ATPase Reaction Research Articles

In Ukrainian

This research is aimed at the investigation the electrosurface and biochemical parameters of bacterial cells B. cereus B4368, L. plantarum, E. coli K-A, P. fluorescens B5040 under the influence of copper in ionic form and as nanoparticles in order to determine the nature and level of their toxic effect on bacteria. Copper nanoparticles synthesized in aqueous solution with NaBH4 and stabilized with dextran were used. Changes in membrane transport parameters were assessed by the value of ATPase activity; changes in transmembrane potential were assessed by the method of penetrating tetraphenylphosphonium cations (TPP+); and bacterial integrity was assessed by UV spectroscopy of cellular metabolites. A concentration-dependent inhibition of the membrane ATPase reaction and dissipation of the transmembrane potential under the action of both forms of copper was found, and the inhibitory effect in the case of the nanoparticles was on average 20 % higher than in the ionic form. As a result of heterocoagulation of dextran-stabilized copper nanoparticles and bacteria, a decrease in the negative ξ - potential of bacteria was observed, which was 40 % more effective under the action of copper nanoparticles compared to Cu2+ ions. The most significant changes in membrane parameters were observed in the range 10–60 μM of copper concentrations. With B. cereus B4368 cells taken as an example, we found a violation of the barrier function of their cell membrane under the influence of both copper preparations. In the case of copper nanoparticles, nucleic acid leakage from the bacterial cytoplasm was detected, which was confirmed by the absorption band at 260 nm. The results obtained indicate a high level of sensitivity of the studied electrosurface and biochemical parameters of bacterial cells to the effects of ionic and nanoparticle copper, which allows them to be used as indicators of the toxicity of metal nanoparticles in the development of metal-containing probiotic preparations.

NH125 Sensitizes Staphylococcus aureus to Cell Wall-Targeting Antibiotics through the Inhibition of the VraS Sensor Histidine Kinase.

Staphylococcus aureus utilizes the two-component regulatory system VraSR to receive and relay environmental stress signals, and it is implicated in the development of bacterial resistance to several antibiotics through the upregulation of cell wall synthesis. VraS inhibition was shown to extend or restore the efficacy of several clinically used antibiotics. In this work, we study the enzymatic activity of the VraS intracellular domain (GST-VraS) to determine the kinetic parameters of the ATPase reaction and characterize the inhibition of NH125 under in vitro and microbiological settings. The rate of the autophosphorylation reaction was determined at different GST-VraS concentrations (0.95 to 9.49 μM) and temperatures (22 to 40°C) as well as in the presence of different divalent cations. The activity and inhibition by NH125, which is a known kinase inhibitor, were assessed in the presence and absence of the binding partner, VraR. The effects of inhibition on the bacterial growth kinetics and gene expression levels were determined. The GST-VraS rate of autophosphorylation increases with temperature and with the addition of VraR, with magnesium being the preferred divalent cation for the metal-ATP substrate complex. The mechanism of inhibition of NH125 was noncompetitive in nature and was attenuated in the presence of VraR. The addition of NH125 in the presence of sublethal doses of the cell wall-targeting antibiotics carbenicillin and vancomycin led to the complete abrogation of Staphylococcus aureus Newman strain growth and significantly decreased the gene expression levels of pbpB, blaZ, and vraSR in the presence of the antibiotics. IMPORTANCE This work characterizes the activity and inhibition of VraS, which is a key histidine kinase in a bacterial two-component system that is involved in Staphylococcus aureus antibiotic resistance. The results show the effect of temperature, divalent ions, and VraR on the activity and the kinetic parameters of ATP binding. The value of the KM of ATP is vital in designing screening assays to discover potent and effective VraS inhibitors with high translational potential. We report the ability of NH125 to inhibit VraS in vitro in a noncompetitive manner and investigate its effect on gene expression and bacterial growth kinetics in the presence and absence of cell wall-targeting antibiotics. NH125 effectively potentiated the effects of the antibiotics on bacterial growth and altered the expression of the genes that are regulated by VraS and are involved in mounting a resistance to antibiotics.

