Juan L. Rendón

Signaling the Signal, Cyclic AMP-dependent Protein Kinase Inhibition by Insulin-formed H2O2 and Reactivation by Thioredoxin

Catecholamines in adipose tissue promote lipolysis via cAMP, whereas insulin stimulates lipogenesis. Here we show that H2O2 generated by insulin in rat adipocytes impaired cAMP-mediated amplification cascade of lipolysis. These micromolar concentrations of H2O2 added before cAMP suppressed cAMP activation of type IIβ cyclic AMP-dependent protein kinase (PKA) holoenzyme, prevented hormone-sensitive lipase translocation from cytosol to storage droplets, and inhibited lipolysis. Similarly, H2O2 impaired activation of type IIα PKA holoenzyme from bovine heart and from that reconstituted with regulatory IIα and catalytic α subunits. H2O2 was ineffective (a) if these PKA holoenzymes were preincubated with cAMP, (b) if added to the catalytic α subunit, which is active independently of cAMP activation, and (c) if the catalytic α subunit was substituted by its C199A mutant in the reconstituted holoenzyme. H2O2 inhibition of PKA activation remained after H2O2 elimination by gel filtration but was reverted with dithiothreitol or with thioredoxin reductase plus thioredoxin. Electrophoresis of holoenzyme in SDS gels showed separation of catalytic and regulatory subunits after cAMP incubation but a single band after H2O2 incubation. These data strongly suggest that H2O2 promotes the formation of an intersubunit disulfide bond, impairing cAMP-dependent PKA activation. Phylogenetic analysis showed that Cys-97 is conserved only in type II regulatory subunits and not in type I regulatory subunits; hence, the redox regulation mechanism described is restricted to type II PKA-expressing tissues. In conclusion, phylogenetic analysis results, selective chemical behavior, and the privileged position in holoenzyme lead us to suggest that Cys-97 in regulatory IIα or IIβ subunits is the residue forming the disulfide bond with Cys-199 in the PKA catalytic α subunit. A new molecular point for cross-talk among heterologous signal transduction pathways is demonstrated.

Hysteresis in thioredoxin-glutathione reductase (TGR) from the adult stage of the liver fluke Fasciola hepatica

Thioredoxin-glutathione reductase (TGR) was purified from the adult stage of the liver fluke Fasciola hepatica. At 38° C and pH 7.8, specific activity values were 10.2U mg(-1) and 64.5U mg(-1), with DTNB or GSSG as substrates, respectively. Under the same conditions, apparent Km values were 46±8 μM (DTNB) and 30 ± 5 μM (GSSG). The enzyme was also able to catalyze thiol/disulfide exchange reactions. A subunit Mr of 61,000 was obtained. Like the homologous enzyme from the tapeworms, a lag time was observed in the enzyme assays at moderate or high concentrations of the substrate GSSG. The hysteretic behavior was reverted in the presence of GSH and was notably dependent on pH, such that the magnitude of the lag time increased with the acidity of the medium. These results strongly suggest that a hysteretic kinetic is a common feature of TGR from any parasitic flatworm. A sequence comparison revealed the structural cysteine residues proposed to be in the origin of the peculiar kinetic behavior of TGR are absent from the F. hepatica enzyme. Based on these observations, the model proposed recently to explain the GSSG-dependent hysteretic kinetic of TGR, which assumes the covalent modification of specific cysteine residues through glutathionylation [Bonilla M. et al. (2008) J Biol Chem 283: 17898] needs to be reevaluated.

Mitochondrial Thioredoxin-Glutathione Reductase from Larval Taenia crassiceps (Cysticerci)

Mitochondrial thioredoxin-glutathione reductase was purified from larval Taenia crassiceps (cysticerci). The preparation showed NADPH-dependent reductase activity with either thioredoxin or GSSG, and was able to perform thiol/disulfide exchange reactions. At 25°C specific activities were 437 ± 27 mU mg−1 and 840 ± 49 mU mg−1 with thioredoxin and GSSG, respectively. Apparent Km values were 0.87 ± 0.04 μM, 41 ± 6 μM and 19 ± 10 μM for thioredoxin, GSSG and NADPH, respectively. Thioredoxin from eukaryotic sources was accepted as substrate. The enzyme reduced H2O2 in a NADPH-dependent manner, although with low catalytic efficiency. In the presence of thioredoxin, mitochondrial TGR showed a thioredoxin peroxidase-like activity. All disulfide reductase activities were inhibited by auranofin, suggesting mTGR is dependent on selenocysteine. The reductase activity with GSSG showed a higher dependence on temperature as compared with the DTNB reductase activity. The variation of the GSSG- and DTNB reductase activities on pH was dependent on the disulfide substrate. Like the cytosolic isoform, mTGR showed a hysteretic kinetic behavior at moderate or high GSSG concentrations, but it was less sensitive to calcium. The enzyme was able to protect glutamine synthetase from oxidative inactivation, suggesting that mTGR is competent to contend with oxidative stress.

