the law of mass action. We studied, in silico, the effect of modifications of the variables on the transient kinetics of the transport reaction. The simulations took into account thermodynamic constraints and provided results regarding initial velocities of transport, maximal velocities in different conditions, apparent influx and efflux affinities, and the turnover number of the transporter. The results are in the range of those experimentally reported. Maximal initial velocities are obtained when the affinities of the ligand for the transporter are the same at the extraand intracellular binding sites and when the equilibrium constants of the transconformation steps are equal among them and equal to 1, independently of the obvious effect of the increase of the rate constant values. The results are well adjusted to Michaelis-Menten kinetics. A larger initial velocity for efflux than for uptake described in human erythrocytes is demonstrated in a model with the same dissociation constants at the outer and inner sites of the membrane. The larger velocities observed for uptake and efflux when transport occurs towards a glucose-containing trans side can also be reproduced with the alternating conformer model, depending on how transport velocities are measured.

Research paper thumbnail of A silver-reducing component in rat striated muscle

Histochemistry, 1989

This study reports the presence of a silver-reducing constituent in rat striated muscle fiber loc... more This study reports the presence of a silver-reducing constituent in rat striated muscle fiber located selectively at the level of the terminal cistern/transverse tubule system. It is related to the T tubule network at or near sites that participate in junctions with terminal cisternae, i.e., at both sides of the T tubule in skeletal muscle (triad) and, predominantly, at one side in the ventricle (dyad). Little reactivity is present in the auricle due to the scarcity of those membrane systems. The longitudinal sarcoplasmic reticulum, the sarcolemma, mitochondria and myofibrils are not outlined by the reaction product. Extraction of low molecular weight substances, nucleic acids and lipids did not suppress the chemical reaction. A new argentaffin (H g-A g) technique is described. Ethanol or aldehyde fixed muscles were passed to water, postfixed 6-24 h with mercuric acetate (5% w/v in 1% acetic acid), washed with 1% acetic acid and distilled water, stained 12-24 h at 43 ~ C with ammoniacal silver nitrate (60% w/v) and washed in 10% sodium sulfite (three changes) and water. All steps were carried out in darkness. Postfixation with mercuric acetate proved to be essential for immobilizing the argentaffin component without interfering with its strong argentaffinity. The procedure also provides a simple method for tracing the pathway of transversally oriented membrane systems in skeletal and cardiac muscle cells.

Research paper thumbnail of Stoichiometry of ATP and metal cofactor interaction with the sarcoplasmic reticulum Ca2+–ATPase: A binding model accounting for radioisotopic and fluorescence results

Biophysical Chemistry, 2006

Sarcoplasmic reticulum Ca-ATPase belongs to the P-type ATPases family and transports calcium at t... more Sarcoplasmic reticulum Ca-ATPase belongs to the P-type ATPases family and transports calcium at the expense of ATP hydrolysis. For years, a complex pattern of activity has been observed as a function of ATP and metal cofactor concentrations, leaving the stoichiometry of both metal and ATP in the active site as an open question. In agreement with recent structural studies we present here-using Mn as analogue of Mg-radioisotopic and fluorescence results showing that two metal ions bind to the Ca-ATPase favoring ATP binding. We further show that low ATP concentration favors the binding of these ions, whereas high ATP concentration is inhibitory. We propose a binding model for ATP and metal ions, which permits simulation of our data. Finally, we suggest that (i) the contribution of two metal ions as cofactors of ATP is essential to get maximal activity; (ii) the contribution of two ATP molecules can activate or inhibit the Ca-ATPase depending on metal concentration.

Research paper thumbnail of Calcium additional to that bound to the transport sites is required for full activation of the sarcoplasmic reticulum Ca-ATPase from skeletal muscle