Identifiability of enzyme kinetic parameters in substrate competition: a case study of CD39/NTPDase1.

CD39 (NTPDase1-nucleoside triphosphate diphosphohydrolase 1) is a membrane-tethered ectonucleotidase that hydrolyzes extracellular ATP to ADP and ADP to AMP. This enzyme is expressed in a variety of cell types and tissues and has broadly been recognized within vascular tissue to have a protective role in converting "danger" ligands (ATP) into neutral ligands (AMP). In this study, we investigate the enzyme kinetics of CD39 using a Michaelis-Menten modeling framework. We show how the unique situation of having a reaction product also serving as a substrate (ADP) complicates the determination of the governing kinetic parameters. Model simulations using values for the kinetic parameters reported in the literature do not align with corresponding time-series data. This dissonance is explained by CD39 kinetic parameters previously being determined by graphical/linearization methods, which have been shown to distort the underlying error structure and lead to inaccurate parameter estimates. Modern methods of estimating these kinetic parameters using nonlinear least squares are still challenging due to unidentifiable parameter interactions. We propose a workflow to accurately determine these parameters by isolating the ADPase and ATPase reactions and estimating the respective ADPase parameters and ATPase parameters with independent data sets. Theoretically, this ensures all kinetic parameters are identifiable and reliable for future prospective model simulations involving CD39. These kinds of mathematical models can be used to understand how circulating purinergic nucleotides affect disease etiology and potentially inform the development of corresponding therapies.

Tracking lipid-induced effects on membrane protein structure in real time.

Lipids in biological membranes can act as allosteric modulators of membrane protein function. P-type ATPase proteins carry out ATP-dependent transport of mostly ions across several different types of membranes. It has been shown that both general properties such as membrane thickness and interactions to lipids at specific protein sites dictate P-type ATPase activity. We have developed a methodology to track P-type ATPase domain rearrangements in real time triggered by laser-induced release of ATP and subsequent monitoring by synchrotron X-ray pulses. This time-resolved X-ray solution scattering (TR-XSS) technique was used to capture the dynamics of an equilibrium state of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA1a), and transient states at 1.5 and 13 milliseconds. The 13-millisecond state showed a previously unresolved actuator (A) domain arrangement that exposed the ADP-binding site after phosphorylation. To identify the effects of protein-lipid interactions, we have now used the TR-XSS technique to register kinetics and structural dynamics in a Ca2+-transporting P-type ATPase from Listeria monocytogenes (LMCA1) in nanodiscs at different lipid compositions. In negatively charged POPG lipids, the LMCA1 reaction kinetics and structural dynamics were reminiscent of the SERCA1a isoform, which is in agreement with the bacterial membranes being predominantly negatively charged. In uncharged POPC nanodiscs, the LMCA1 reaction was significantly different with slow buildup of the rate-limiting state. The results enable a mechanistic view of how protein-lipid interactions regulate the P-type ATPase reaction cycle. The TR-XSS technique presents a unique possibility to simultaneously determine kinetics and structural rearrangements in P-type ATPases to better understand the transport reactions and their regulation mechanisms.

Role of the reaction-structure coupling in temperature compensation of the KaiABC circadian rhythm.

When the mixture solution of cyanobacterial proteins, KaiA, KaiB, and KaiC, is incubated with ATP in vitro, the phosphorylation level of KaiC shows stable oscillations with the temperature-compensated circadian period. Elucidating this temperature compensation is essential for understanding the KaiABC circadian clock, but its mechanism has remained a mystery. We analyzed the KaiABC temperature compensation by developing a theoretical model describing the feedback relations among reactions and structural transitions in the KaiC molecule. The model showed that the reduced structural cooperativity should weaken the negative feedback coupling among reactions and structural transitions, which enlarges the oscillation amplitude and period, explaining the observed significant period extension upon single amino-acid residue substitution. We propose that an increase in thermal fluctuations similarly attenuates the reaction-structure feedback, explaining the temperature compensation in the KaiABC clock. The model explained the experimentally observed responses of the oscillation phase to the temperature shift or the ADP-concentration change and suggested that the ATPase reactions in the CI domain of KaiC affect the period depending on how the reaction rates are modulated. The KaiABC clock provides a unique opportunity to analyze how the reaction-structure coupling regulates the system-level synchronized oscillations of molecules.

