Rodolfo M. González-Lebrero

Recombinant plant gamma carbonic anhydrase homotrimers bind inorganic carbon

MINT‐7266036: gamma CA2 (uniprotkb:Q9C6B3) and gamma CA2 (uniprotkb:Q9C6B3) physically interact (MI:0914) by molecular sieving (MI:0071)

Are the States That Occlude Rubidium Obligatory Intermediates of the Na+/K+-ATPase Reaction?

In the Albers-Post model, occlusion of K+ in the E 2 conformer of the enzyme (E) is an obligatory step of Na+/K+-ATPase reaction. If this were so the ratio (Na+/K+-ATPase activity)/(concentration of occluded species) should be equal to the rate constant for deocclusion. We tested this prediction in a partially purified Na+/K+-ATPase from pig kidney by means of rapid filtration to measure the occlusion using the K+ congener Rb+. Assuming that always two Rb+ are occluded per enzyme, the steady-state levels of occluded forms and the kinetics of deocclusion were adequately described by the Albers-Post model over a very wide range of [ATP] and [Rb+]. The same happened with the kinetics of ATP hydrolysis. However, the value of the parameters that gave best fit differed from those for occlusion in such a way that the ratio (Na+/K+-ATPase activity)/(concentration of occluded species) became much larger than the rate constant for deocclusion when [Rb+] <10 mm. This points to the presence of an extra ATP hydrolysis that is not Na+-ATPase activity and that does not involve occlusion. A possible way of explaining this is to posit that the binding of a single Rb+ increases ATP hydrolysis without occlusion.

Calcium Occlusion in Plasma Membrane Ca2+-ATPase

In this work, we set out to identify and characterize the calcium occluded intermediate(s) of the plasma membrane Ca2+-ATPase (PMCA) to study the mechanism of calcium transport. To this end, we developed a procedure for measuring the occlusion of Ca2+ in microsomes containing PMCA. This involves a system for overexpression of the PMCA and the use of a rapid mixing device combined with a filtration chamber, allowing the isolation of the enzyme and quantification of retained calcium. Measurements of retained calcium as a function of the Ca2+ concentration in steady state showed a hyperbolic dependence with an apparent dissociation constant of 12 ± 2.2 μm, which agrees with the value found through measurements of PMCA activity in the absence of calmodulin. When enzyme phosphorylation and the retained calcium were studied as a function of time in the presence of LaIII (inducing accumulation of phosphoenzyme in the E1P state), we obtained apparent rate constants not significantly different from each other. Quantification of EP and retained calcium in steady state yield a stoichiometry of one mole of occluded calcium per mole of phosphoenzyme. These results demonstrate for the first time that one calcium ion becomes occluded in the E1P-phosphorylated intermediate of the PMCA.

Binding of a Single Rb+ Increases Na+/K+-ATPase, Activating Dephosphorylation without Stoichiometric Occlusion

We used partially purified Na+/K+-ATPase from pig kidney to study dephosphorylation, occlusion, and ATPase activity in the same enzyme preparation and in media of identical composition containing 10 μm ATP and different concentrations of Rb+, used as a K+ congener. The experiments were performed using a rapid-mixing apparatus with a time resolution of 3.5 ms. The main findings were as follows. (i) At sufficiently low Rb+ concentration the initial rate of dephosphorylation was higher than that of occlusion, (ii) as [Rb+] tended to zero the slope of the time course of occlusion but not that of the time course of dephosphorylation approached zero and, (iii) as Rb+ concentration increased, ATPase activity first increased and, after passing through a maximum, tended to a value that was lower than that observed in media without Rb+. None of these results is compatible with the currently held idea that binding of a single Rb+ to the E2P conformer of the ATPase does not modify the rate of dephosphorylation and strongly suggest that a single Rb+ does promote dephosphorylation through a mechanism that is not stoichiometrically coupled to Rb+ occlusion. If this mechanism is included in the currently accepted scheme for ATP hydrolysis by the Na+/K+-ATPase, a reasonable prediction of the experimental results is obtained.

Steady-State NTPase Activity of Dengue Virus NS3: Number of Catalytic Sites, Nucleotide Specificity and Activation by ssRNA

Dengue virus nonstructural protein 3 (NS3) unwinds double stranded RNA driven by the free energy derived from the hydrolysis of nucleoside triphosphates. This paper presents the first systematic and quantitative characterization of the steady-state NTPase activity of DENV NS3 and their interaction with ssRNA. Substrate curves for ATP, GTP, CTP and UTP were obtained, and the specificity order for these nucleotides - evaluated as the ratio (kcat/KM)- was GTPATPCTP UTP, which showed that NS3 have poor ability to discriminate between different NTPs. Competition experiments between the four substrates indicated that all of them are hydrolyzed in one and the same catalytic site of the enzyme. The effect of ssRNA on the ATPase activity of NS3 was studied using poly(A) and poly(C). Both RNA molecules produced a 10 fold increase in the turnover rate constant (kcat) and a 100 fold decrease in the apparent affinity (KM) for ATP. When the ratio [RNA bases]/[NS3] was between 0 and 20 the ATPase activity was inhibited by increasing both poly(A) and poly(C). Using the theory of binding of large ligands (NS3) to a one-dimensional homogeneous lattice of infinite length (RNA) we tested the hypothesis that inhibition is the result of crowding of NS3 molecules along the RNA lattices. Finally, we discuss why this hypothesis is consistent with the idea that the ATPase catalytic cycle is tightly coupled to the movement of NS3 helicase along the RNA.