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1998

The sarcoplasmic reticulum Ca-ATPase is fully activated when v1 WM [Ca 2 ] saturates the two tran... more The sarcoplasmic reticulum Ca-ATPase is fully activated when v1 WM [Ca 2 ] saturates the two transport sites; higher [Ca] inhibits the ATPase by competition of Ca-ATP with Mg-ATP as substrates. Here we describe a novel effect of EGTA and other chelators, raising the possibility of an additional activating effect of Ca in the sub-or low WM range. Sarcoplasmic reticulum membranes were isolated from rabbit skeletal muscles. The ATPase activity was measured after incubation at 37³C in 3 mM ATP, 3 mM MgCl 2 , 50 mM MOPS-Tris (pH 7.2), 100 mM KCl, and variable CaCl 2 , EGTA and calcimycin. In the absence of added EGTA and Ca the ATPase activity is high due to contaminant Ca. The determination of the ATPase activity in the presence of increasing amounts of EGTA, without added Ca, yields a decreasing sigmoidal function. K i ranged between 20 and 100 WM, depending on the enzyme concentration. P i production is linear with time for several [EGTA] yielding suboptimal ATPase activities, which are inhibited by thapsigargin. These suboptimal Ca-ATPase activities are inhibited by preincubation of the enzyme in EGTA, at pH 7.2. This effect increases upon increasing EGTA concentration and preincubation time. The inhibitory effect of the previous exposure of the enzyme to EGTA is partially but significantly reverted by increasing [Ca 2 ] during incubations. Calcimycin and EDTA have similar effects as EGTA when added in preincubations. The effect of calcimycin is fully reverted by optimal [Ca 2 ] in incubations. The effects of EGTA, EDTA and calcimycin in preincubation are not additive. The results suggest that an additional calcium, lost during preincubations from a site with affinity near 1 WM, is necessary for full activation of the ATPase.

Research paper thumbnail of Demonstration of the simultaneous activation of Ca2+-independent and Ca2+-dependent ATPases from rat skeletal muscle microsomes

Biochimica et Biophysica Acta (BBA) - Biomembranes, 1988

The activation of the Ca2+-independent (basal) ATPase from rat skeletal muscle microsomes is demo... more The activation of the Ca2+-independent (basal) ATPase from rat skeletal muscle microsomes is demonstrated in the presence of enough Ca 2+ to provide the simultaneous activation of the (Ca 2+ + Mg2+)-ATPase. It was achieved taking advantage of the delayed inorganic phosphate (Pi) release due to the formation of a phosphoenzyme complex during the Ca2+-dependent enzymatic cycle, which is evidenced in fast experiments. The microsomes were immobilized on a filter and perfused at constant flow with an incubation medium which was briefly interrupted with a pulse of appropriate reactants to activate the ATPases, at 2 o C. Successive samples were collected after passing through the filter, at approx. 0.1 s intervals. The Pi effluent profile coincides with the pattern of the pulse when it activates only the Ca2+-independent ATPase, it appears delayed when the pulse activates only extra Pi production by the (Ca2+ + Mg2+)-ATPase, and it includes a rapid and a delayed component when both Ca2+-independent and Ca2+-dependent ATPases are activated simultaneously by the pulse.

Research paper thumbnail of Manganese as a cosubstrate for the phosphorylation of the sarcoplasmic reticulum Ca-dependent adenosine triphosphatase with orthophosphate

Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1996

The phosphorylation of the sarcoplasmic reticulum Ca-ATPase (EC 3.6.1.38) with P~ was characteriz... more The phosphorylation of the sarcoplasmic reticulum Ca-ATPase (EC 3.6.1.38) with P~ was characterized using Mn as a Mg analogue. Steady state and transient fluorescence and radioisotopic techniques were used; the affinities of Mn and Pi for the enzyme and the rate constants of the phosphorylation and dephosphorylation reactions were determined, under several conditions. The reactions were carried out at pH 5.5 to minimize the binding of contaminant Ca to the transport sites, thus avoiding the use of Ca chelators. The apparent affinity of Mn binding at low [Mn] is larger in the absence of Pi (35 txM) than in the presence of saturating Pi (70 p.M). On the contrary, the apparent affinity of Mn for the formation of the phosphoenzyme increases, from 1.5 mM to 0.15 mM, upon increasing [Pi] in the millimolar range. The apparent affinty of Pi for the formation of the phosphoenzyme also increases, from 2.2 mM to 0.2 mM, upon increasing [Mn] in the millimolar range. The equilibrium of the phosphoenzyme with the noncovalent Mn-Pi ' Enzyme complex favors the covalent species. The simulation of a reaction model including the random binding of 2 Mn and 1 Pi per mol of ATPase and a noncovalent complex in equilibrium with the phosphoenzyme, using a set of equilibrium constants deduced from the results, agree with the experimental data.

Research paper thumbnail of A Model Accounting for the Simultaneous Transport of Calcium and Manganese in Sarcoplasmic Reticulum Membranes

Annals of the New York Academy of Sciences, 2003

Skip to Content. If you are seeing this message, you may be experiencing temporary network proble... more Skip to Content. If you are seeing this message, you may be experiencing temporary network problems. Please wait a few minutes and refresh the page. If the problem persists, you may wish to report it to your local Network Manager. ...