Thermal sensitivity of gill Na+/K+ ATPase activity in juvenile red drum

The red drum is a valuable coastal species distributed throughout the southeastern US where winter cold stress can vary geographically and impact survivorship. We studied the thermal sensitivity of red drum gill Na+/K+ ATPase in order to gain insight regarding temperature effects on osmoregulatory capabilities with potential implications for overwinter survival. We collected gill tissue from age-0 juvenile red drum acclimated to a temperature of 15 °C and measured Na+/K+ ATPase activity at 1, 5, 10, 15, and 25 °C to assess function over a broad thermal range. The Arrhenius plot for Na+/K+ ATPase activity indicated a breakpoint at 8.6 °C, below which greater activation energy was required for the Na+/K+ ATPase reaction. Increased gill Na+/K+ ATPase temperature sensitivity below 8.6 °C may limit the ability of age-0 red drum to maintain ionic and osmotic balance during acute exposure to extreme cold temperatures.

Effect of Binding-Affinity and ATPase Activity on the Velocities of Kinesins Using Ratchet Models.

We use two-state ratchet models containing single and coupled Brownian motors to understand the role of motor-microtubule binding, ATPase reaction rate and dimerisation on the translational velocities of Kinesin motors. We use model parameters derived from the experimental measurements on KIF1A, KIF13A, KIF13B, and KIF16B motors to compute velocities in μm/s. We observe that both the models show the same trend in velocities (KIF1A > KIF13A > KIF13B > KIF16B) as the experimental results. However, the models significantly underpredict the velocities when compared with the experiments. The predictions of the coupled-motor model are closer to the experiments than those of the single-motor model. Our results indicate that the variation of ATPase reaction rate governs the trend in velocities for the above four motors. The variation of motor-microtubule binding affinity and the coupling strength between the motor domains may only have a secondary effect. More rigorous models that incorporate the power-stroke mechanism are necessary for better quantitative compliance with the experiments.

The first application of sensory structures based on photoelectric transducer for the study of enzymatic reactions

Background. The development of highly sensitive sensor equipment with the possibility of registering analytes in real time is a fast growing research area and a promising diagnostic biomedical technology. Currently, the standard laboratory method for determining the activities of ATPses is an indirect spectroscopic study of the concentration of inorganic phosphate formed as a result of ATP hydrolysis by these enzymes. However, there is no commercially available phosphate sensor with satisfactory parameters of sensitivity, selectivity and stability over time. The purpose of our research was the deve­lopment of a photoelectric recombination sensor system for the real-time detection of biochemical markers and its testing on the example of screening ATPase activity of rat erythrocyte plasma membrane suspension preparations. Materials and Methods. Experiments were performed on suspension preparations of plasma membranes of erythrocytes of Wistar rats. Preparations of plasma membrane suspensions obtained by Dodge method from each animal were divided into aliquots and used for the simultaneous study of ATPase activity by the reference method of Rathbun & Betlach, as well as the registration of photocurrents induced during the passage of the ATPase reaction using the photoelectric recombination multisensor system of our own design. Results. The application of silicon sensory structures based on photoelectrical transducer principle for detecting the activity of adenosine triphosphate hydrolases on the example of total Mg2+,Na+,K+-ATPases preparations of plasma membranes of rat erythro­cytes has been experimentally tested. The directly measured analytic parameter is the photocurrent of the deep silicon barrier structure under illumination with high absorption coefficient. The physical features of the device operation have been examined. Detection of such metabolites becomes possible due to reactions intermediates with their own dipole moment (inorganic phosphate, which is one of the products of ATP hydrolysis). The drastic change of photocurrent that characterizes the course of biochemical reaction was observed in real time. The effect is explained by local electrostatic influence on the parameters of recombination centers at the silicon surface that results in surface recombination velocity change. The sensor operation is qualitatively explained in the frame of Stevenson-Keyes’s theory. Conclusions. Our approach can be regarded as a promising way to elaborate technically simple and highly sensitive method for detection of quantitative behavior of enzymatic reactions. Moreover, the local modification of silicon surface allows obtaining time depending scenarios of the adsorption and thus improving the sensitivity of the sensor. These circumstances open up the possibility of elaborating the complex sensory structures with optimized parameters for certain enzymatic reactions.