The alternative NADH dehydrogenase is present in mitochondria of some animal taxa

The distribution of the alternative NADH dehydrogenase (NDH-2) in the living world was explored. The enzyme, although present in representatives of all living kingdoms, does not have a universal distribution. With the exception of ε-proteobacteria, the enzyme was found in all eubacterial groups. In contrast with the known presence of the NDH-2 in Archaea, the alternative oxidase (AOX) is absent in this group. With regard to the Eukarya domain, the NDH-2 was found in representatives of Protista, Fungi, Plantae, and Animalia. In the latter, however, the presence of the enzyme was restricted to some primitive Metazoa (Placozoa and Cnidaria), and two members of the Deuterostomate lineage of the Bilateria (Echinodermata and Urochordata). No evidence for the presence of the NDH-2 was found in any representative of the Protostomate branch of the Bilateria, contrasting with the existence of the AOX in this same group. It is worth mentioning that those animal species containing the NDH-2 also have an AOX. The actual distribution of the NDH-2 in the various living kingdoms is discussed within the framework of the endosymbiotic theory; in addition, a hypothesis is proposed to explain the disappearance of the alternative NDH-2 and AOX from the majority of the animals.

Auranofin-induced oxidative stress causes redistribution of the glutathione pool in Taenia crassiceps cysticerci.

Previously, we have studied the effect of the gold-compound auranofin (AF) on both thioredoxin-glutathione reductasa (TGR) activity and viability of Taenia crassiceps cysticerci. It was demonstrated that micromolar concentrations of AF were high enough to fully inhibit TGR and kill the parasites. In this work, the dynamics of changes in the glutathione pool of T. crassiceps cysticerci following the addition of AF, was analyzed. A dose-dependent decrease in the internal glutathione concentration, concomitant with an increase in ROS production was observed. These changes were simultaneous with the formation of glutathione-protein complexes and the export of glutathione disulfide (GSSG) to the culture medium. Incubation of cysticerci in the presence of both AF and N-acetyl cysteine (NAC) prevents all the above changes, maintaining cysticerci viability. By contrast, the presence of both AF and buthionine sulfoximine (BSO) resulted in a potentiation of the effects of the gold compound, jeopardizing cysticerci viability. These results suggest the lethal effect of AF on T. crassiceps cysticerci, observed at micromolar concentrations, can be explained as a consequence of major changes in the glutathione status, which results in a significant increase in the oxidative stress of the parasites.

Presence of two enzymes, different from the F1F0-ATPase, hydrolyzing nucleotides in human term placental mitochondria.

The hydrolysis of ATP, ADP or GTP was characterized in mitochondria and submitochondrial particles since a tightly-bound ATPase associated with the inner mitochondrial membrane from the human placenta has been described. Submitochondrial particles, which are basically inner membranes, were used to define the location of this enzyme. Mitochondria treated with trypsin and specific inhibitors were also used. The oxygen consumption stimulated by ATP or ADP was 100% inhibited in intact mitochondria by low concentrations of oligomycin (0.5 microgram/mg) or venturicidine (0.1 microgram/mg), while the hydrolysis of ATP or ADP was insensitive to higher concentrations of these inhibitors but it was inhibited by vanadate. Oligomycin or venturicidine showed a different inhibition pattern in intact mitochondria in relation to the hydrolysis of ATP, ADP or GTP. When submitochondrial particles were isolated from mitochondria incubated with oligomycin or venturicidine, no further inhibition of the nucleotide hydrolysis was observed, contrasting with the partial inhibition observed in the control. By incubating the placental mitochondria with trypsin, a large fraction of the hydrolysis of nucleotides was eliminated. In submitochondrial particles obtained from mitochondria treated with trypsin or trypsin plus oligomycin, the hydrolysis of ATP was 100% sensitive to oligomycin at low concentrations, resembling the oxygen consumption; however, this preparation still showed some ADP hydrolysis. Native gel electrophoresis showed two bands hydrolyzing ADP, suggesting at least two enzymes involved in the hydrolysis of nucleotides, besides the F1F0-ATPase. It is concluded that human placental mitochondria possesses ADPase and ATP-diphosphohydrolase activities (247).

Dimer-tetramer equilibrium of glutathione reductase from the cyanobacterium Spirulina maxima

Glutathione reductase [NAD(P)H:GSSG oxidoreductase; EC 1.6.4.2] from cyanobacterium Spirulina maxima exists as an equilibrium system between a dimer (S20,W = 5.96) and a tetramer (S20,W = 8.49) which has a very slow interconversion rate at neutral pH. Our results showed that the apparent dissociation constant (kd) was 4.61 X 10(-7) M. The proportion of both forms at pH 7.0 did not alter at either 4 or 25 degrees C. However, electrophoretic analysis at various pH values showed that at 25 degrees C a gradual transition takes place between oligomers with an apparent pKa of 7.55. When dimers aggregate to form tetramers, the reaction involves the uptake of eight protons (K = 1.58 X 10(-64) M9). At pH 7.7, the equilibrium shifts completely from dimers-tetramers to dimers when temperature is increased, which would suggest that the dissociation is an endothermic process. Thermodynamic parameters obtained from the temperature study show that the dissociation of glutathione reductase is characterized by positive entropy and enthalpy changes. Neither NADPH nor GSSG have any effect on the dimer-tetramer equilibrium. Measurements of reductase activity indicate that the tetramer is almost certainly active, whereas the dimer is either less active or inactive.

