Julio A. Mignaco

Subunit-subunit interactions and overall topology of the dimeric mitochondrial ATP synthase of Polytomella sp.

Mitochondrial F1F0-ATP synthase of chlorophycean algae is a dimeric complex of 1600 kDa constituted by 17 different subunits with varying stoichiometries, 8 of them conserved in all eukaryotes and 9 that seem to be unique to the algal lineage (subunits ASA1-9). Two different models proposing the topological assemblage of the nine ASA subunits in the ATP synthase of the colorless alga Polytomella sp. have been put forward. Here, we readdressed the overall topology of the enzyme with different experimental approaches: detection of close vicinities between subunits based on cross-linking experiments and dissociation of the enzyme into subcomplexes, inference of subunit stoichiometry based on cysteine residue labelling, and general three-dimensional structural features of the complex as obtained from small-angle X-ray scattering and electron microscopy image reconstruction. Based on the available data, we refine the topological arrangement of the subunits that constitute the mitochondrial ATP synthase of Polytomella sp.

Effects of Photo-oxidizing Analogs of Fluorescein on the Sarcoplasmic Reticulum Ca2+-ATPase FUNCTIONAL CONSEQUENCES FOR SUBSTRATE HYDROLYSIS AND EFFECTS ON THE PARTIAL REACTIONS OF THE HYDROLYTIC CYCLE

Erythrosin B was used to photo-oxidize the sarcoplasmic reticulum Ca2+-ATPase. The ATPase activity is rapidly and irreversibly inhibited by photo-oxidation with erythrosin. This inhibition is protected by the presence of ATP during the photo-oxidation period. After photo-oxidation, the steady-state phosphorylation by ATP remains almost unchanged, whereas phosphorylation by inorganic phosphate is impaired. The pseudo-first order rate constants for phosphorylation by 15 μM ATP at 25°C are strongly inhibited when starting from either a Ca2+-bound or a Ca2+-free enzyme form, decreasing from 145 to 23 s−1 for the Ca2+-bound form and from 50 to 18 s−1 for the Ca2+-free form. Concurrently, the rate constants for dephosphorylation are also severely inhibited, changing from a fast double exponential to a very slow single exponential decay in the reverse direction and from a moderately slow single to a very slow single exponential decay in the forward direction. Ca2+ binding data show that the phosphorylated intermediate formed by the photo-oxidized enzyme contains two occluded Ca2+, and TNP-ATP fluorescence measurements indicate that it accumulates in a E1-P·;Ca2-like conformation. Protection by ADP against glutaraldehyde-induced cross-linking indicates that ADP binding to Ca2+-ATPase is not impaired by photo-oxidation nor by free erythrosin. These data support the view that an ADP-insensitive, Ca2+-bound, slowly interconverting phosphoenzyme is formed. Thus, photo-oxidation with erythrosin B leads to impairment of phosphoryl transfer reactions and related conformational changes.

Could Na,K-ATPase play a role in potassium leakage from irradiated erythrocytes?

BACKGROUND In developing countries, the access to red blood cell (RBC) irradiators is restricted. Thus, it is a common practice in blood banks to stock irradiated RBC units until they expire. The aim of this work is to elucidate the involvement of Na,K-ATPase in potassium leakage from prophylactically irradiated RBCs. METHODS Whole blood was collected from healthy donors, and blood concentrates were irradiated with 25Gy of γ-radiation within 24h of collection. At days 3, 5, 7, 9, 11, 14 and 28 post-irradiation, fractions were removed and centrifuged and Na,K-ATPase activity from ghost membranes was determined. RESULTS The inhibition of Na,K-ATPase activity in RBCs reached 12.6% by day 7 of storage and up to 50% by day 14 of storage. The addition of vitamin C prevented the irradiation-induced loss of Na,K-ATPase activity. The irradiation of RBCs provoked an increase in potassium plasma levels and a decrease in sodium plasma levels. The incubation of RBCs with ouabain did not change the sodium or potassium levels in the plasma, and the addition of vitamin C only partially prevented a decrease in sodium levels caused by irradiation. CONCLUSION Because the inhibition of Na,K-ATPase by ouabain did not cause potassium accumulation in the plasma, we conclude that the irradiation-induced inhibition of the pump is not a key factor driving this effect.

Chelerythrine inhibits the sarco/endoplasmic reticulum Ca2+-ATPase and results in cell Ca2+ imbalance

The isoquinoline alkaloid chelerythrine is described as an inhibitor of SERCA. The ATPase inhibition presented two non-competitive components, Ki1=1, 2 μM and Ki2=26 μM. Conversely, chelerythrine presented a dual effect on the p-nitrophenylphosphatase (pNPPase) of SERCA. Ca(2+)-dependent pNPPase was activated up to ∼5 μM chelerythrine with inhibition thereafter. Ca(2+)-independent pNPPase was solely inhibited. The phosphorylation of SERCA with ATP reached half-inhibition with 10 μM chelerythrine and did not parallel the decrease of ATPase activity. In contrast, chelerythrine up to 50 μM increased the phosphorylation by Pi. Cross-linking of SERCA with glutaraldehyde was counteracted by high concentrations of chelerythrine. The controlled tryptic digestion of SERCA shows that the low-affinity binding of chelerythrine evoked an E2-like pattern. Our data indicate a non-competitive inhibition of ATP hydrolysis that favors buildup of the E2-conformers of the enzyme. Chelerythrine as low as 0.5-1.5 μM resulted in an increase of intracellular Ca(2+) on cultured PBMC cells. The inhibition of SERCA and the loss of cell Ca(2+) homeostasis could in part be responsible for some described cytotoxic effects of the alkaloid. Thus, the choice of chelerythrine as a PKC-inhibitor should consider its potential cytotoxicity due to the alkaloids effects on SERCA.