Measurement of Nucleotide Hydrolysis Using Fluorescent Biosensors for Phosphate.

Assays for the detection of inorganic phosphate (Pi) are widely used to measure the activity of nucleotide hydrolyzing enzymes, such as ATPases and GTPases. The fluorescent biosensors for Pi, described here, are based on fluorescently labeled versions of E. coli phosphate-binding protein (PBP), which translates Pi binding into a large change in fluorescence intensity. In comparison with other Pi-detection systems, these biosensors are characterized by a high sensitivity (sub-micromolar Pi concentrations) and high time resolution (tens of milliseconds), and they are therefore particularly well suited for measurements of phosphate ester hydrolysis in real time. In this chapter, it is described how the Pi biosensors can be used to measure kinetics of ATPase and GTPase reactions, both under steady state and pre-steady state conditions. An example protocol is given for determining steady state kinetic parameters, Km and kcat, of the ATP-dependent chromatin remodeler Chd1, in a plate reader format. In addition, the measurement of Pi release kinetics under pre-steady state conditions is described, including a detailed experimental procedure for a singleturnover measurement of ATP hydrolysis by the ABC-type ATPase SufBC using rapid mixing.

Structural and mechanistic analysis of ATPase inhibitors targeting mycobacterial DNA gyrase.

ObjectivesTo evaluate the efficacy of two novel compounds against mycobacteria and determine the molecular basis of their action on DNA gyrase using structural and mechanistic approaches.MethodsRedx03863 and Redx04739 were tested in antibacterial assays, and also against their target, DNA gyrase, using DNA supercoiling and ATPase assays. X-ray crystallography was used to determine the structure of the gyrase B protein ATPase sub-domain from Mycobacterium smegmatis complexed with the aminocoumarin drug novobiocin, and structures of the same domain from Mycobacterium thermoresistibile complexed with novobiocin, and also with Redx03863.ResultsBoth compounds, Redx03863 and Redx04739, were active against selected Gram-positive and Gram-negative species, with Redx03863 being the more potent, and Redx04739 showing selectivity against M. smegmatis. Both compounds were potent inhibitors of the supercoiling and ATPase reactions of DNA gyrase, but did not appreciably affect the ATP-independent relaxation reaction. The structure of Redx03863 bound to the gyrase B protein ATPase sub-domain from M. thermoresistibile shows that it binds at a site adjacent to the ATP- and novobiocin-binding sites. We found that most of the mutations that we made in the Redx03863-binding pocket, based on the structure, rendered gyrase inactive.ConclusionsRedx03863 and Redx04739 inhibit gyrase by preventing the binding of ATP. The fact that the Redx03863-binding pocket is distinct from that of novobiocin, coupled with the lack of activity of resistant mutants, suggests that such compounds could have potential to be further exploited as antibiotics.

Regulation of the Asymmetric Distribution of Lipids in Human Erythrocyte Membrane in the Presence of Glycerol and Polyethylene Glycol