Research paper thumbnail of Mn as Cosubstrate for the Phosphorylation of the Sarcoplasmic Reticulum Ca-ATPase by P i

Annals of the New York Academy of Sciences, 1997

Research paper thumbnail of Direct demonstration of an acid-labile phosphoenzyme in the cycle of the sarcoplasmic reticulum Ca2+-dependent adenosinetriphosphatase

Biochimica et Biophysica Acta (BBA) - Biomembranes, 1990

The CaZ+-dependent adenosinetriphosphatase (CaZ+-ATPase) from the sarcoplasmic reticulum (SR) of ... more The CaZ+-dependent adenosinetriphosphatase (CaZ+-ATPase) from the sarcoplasmic reticulum (SR) of rat skeletal muscles is pbosphorylated by inorganic phosphate (Pi) in the absence of Ca z +. The reaction can be described by the following simplified scheme: 1 2 E+ Pi~E-Pi~E-P+ H20 where E-P is a covalent, acid-stable and ADP-insensitive phosphoenzyme, and E. Pi is a noncovalent and acid-labile complex. The reaction is Mg2+-dependent. Membrane fragments deposited on Miilipore filters were successively pertused with two solutions, at constant flow. The effluent samples were analyzed. The pertused solutions were Ca z÷ free and always contained 40% dimethylsulfoxide (DMSO), plus other reactants. Following the successive perfusion of solutions without and with [3ZpIPi, 32p binding is only detected in the presence of Mg 2+, indicating the formation of the phosphoenzymes (E. Pi and E-P). Following pertusions of the phosphoenzymes with 5% trichluroacetic acid, 32p release indicates the mount of the acid-labile moiety (E. Pi). After pbosphorylations, the filters were washed with acid and unlabeled Pi, and the remaining radioactivity was measured to evaluate the acid-stable phosphoenzyme (E-P). The acid-labile and acid-stable phosphoenzymes amounted, respectively, 0.72 5: 0.12, and 1.48 + 0.10 nmol of Pi/mg of protein (+S.E., n = 5), after pbosphorylations with 20 pM Pi. The results indicate: (1) The method allowed the evaluation of the acid-labile intermediate of the SR Ca2+-ATPase cycle. K~=kz/k2, in the above scheme, approaches 2.0. (2) The substrate of the phosphorylation reaction, in the presence of DMSO, is likely to be the Mg • Pi complex, since Mg 2+ is necessary for step 1 in the above scheme.

Research paper thumbnail of the Transient States of the Reaction

distributed under the Creative Commons Attribution License, which permits unrestricted use, distr... more distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The step-by-step free energy (ΔG) profile of the sarcoplasmic reticulum Ca-ATPase cycle during the transient state of simulated reactions was analyzed previously by Alonso and Hecht (1990). ΔG was negative at all the steps. Here we calculated the step-by-step activation energies (ΔG) in the same model according the transition state theory. Transient species were introduced as chemical intermediates at each step; they react with enzyme species accordingly with the mass law, decaying at 6.56x10

Research paper thumbnail of Salivary extracellular vesicles can modulate purinergic signalling in oral tissues by combined ectonucleoside triphosphate diphosphohydrolases and ecto-5′-nucleotidase activities

Molecular and Cellular Biochemistry

Research paper thumbnail of Histamine stimulates secretion of extracellular vesicles with nucleotidase activity in rat submandibular gland

Archives of oral biology, 2018

Extracellular vesicles released by different cells have been isolated from diverse fluids includi... more Extracellular vesicles released by different cells have been isolated from diverse fluids including saliva. We previously reported that rat submandibular glands secrete nanovesicles that catalyze hydrolysis of ATP, ADP and AMP, which are actors of the purinergic signaling system along with adenosine. Extracellular nucleotides like ATP and adenosine are involved in the regulation of inflammatory processes and apoptosis. Histamine, a widely distributed biogenic amine, is involved in inflammatory response. To test if activation of histamine receptors in rat submandibular gland promotes changes in the release of vesicles with nucleotidase activity that could modulate purinergic signaling. Rat submandibular glands were incubated in the absence or presence of histamine and JNJ7777120, an antagonist for H4 receptors. Extracellular vesicles were isolated from incubation media by differential centrifugation. Vesicular nucleotidase activity was measured following Pi release by 3mM MgATP, MgAD...