Membrane potential regulates mitochondrial ATP-diphosphohydrolase activity but is not involved in progesterone biosynthesis in human syncytiotrophoblast cells.

ATP-diphosphohydrolase is associated with human syncytiotrophoblast mitochondria. The activity of this enzyme is implicated in the stimulation of oxygen uptake and progesterone synthesis. We reported previously that: (1) the detergent-solubilized ATP-diphosphohydrolase has low substrate specificity, and (2) purine and pyrimidine nucleosides, tri- or diphosphates, are fully dephosphorylated in the presence of calcium or magnesium (Flores-Herrera 1999, 2002). In this study we show that ATP-diphosphohydrolase hydrolyzes first the nucleoside triphosphate to nucleoside diphosphate, and then to nucleotide monophosphate, in the case of all tested nucleotides. The activation energies (Ea) for ATP, GTP, UTP, and CTP were 6.06, 4.10, 6.25, and 5.26 kcal/mol, respectively; for ADP, GDP, UDP, and CDP, they were 4.67, 5.42, 5.43, and 6.22 kcal/mol, respectively. The corresponding Arrhenius plots indicated a single rate-limiting step for each hydrolyzed nucleoside, either tri- or diphosphate. In intact mitochondria, the ADP produced by ATP-diphosphohydrolase activity depolarized the membrane potential (ΔΨm) and stimulated oxygen uptake. Mitochondrial respiration showed the state-3/state-4 transition when ATP was added, suggesting that ATP-diphosphohydrolase and the F1F0-ATP synthase work in conjunction to avoid a futile cycle. Substrate selectivity of the ATP-diphosphohydrolase was modified by ΔΨm (i.e. ATP was preferred over GTP when the inner mitochondrial membrane was energized). In contrast, dissipation of ΔΨm by CCCP produced a loss of substrate specificity and so the ATP-diphosphohydrolase was able to hydrolyze ATP and GTP at the same rate. In intact mitochondria, ATP hydrolysis increased progesterone synthesis as compared with GTP. Although dissipation of ΔΨm by CCCP decreased progesterone synthesis, NADPH production restores steroidogenesis. Overall, our results suggest a novel physiological role for ΔΨm in steroidogenesis.

Purification and characterization of a lectin from Erythrina americana by affinity chromatography

Abstract A lectin from the seeds of Erythrina americana Mill has been purified by affinity chromatography with a human type O red blood cell stroma column. In its monomeric form the lectin, which is a glycoprotein with a 7% sugar content by weight, has a molecular weight of 30 000. Ultracentrifugation analysis indicates that the lectin is a dimer with a molecular weight of 57 000 and a Sw,20 value of 4.1. Isoelectric focusing reveals the presence of two major molecular components with pI values of 6.3 and 6.6. The lectin is characterized by a high content of leucine, tyrosine, phenylalanine and lysine, a low content of histidine, arginine and methionine, and the absence of cysteine. The composition of the lectin saacharidic portion shows N- acetyl - d - glucosamine , mannose, fusoce and xylose in a molar ratio of 4:3:1:1, respectively. The hemagglutinating activity of the lectin lacks species-specificity, is not modified by treatment with 0.1 M ethylenediamine tetraacetic acid in 1 M acetic acid, and is abolished by galactose, lactose and lactosaminic-containing oligosaccharides or glycosylpeptides. Moreover, N-glycosidically bonded oligosaccharides composed of tri or tetra-antennary structures, derived from fetuin and orosomucoid, with galactose residues in terminal position, are much better inhibitors.

Thermal denaturation of glutathione reductase from cyanobacterium Spirulina maxima

The thermal unfolding of glutathione reductase (NAD[P]H:GSSG oxidoreductase EC 1.6.4.2.) from cyanobactefium Spirulina maxima was monitored by differential scanning calorimetry and circular dichroism at neutral pH. Covalent cross‐linking of enzyme at different temperatures revealed dimer as the species undergoing the thermal transition. A single endotherm was observed, but its thermodynamic parameters showed dependence on the scan rate. In the transition zone, aggregation of the dimeric species was observed. Analysis of the enzyme heated at 80°C revealed that the resultant species retained a high content of secondary structure. The addition of low concentrations of guanidinium hydrochlofide resulted in a full cooperative thermal transition. A model in which the dimeric protein undergoes a partial unfolding in a kinetically controlled fashion is proposed, such that the experimental value of ΔHcal results from the simultaneous occurrence of endothermic and exothermic events.