Cryoprotective agents (CPAs) used to protect erythrocytes during freezing can affect the structural and functional parameters of the membrane, which have an impact on cell viability. The purpose of the study was to examine the effect of glycerol and PEG on the externalization of phosphatidylserine (PS) on the surface of erythrocytes, as well as to determine the role of Ca2+ ions and ATP-dependent processes in the regulation of transmembrane lipid asymmetry in the presence of these CPAs. It was found that the glycerol effect on erythrocytes does not lead to PS externalization. Maintaining the activity of scramblases and flipases in erythrocytes in the presence of glycerol at the level of control parameters allows them to respond similar to native cells to an increase in [Ca2+]in caused by the ionophore A23187 and blocking of ATPase reactions caused by vanadate. The PEG effect contributes to the disruption of the PS distribution in the membrane due to changes in the activity of lipid translocases. However, activation of scramblases and/or inhibition of flipases under the PEG effect does not reach maximum values, as evidenced by an increase in the amount of erythrocytes with externalized PS under cell loading with Ca2+ by ionophore and inhibition of ATP-dependent reactions by vanadate. The effect of PEG on the lipid asymmetry of erythrocyte membranes is apparently mediated by its impact on the Ca2+ level in the cells. Disturbance of the structural parameters of the membrane due to the PS redistribution in the PEG presence, that distinguish it from the glycerol effect, can cause instability of cryopreserved erythrocytes under physiological conditions in vitro.

How Cytoplasmic Dynein Couples ATP Hydrolysis Cycle to Diverse Stepping Motions: Kinetic Modeling

Cytoplasmic dynein is a two-headed molecular motor that moves to the minus end of a microtubule by ATP hydrolysis free energy. By employing its two heads (motor domains), cytoplasmic dynein exhibits various bipedal stepping motions: inchworm and hand-over-hand motions, as well as nonalternating steps of one head. However, the molecular basis to achieve such diverse stepping manners remains unclear because of the lack of an experimental method to observe stepping and the ATPase reaction of dynein simultaneously. Here, we propose a kinetic model for bipedal motions of cytoplasmic dynein and perform Gillespie Monte Carlo simulations that qualitatively reproduce most experimental data obtained to date. The model represents the status of each motor domain as five states according to conformation and nucleotide- and microtubule-binding conditions of the domain. In addition, the relative positions of the two domains were approximated by three discrete states. Accompanied by ATP hydrolysis cycles, the model dynein stochastically and processively moved forward in multiple steps via diverse pathways, including inchworm and hand-over-hand motions, similarly to experimental data. The model reproduced key experimental motility-related properties, including velocity and run length, as functions of the ATP concentration and external force, therefore providing a plausible explanation of how dynein achieves various stepping manners with explicit characterization of nucleotide states. Our model highlights the uniqueness of dynein in the coupling of ATPase with its movement during both inchworm and hand-over-hand stepping.

Conformational dynamics modulate the catalytic activity of the molecular chaperone Hsp90

The heat shock protein 90 (Hsp90) is a molecular chaperone that employs the free energy of ATP hydrolysis to control the folding and activation of several client proteins in the eukaryotic cell. To elucidate how the local ATPase reaction in the active site couples to the global conformational dynamics of Hsp90, we integrate here large-scale molecular simulations with biophysical experiments. We show that the conformational switching of conserved ion pairs between the N-terminal domain, harbouring the active site, and the middle domain strongly modulates the catalytic barrier of the ATP-hydrolysis reaction by electrostatic forces. Our combined findings provide a mechanistic model for the coupling between catalysis and protein dynamics in Hsp90, and show how long-range coupling effects can modulate enzymatic activity.

High Speed AFM Imaging of Structure and Dynamics of Bacterial ABC Transporter MsbA During Lipid Transport

All ATP-binding cassette (ABC) transporters that operate in all species from bacteria to human share a common structural organization, consisting of two distinct domains (i.e., transmembrane domains (TMDs) and cytosolic nucleotide binding domains (NBDs)). MsbA, an ABC transporter, exports lipid A across the cytoplasmic membrane of Gram-negative bacteria. MsbA also exports drugs, conferring multidrug resistance. The crystal and cryo-EM snapshot structures of MsbA bound with different nucleotides have suggested that large conformational changes possibly take place in the TMDs accompanied the ATPase reaction in two NBDs. Therefore, the two NBDs must repeat coupling and uncoupling during multiple ATPase cycles. These dynamic events very likely transmit to the two α-helical bundles connected with the NBDs, so that the TMDs (major parts of the α-helical bundles) undergo different conformations including inward-facing and outward-facing conformations that facilitate substrate entry, translocation and export. However, this structure and dynamics of MsbA during these functional activities have not been verified simultaneously. Using HS-AFM advantage, the structures and dynamics of detergent-solubilized MsbA bound with different nucleotides were successfully real-time imaged to follow their conformational changes. The kinetics of open and closed conformations in NBDs and TMDs was analyzed to evaluate how coupling and uncoupling NBDs could change structural dynamics in TMDs. Next, structural dynamics of TMDs channel and NBDs of MsbA embedded within lipid membrane containing lipid A was also real-time imaged in the presence of different nucleotides and drugs. The translocation of lipid A across TMDs channel was successfully recorded in the controllably oriented MsbA molecules within lipid membrane. Taken together, the dynamic conformational changes in TMDs accompanying ATP hydrolysis in NBDs during lipid A transport unveil a very important operational mechanism in bacterial ABC transporters.