Research paper thumbnail of Characterization of a functional NTPDase in the endoplasmic reticulum of rat submandibular salivary gland

Physiological research / Academia Scientiarum Bohemoslovaca, 2009

Nucleotidase activity and Ca-uptake were characterized in endoplasmic reticulum (ER) enriched rat... more Nucleotidase activity and Ca-uptake were characterized in endoplasmic reticulum (ER) enriched rat submandibular gland (SMG) microsomal preparations. (i) Ca-uptake had characteristics of an ER Ca-ATPase. (ii) Nucleotidase activity was equally stimulated by calcium, magnesium and manganese, but with different Km values. (iii) Specific inhibitors of P-type Ca-ATPases were ineffective on nucleotidase activity, demonstrating that this activity was not related to calcium uptake and did not correspond to classical Ca(2+) pumps. (iv) ATP and UTP were more efficient substrates, whereas ADP and UDP were hydrolyzed at significantly slower rate. (v) Nucleotidase activity was sensitive to mild detergent solubilization and insensitive to ionophore addition. (vi) Nucleotidase activity was strongly inhibited by suramin, a nucleoside triphosphate diphosphohydrolase (NTPDase) inhibitor. (vii) Nucleotidase activity exponentially diminished as function of time. All these observations are consistent wit...

Research paper thumbnail of Activation free energies in partial steps of the sarcoplasmic reticulum Ca-ATPase cycle during the transient states of the reaction

Advanced Studies in Biology, 2014

The step-by-step free energy (ΔG) profile of the sarcoplasmic reticulum Ca-ATPase cycle during th... more The step-by-step free energy (ΔG) profile of the sarcoplasmic reticulum Ca-ATPase cycle during the transient state of simulated reactions was analyzed previously by Alonso and Hecht (1990). ΔG was negative at all the steps. Here we calculated the step-by-step activation energies (ΔG) in the same model according the transition state theory. Transient species were introduced as chemical intermediates at each step; they react with enzyme species accordingly with the mass law, decaying at 6.56x10 12 s-1. Other rate constants are as in the referenced paper. The transient evolution of the model was followed with a computer program. The evolution of the ligands concentrations agree with experimental results. Enzymatic species concentrations lie within 10-10-10-6 M, transient species lie within 10-19-10-17 M, at 0.1 s, when the reaction is in a cuasi-steady-state. These differences preclude the use of the standard (ΔG o) or basic (ΔG basic) free energies, to describe ΔG fall along the cycle. Forward and backward ΔG are shown for 10 and 100 ms of simulated reaction; both are negative in forward cycles, at any reaction time, and their sum equals the partial ΔG. The results show the absence of any activation energy hill to be surmounted upon the reaction advance. Similar results were obtained for the successive binding of 2 Ca ions to the enzyme through (+)cooperative reactions.

Research paper thumbnail of Rat submandibular glands secrete nanovesicles with NTPDase and 5′-nucleotidase activities

Purinergic Signalling, 2014

Extracellular nucleotides modulate a wide number of biological processes such as neurotransmissio... more Extracellular nucleotides modulate a wide number of biological processes such as neurotransmission, platelet aggregation, muscle contraction, and epithelial secretion acting by the purinergic pathway. Nucleotidases as NTPDases and ecto-5'-nucleotidase are membrane-anchored proteins that regulate extracellular nucleotide concentrations. In a previous work, we have partially characterized an NTPDase-like activity expressed by rat submandibular gland microsomes, giving rise to the hypothesis that membrane NTPDases could be released into salivary ducts to regulate luminal nucleotide concentrations as was previously proposed for ovarian, prostatic, and pancreatic secretions. Present results show that rat submandibular glands incubated in vitro release membrane-associated NTPDase and ecto-5'-nucleotidase activities. Electron microscopy images show that released membranes presenting nucleotidase activity correspond to exosome-like vesicles which are also present at microsomal fraction. Both exosome release and nucleotidase activities are raised by adrenergic stimulation. Nucleotidase activities present the same kinetic characteristics than microsomal nucleotidase activity, corresponding mainly to the action of NTPDase2 and NTPDase3 isoforms as well as 5'-nucleotidase. This is consistent with Western blot analysis revealing the presence of these enzymes in the microsomal fraction.