Conformational changes produced by ATP binding to the plasma membrane calcium pump

Background: The plasma membrane calcium ATPase (PMCA) reaction cycle is associated with conformational changes. Results: We identified different conformations after the association of Ca2+, ATP, and vanadate to PMCA. Conclusion: PMCA forms a stable complex with Ca2+ and vanadate; ATP can bind to all pump conformations. Significance: This study found a new intermediate in the PMCA reaction cycle; all of the intermediates interact with ATP. The aim of this work was to study the plasma membrane calcium pump (PMCA) reaction cycle by characterizing conformational changes associated with calcium, ATP, and vanadate binding to purified PMCA. This was accomplished by studying the exposure of PMCA to surrounding phospholipids by measuring the incorporation of the photoactivatable phosphatidylcholine analog 1-O-hexadecanoyl-2-O-[9-[[[2-[125I]iodo-4-(trifluoromethyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine to the protein. ATP could bind to the different vanadate-bound states of the enzyme either in the presence or in the absence of Ca2+ with high apparent affinity. Conformational movements of the ATP binding domain were determined using the fluorescent analog 2′(3′)-O-(2,4,6-trinitrophenyl)adenosine 5′-triphosphate. To assess the conformational behavior of the Ca2+ binding domain, we also studied the occlusion of Ca2+, both in the presence and in the absence of ATP and with or without vanadate. Results show the existence of occluded species in the presence of vanadate and/or ATP. This allowed the development of a model that describes the transport of Ca2+ and its relation with ATP hydrolysis. This is the first approach that uses a conformational study to describe the PMCA P-type ATPase reaction cycle, adding important features to the classical E1-E2 model devised using kinetics methodology only.

Kinetic characterization of human thyroperoxidase. Normal and pathological enzyme expression in Baculovirus system: a molecular model of functional expression.

BACKGROUND Human thyroperoxidase (hTPO) is a membrane-bound glycoprotein located at the apical membrane of the thyroid follicular cells which catalyzes iodide oxidation and organification in the thyroglobulin (TG) tyrosine residues, leading to the thyroid hormone synthesis by coupling of iodotyrosine residues. Mutations in hTPO gene are the main cause of iodine organification defects (IOD) in infants. METHODS We investigated the functional impact of hTPO gene missense mutations previously identified in our laboratory (p.C808R, p.G387R and p.P499L). In order to obtain the whole wild-type (WT) coding sequence of hTPO, sequential cloning strategy in pGEMT vector was carried out. Then, site-directed mutagenesis was performed. WT and mutant hTPOs were cloned into the pAcGP67B transfer vector and the recombinant proteins were expressed in Baculovirus System, purified and characterized by SDS-PAGE and Western blot. Moreover, we report for the first time the kinetic constants of hTPO, of both WT and mutant enzymes. RESULTS The functional evaluation of the recombinant hTPOs showed decreased activity in the three mutants with respect to WT. Regarding to the affinity for the substrate, the mutants showed higher Km values with respect to the WT. Additionally, the three mutants showed lower reaction efficiencies (Vmax/Km) with respect to WT hTPO. CONCLUSIONS We optimize the expression and purification of recombinant hTPOs using the Baculovirus System and we report for the first time the kinetic characterization of hTPOs.

Binding of 1 Rb+ Accelerates Dephosphorylation of the Na+,K+-ATPase without Leading to Rb+ Occlusion

Abstract: In steady‐state conditions and for concentrations of the K+‐congener Rb+ less than 2.5 mM, Rb+‐dependent ATPase activity is significantly higher than the steady‐state rate of breakdown of Rb+‐occluded states, a discrepancy that disappears at sufficiently high [Rb+]. Direct experimental evidence is provided that supports the explanation that the binding of a single Rb+ to the phosphoenzyme conformer E2P accelerates dephosphorylation without leading to the occlusion of the cation.

The pathway for spontaneous occlusion of Rb+ in the Na+/K+-ATPase.

Occlusion of K (+) in the Na (+)/K (+)-ATPase can be achieved under two conditions: during hydrolysis of ATP, in media with Na (+) and Mg (2+), after the K (+)-stimulated dephosphorylation of E2P (physiological route) or spontaneously, after binding of K (+) to the enzyme (direct route). We investigated the sidedness of spontaneous occlusion and deocclusion of Rb (+) in an unsided, purified preparation of Na (+)/K (+)-ATPase. Our studies were based on two propositions: (i) in the absence of ATP, deocclusion of K (+) and its congeners is a sequential process where two ions are released according to a single file mechanism, both in the absence and in the presence of Mg (2+) plus inorganic orthophosphate (Pi), and (ii) in the presence of Mg (2+) plus Pi, exchange of K (+) would take place through sites exposed to the extracellular surface of the membrane. The experiments included a double incubation sequence where one of the two Rb (+) ions was labeled as (86)Rb (+). We found that, when the enzyme is in the E2 conformation, the first Rb (+) that entered the enzyme in media without Mg (2+) and Pi was the last to leave after addition of Mg (2+) plus Pi, and vice-versa. This indicates that spontaneous exchange of Rb (+) between E2(Rb 2) and the medium takes place when the transport sites are exposed to the extracellular surface of the membrane. Our results open the question if occlusion and deocclusion via the direct route participates in any significant degree in the transport of K (+) during the ATPase activity.