ATPase reaction cycle of P4-ATPases affects their transport from the endoplasmic reticulum.

P4-ATPases belonging to the P-type ATPase superfamily mediate active transport of phospholipids across cellular membranes. Most P4-ATPases, except ATP9A and ATP9B proteins, form heteromeric complexes with CDC50 proteins, which are required for transport of P4-ATPases from the endoplasmic reticulum (ER) to their final destinations. P-type ATPases form autophosphorylated intermediates during the ATPase reaction cycle. However, the association of the catalytic cycle of P4-ATPases with their transport from the ER and their cellular localization has not been studied. Here, we show that transport of ATP9 and ATP11 proteins as well as that of ATP10A from the ER depends on the ATPase catalytic cycle, suggesting that conformational changes in P4-ATPases during the catalytic cycle are crucial for their transport from the ER.

Interplay of actin, ADP and Mg2+ interactions with striated muscle myosin: Implications of their roles in ATPase

The effects of Mg2+ on the interaction between ADP, a product of the ATPase reaction, and striated muscle myosin-subfragment 1 (S1) were investigated with both functional and spectroscopic methods. Mg2+ inhibited striated muscle myosin ATPase in the presence of F-actin. Significant effects of Mg2+ were observed in both rate constants of NOE build-up and maximal intensities in WaterLOGSY NMR experiments as F-actin concentration increased. In the absence of F-actin, myosin S1 with Mg2+ bound to a fluorescent ADP analog about five-times tighter than without Mg2+. In the presence of F-actin, the affinity of myosin S1 toward the ADP analog significantly decreased both with and without Mg2+. The equilibrium titration of myosin-S1 into F-actin revealed that in the presence of ADP the apparent dissociation constant (Kd) without Mg2+ was more than five-fold smaller than with Mg2+. Further, we examined effects of F-actin, ADP and Mg2+ binding to myosin on the tertiary structure of myosin-S1 using near UV circular dichroism (CD) spectroscopy. Both in the presence and absence of ADP, there was a Mg2+-dependent difference in the near UV CD spectra of actomyosin. Our results show that Mg2+ affects myosin-ADP and actin-myosin interactions which may be reflected in myosin ATPase activity.

The bacterial Mre11-Rad50 homolog SbcCD cleaves opposing strands of DNA by two chemically distinct nuclease reactions.

The Mre11–Rad50 complex is a DNA double-strand break sensor that cleaves blocked DNA ends and hairpins by an ATP-dependent endo/exonuclease activity for subsequent repair. For that, Mre11–Rad50 complexes, including the Escherichia coli homolog SbcCD, can endonucleolytically cleave one or both strands near a protein block and process free DNA ends via a 3′-5′ exonuclease, but a unified basis for these distinct activities is lacking. Here we analyzed DNA binding, ATPase and nuclease reactions on different DNA substrates. SbcCD clips terminal bases of both strands of the DNA end in the presence of ATPγS. It introduces a DNA double-strand break around 20–25 bp from a blocked end after multiple rounds of ATP hydrolysis in a reaction that correlates with local DNA meltability. Interestingly, we find that nuclease reactions on opposing strands are chemically distinct, leaving a 5′ phosphate on one strand, but a 3′ phosphate on the other strand. Collectively, our results identify an unexpected chemical variability of the nuclease, indicating that the complex is oriented at a free DNA end and facing a block with opposite polarity. This suggests a unified model for ATP-dependent endo- and exonuclease reactions at internal DNA near a block and at free DNA ends.