Research paper thumbnail of Kinetic Analysis of a Model of the Sarcoplasmic Reticulum Ca-ATPase, with Variable Stoichiometry, which Enhances the Amount and the Rate of Ca Transport

Journal of Theoretical Biology, 2001

The sarcoplasmic reticulum (SR) Ca-dependent adenosinetriphosphatase (Ca-ATPase) actively transpo... more The sarcoplasmic reticulum (SR) Ca-dependent adenosinetriphosphatase (Ca-ATPase) actively transports Ca2+ from the myoplasm to the SR lumen. Under optimal conditions a 2:1 stoichiometry of Ca transport/ATP hydrolysis has been observed, but lower stoichiometries have been reported under several circumstances. A lower stoichiometry under conditions of high Ca2+ load, although thermodynamically less efficient, could in theory increase the rate and the maximal amount of Ca uptake. We analysed, by computing simulation, the transient kinetics of a model of the SR Ca-ATPase with variable stoichiometry. The model is based on current experimental reports and includes the most relevant properties of the system. The results show an acceleration in the rate of Ca uptake, an increase in the net Ca transport, and an increase in the rate of [Ca2+] reduction in the medium, which might be physiologically useful to increase the rate of Ca pumping at high Ca load of the sarcoplasmic reticulum.

Research paper thumbnail of In Silico Kinetic Study of the Glucose Transporter

Journal of Biological Physics, 2007

Glucose transport in plasma membranes is the prototypic example of facilitated diffusion through ... more Glucose transport in plasma membranes is the prototypic example of facilitated diffusion through biological membranes, and transport in erythrocytes is the most widely studied. One of the oldest and simplest models describing the kinetics of the transport reaction is that of alternating conformers, schematized in a cycle of four partial reactions where glucose binds and dissociates at two opposite steps, and the transporter undergoes transconformations at the other two opposite steps. The transport kinetics is entirely defined by the forward and backward rate constants of the partial reactions and the glucose and transporter concentrations at each side of the membrane, related by the law of mass action. We studied, in silico, the effect of modifications of the variables on the transient kinetics of the transport reaction. The simulations took into account thermodynamic constraints and provided results regarding initial velocities of transport, maximal velocities in different conditions, apparent influx and efflux affinities, and the turnover number of the transporter. The results are in the range of those experimentally reported. Maximal initial velocities are obtained when the affinities of the ligand for the transporter are the same at the extraand intracellular binding sites and when the equilibrium constants of the transconformation steps are equal among them and equal to 1, independently of the obvious effect of the increase of the rate constant values. The results are well adjusted to Michaelis-Menten kinetics. A larger initial velocity for efflux than for uptake described in human erythrocytes is demonstrated in a model with the same dissociation constants at the outer and inner sites of the membrane. The larger velocities observed for uptake and efflux when transport occurs towards a glucose-containing trans side can also be reproduced with the alternating conformer model, depending on how transport velocities are measured.

Research paper thumbnail of A silver-reducing component in rat striated muscle

Histochemistry, 1989

This study reports the presence of a silver-reducing constituent in rat striated muscle fiber loc... more This study reports the presence of a silver-reducing constituent in rat striated muscle fiber located selectively at the level of the terminal cistern/transverse tubule system. It is related to the T tubule network at or near sites that participate in junctions with terminal cisternae, i.e., at both sides of the T tubule in skeletal muscle (triad) and, predominantly, at one side in the ventricle (dyad). Little reactivity is present in the auricle due to the scarcity of those membrane systems. The longitudinal sarcoplasmic reticulum, the sarcolemma, mitochondria and myofibrils are not outlined by the reaction product. Extraction of low molecular weight substances, nucleic acids and lipids did not suppress the chemical reaction. A new argentaffin (H g-A g) technique is described. Ethanol or aldehyde fixed muscles were passed to water, postfixed 6-24 h with mercuric acetate (5% w/v in 1% acetic acid), washed with 1% acetic acid and distilled water, stained 12-24 h at 43 ~ C with ammoniacal silver nitrate (60% w/v) and washed in 10% sodium sulfite (three changes) and water. All steps were carried out in darkness. Postfixation with mercuric acetate proved to be essential for immobilizing the argentaffin component without interfering with its strong argentaffinity. The procedure also provides a simple method for tracing the pathway of transversally oriented membrane systems in skeletal and cardiac muscle cells.