Competitive cation binding computations of proton balance for reactions of the phosphagen and glycolytic energy systems within skeletal muscle.

Limited research and data has been published for the H+ coefficients for the metabolites and reactions involved in non-mitochondrial energy metabolism. The purpose of this investigation was to compute the fractional binding of H+, K+, Na+ and Mg2+ to 21 metabolites of skeletal muscle non-mitochondrial energy metabolism, resulting in 104 different metabolite-cation complexes. Fractional binding of H+ to these metabolite-cation complexes were applied to 17 reactions of skeletal muscle non-mitochondrial energy metabolism, and 8 conditions of the glycolytic pathway based on the source of substrate (glycogen vs. glucose), completeness of glycolytic flux, and the end-point of pyruvate vs. lactate. For pH conditions of 6.0 and 7.0, respectively, H+ coefficients (-‘ve values = H+ release) for the creatine kinase, adenylate kinase, AMP deaminase and ATPase reactions were 0.8 and 0.97, -0.13 and -0.02, 1.2 and 1.09, and -0.01 and -0.66, respectively. The glycolytic pathway is net H+ releasing, regardless of lactate production, which consumes 1 H+. For glycolysis fueled by glycogen and ending in either pyruvate or lactate, H+ coefficients for pH 6.0 and 7.0 were -3.97 and -2.01 (pyruvate), and -1.96 and -0.01 (lactate), respectively. When starting with glucose, the same conditions result in H+ coefficients of -3.98 and -2.67, and -1.97 and –0.67, respectively. The most H+ releasing reaction of glycolysis is the glyceraldehyde-3-phosphate dehydrogenase reaction, with H+ coefficients for pH 6.0 and 7.0 of -1.58 and -0.76, respectively. Incomplete flux of substrate through glycolysis would increase net H+ release due to the absence of the pyruvate kinase and lactate dehydrogenase reactions, which collectively result in H+ coefficients for pH 6.0 and 7.0 of 1.35 and 1.88, respectively. The data presented provide an extensive reference source for academics and researchers to accurately profile the balance of protons for all metabolites and reactions of non-mitochondrial energy metabolism, and reveal the greater role of glycolysis in net H+ release than previously assumed. The data can also be used to improve the understanding of the cause of metabolic acidosis, and reveal mechanistic connections between H+ release within and from muscle and the electrochemical neutrality concepts that further refine acid-base balance in biological solutions.

Mg 2+ -dependent ATPase activity in triatomine salivary glands (Heteroptera, Triatominae)

ABSTRACT Mg2+-ATPase activity was detected in the three salivary glands of adult triatomines, males and females, of Triatoma infestans (Klug, 1834) and Panstrongylus megistus (Burmeister, 1835) (Heteroptera, Triatominae). A predominance of binucleated cells in D1 and D2 and mononucleated in D3 was observed, with bulky and polyploidy nuclei. ATPase activity was detected in the nuclei, possibly in euchromatin and nucleolus, where this enzyme probably acts in the transcription process. ATPase reaction was also evidenced in the nuclear membrane, which is probably associated with nuclear-cytoplasmatic transport. These characteristics indicate a high metabolism and protein synthesis, which must be essential to saliva production as well as in maintaining the hematophagy of triatomines.

Sequential allosteric mechanism of ATP hydrolysis by the CCT/TRiC chaperone is revealed through Arrhenius analysis

Knowing the mechanism of allosteric switching is important for understanding how molecular machines work. The CCT/TRiC chaperonin nanomachine undergoes ATP-driven conformational changes that are crucial for its folding function. Here, we demonstrate that insight into its allosteric mechanism of ATP hydrolysis can be achieved by Arrhenius analysis. Our results show that ATP hydrolysis triggers sequential ‟conformational waves." They also suggest that these waves start from subunits CCT6 and CCT8 (or CCT3 and CCT6) and proceed clockwise and counterclockwise, respectively.