Research paper thumbnail of Stoichiometry of ATP and metal cofactor interaction with the sarcoplasmic reticulum Ca2+–ATPase: A binding model accounting for radioisotopic and fluorescence results

Biophysical Chemistry, 2006

Sarcoplasmic reticulum Ca-ATPase belongs to the P-type ATPases family and transports calcium at t... more Sarcoplasmic reticulum Ca-ATPase belongs to the P-type ATPases family and transports calcium at the expense of ATP hydrolysis. For years, a complex pattern of activity has been observed as a function of ATP and metal cofactor concentrations, leaving the stoichiometry of both metal and ATP in the active site as an open question. In agreement with recent structural studies we present here-using Mn as analogue of Mg-radioisotopic and fluorescence results showing that two metal ions bind to the Ca-ATPase favoring ATP binding. We further show that low ATP concentration favors the binding of these ions, whereas high ATP concentration is inhibitory. We propose a binding model for ATP and metal ions, which permits simulation of our data. Finally, we suggest that (i) the contribution of two metal ions as cofactors of ATP is essential to get maximal activity; (ii) the contribution of two ATP molecules can activate or inhibit the Ca-ATPase depending on metal concentration.

Research paper thumbnail of Calcium additional to that bound to the transport sites is required for full activation of the sarcoplasmic reticulum Ca-ATPase from skeletal muscle

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1998

The sarcoplasmic reticulum Ca-ATPase is fully activated when v1 WM [Ca 2 ] saturates the two tran... more The sarcoplasmic reticulum Ca-ATPase is fully activated when v1 WM [Ca 2 ] saturates the two transport sites; higher [Ca] inhibits the ATPase by competition of Ca-ATP with Mg-ATP as substrates. Here we describe a novel effect of EGTA and other chelators, raising the possibility of an additional activating effect of Ca in the sub-or low WM range. Sarcoplasmic reticulum membranes were isolated from rabbit skeletal muscles. The ATPase activity was measured after incubation at 37³C in 3 mM ATP, 3 mM MgCl 2 , 50 mM MOPS-Tris (pH 7.2), 100 mM KCl, and variable CaCl 2 , EGTA and calcimycin. In the absence of added EGTA and Ca the ATPase activity is high due to contaminant Ca. The determination of the ATPase activity in the presence of increasing amounts of EGTA, without added Ca, yields a decreasing sigmoidal function. K i ranged between 20 and 100 WM, depending on the enzyme concentration. P i production is linear with time for several [EGTA] yielding suboptimal ATPase activities, which are inhibited by thapsigargin. These suboptimal Ca-ATPase activities are inhibited by preincubation of the enzyme in EGTA, at pH 7.2. This effect increases upon increasing EGTA concentration and preincubation time. The inhibitory effect of the previous exposure of the enzyme to EGTA is partially but significantly reverted by increasing [Ca 2 ] during incubations. Calcimycin and EDTA have similar effects as EGTA when added in preincubations. The effect of calcimycin is fully reverted by optimal [Ca 2 ] in incubations. The effects of EGTA, EDTA and calcimycin in preincubation are not additive. The results suggest that an additional calcium, lost during preincubations from a site with affinity near 1 WM, is necessary for full activation of the ATPase.

Research paper thumbnail of Demonstration of the simultaneous activation of Ca2+-independent and Ca2+-dependent ATPases from rat skeletal muscle microsomes

Biochimica et Biophysica Acta (BBA) - Biomembranes, 1988

The activation of the Ca2+-independent (basal) ATPase from rat skeletal muscle microsomes is demo... more The activation of the Ca2+-independent (basal) ATPase from rat skeletal muscle microsomes is demonstrated in the presence of enough Ca 2+ to provide the simultaneous activation of the (Ca 2+ + Mg2+)-ATPase. It was achieved taking advantage of the delayed inorganic phosphate (Pi) release due to the formation of a phosphoenzyme complex during the Ca2+-dependent enzymatic cycle, which is evidenced in fast experiments. The microsomes were immobilized on a filter and perfused at constant flow with an incubation medium which was briefly interrupted with a pulse of appropriate reactants to activate the ATPases, at 2 o C. Successive samples were collected after passing through the filter, at approx. 0.1 s intervals. The Pi effluent profile coincides with the pattern of the pulse when it activates only the Ca2+-independent ATPase, it appears delayed when the pulse activates only extra Pi production by the (Ca2+ + Mg2+)-ATPase, and it includes a rapid and a delayed component when both Ca2+-independent and Ca2+-dependent ATPases are activated simultaneously by the pulse.

Research paper thumbnail of Manganese as a cosubstrate for the phosphorylation of the sarcoplasmic reticulum Ca-dependent adenosine triphosphatase with orthophosphate

Biochimica et Biophysica Acta (BBA) - Bioenergetics, 1996

The phosphorylation of the sarcoplasmic reticulum Ca-ATPase (EC 3.6.1.38) with P~ was characteriz... more The phosphorylation of the sarcoplasmic reticulum Ca-ATPase (EC 3.6.1.38) with P~ was characterized using Mn as a Mg analogue. Steady state and transient fluorescence and radioisotopic techniques were used; the affinities of Mn and Pi for the enzyme and the rate constants of the phosphorylation and dephosphorylation reactions were determined, under several conditions. The reactions were carried out at pH 5.5 to minimize the binding of contaminant Ca to the transport sites, thus avoiding the use of Ca chelators. The apparent affinity of Mn binding at low [Mn] is larger in the absence of Pi (35 txM) than in the presence of saturating Pi (70 p.M). On the contrary, the apparent affinity of Mn for the formation of the phosphoenzyme increases, from 1.5 mM to 0.15 mM, upon increasing [Pi] in the millimolar range. The apparent affinty of Pi for the formation of the phosphoenzyme also increases, from 2.2 mM to 0.2 mM, upon increasing [Mn] in the millimolar range. The equilibrium of the phosphoenzyme with the noncovalent Mn-Pi ' Enzyme complex favors the covalent species. The simulation of a reaction model including the random binding of 2 Mn and 1 Pi per mol of ATPase and a noncovalent complex in equilibrium with the phosphoenzyme, using a set of equilibrium constants deduced from the results, agree with the experimental data.

Research paper thumbnail of A Model Accounting for the Simultaneous Transport of Calcium and Manganese in Sarcoplasmic Reticulum Membranes

Annals of the New York Academy of Sciences, 2003

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Research paper thumbnail of Mn as Cosubstrate for the Phosphorylation of the Sarcoplasmic Reticulum Ca-ATPase by P i

Annals of the New York Academy of Sciences, 1997

Research paper thumbnail of Direct demonstration of an acid-labile phosphoenzyme in the cycle of the sarcoplasmic reticulum Ca2+-dependent adenosinetriphosphatase

Biochimica et Biophysica Acta (BBA) - Biomembranes, 1990

The CaZ+-dependent adenosinetriphosphatase (CaZ+-ATPase) from the sarcoplasmic reticulum (SR) of ... more The CaZ+-dependent adenosinetriphosphatase (CaZ+-ATPase) from the sarcoplasmic reticulum (SR) of rat skeletal muscles is pbosphorylated by inorganic phosphate (Pi) in the absence of Ca z +. The reaction can be described by the following simplified scheme: 1 2 E+ Pi~E-Pi~E-P+ H20 where E-P is a covalent, acid-stable and ADP-insensitive phosphoenzyme, and E. Pi is a noncovalent and acid-labile complex. The reaction is Mg2+-dependent. Membrane fragments deposited on Miilipore filters were successively pertused with two solutions, at constant flow. The effluent samples were analyzed. The pertused solutions were Ca z÷ free and always contained 40% dimethylsulfoxide (DMSO), plus other reactants. Following the successive perfusion of solutions without and with [3ZpIPi, 32p binding is only detected in the presence of Mg 2+, indicating the formation of the phosphoenzymes (E. Pi and E-P). Following pertusions of the phosphoenzymes with 5% trichluroacetic acid, 32p release indicates the mount of the acid-labile moiety (E. Pi). After pbosphorylations, the filters were washed with acid and unlabeled Pi, and the remaining radioactivity was measured to evaluate the acid-stable phosphoenzyme (E-P). The acid-labile and acid-stable phosphoenzymes amounted, respectively, 0.72 5: 0.12, and 1.48 + 0.10 nmol of Pi/mg of protein (+S.E., n = 5), after pbosphorylations with 20 pM Pi. The results indicate: (1) The method allowed the evaluation of the acid-labile intermediate of the SR Ca2+-ATPase cycle. K~=kz/k2, in the above scheme, approaches 2.0. (2) The substrate of the phosphorylation reaction, in the presence of DMSO, is likely to be the Mg • Pi complex, since Mg 2+ is necessary for step 1 in the above scheme.