Mechanism of modulation of the plasma membrane Ca2+-ATPase by arachidonic acid

The intracellular level of long chain fatty acids controls the Ca(2+) concentration in the cytoplasm. The molecular mechanisms underlying this Ca(2+) mobilization are not fully understood. We show here that the addition of low micromolar concentrations of fatty acids directly to the purified plasma membrane Ca(2+)-ATPase enhance ATP hydrolysis, while higher concentration decrease activity, exerting a dual effect on the enzyme. The effect of arachidonic acid is similar in the presence or absence of calmodulin, acidic phospholipids or ATP at the regulatory site, thereby precluding these sites as probable acid binding sites. At low arachidonic acid concentrations, neither the affinity for calcium nor the phosphoenzyme levels are significantly modified, while at higher concentrations both are decreased. The action of arachidonic acid is isoenzyme specific. The increase on ATP hydrolysis, however, is uncoupled from calcium transport, because arachidonic acid increases the permeability of erythrocyte membranes to calcium. Oleic acid has no effect on membrane permeability while linoleic acid shows an effect similar to that of arachidonic acid. Such effects might contribute to the entry of extracellular Ca(2+) following to fatty acid release.

3-O-Methylfluorescein phosphate as a fluorescent substrate for plasma membrane Ca2+-ATPase

3-O-methylfluorescein phosphate hydrolysis, catalyzed by purified erythrocyte Ca2+-ATPase in the absence of Ca2+, was slow in the basal state, activated by phosphatidylserine and controlled proteolysis, but not by calmodulin. p-Nitrophenyl phosphate competitively inhibits hydrolysis in the absence of Ca2+, while ATP inhibits it with a complex kinetics showing a high and a low affinity site for ATP. Labeling with fluorescein isothiocyanate impairs the high affinity binding of ATP, but does not appreciably modify the binding of any of the pseudosubstrates. In the presence of calmodulin, an increase in the Ca2+ concentration produces a bell-shaped curve with a maximum at 50 microM Ca2+. At optimal Ca2+ concentration, hydrolysis of 3-O-methylfluorescein phosphate proceeds in the presence of fluorescein isothiocyanate, is competitively inhibited by p-nitrophenyl phosphate and, in contrast to the result observed in the absence of Ca2+, it is activated by calmodulin. In marked contrast with other pseudosubstrates, hydrolysis of 3-O-methylfluorescein phosphate supports Ca2+ transport. This highly specific activity can be used as a continuous fluorescent marker or as a tool to evaluate partial steps from the reaction cycle of plasma membrane Ca2+-ATPases.

ATPase and phosphatase activities are differentially inhibited by photo-oxidation of the sarcoplasmic reticulum Ca2+-ATPase

We have already described that photo-oxidation of the sarcoplasmic reticulum Ca(2+)-ATPase with the halogenated dye erythrosin B produces inhibition of the ATPase activity (J.A. Mignaco et al., Biochemistry 35 (1996) 3886-3891). We now show that the Ca(2+)-dependent and Ca(2+)-independent p-nitrophenylphosphatase activities are also inhibited by this treatment. Modification of rapidly (< 10 min) oxidized residue(s) is responsible for the major loss of ATPase activity, whereas photo-inhibition of the phosphatase activities occurs more slowly (t1/2 20-30 min). Here we have focused on photo-inhibition of the Ca(2+)-independent pNPPase activity, and the counteracting effects of ATP and FITC. Following photo-oxidation, the Ca(2+)-independent pNPPase activity decreases monotonically. ATP partially protects against the inactivation of the pNPPase, whereas labeling the enzyme with FITC does not. However, the protective effect of ATP is completely abolished by the attached FITC. These data are interpreted in terms of two different sites that are susceptible to photo-oxidation and are involved in different events related to substrate hydrolysis.

Ouabain attenuates the oxidative stress induced by lipopolysaccharides in the cerebellum of rats

Our study aimed to analyze the effect of ouabain administration on lipopolysaccharide (LPS)‐induced changes in oxidative parameters, membrane lipid composition, and the activities of some important enzymes of the nervous system. The content of phospholipids, cholesterol, and gangliosides were analyzed in Wistar rats after intraperitoneal injection of ouabain (1.8 μg/kg), LPS (200 μg/kg), or saline. Oxidative parameters were also evaluated, including the activities of superoxide dismutase, catalase and glutathione peroxidase, the levels of glutathione and lipid peroxidation, as well as Na,K‐ATPase activity and the level of glutamate transporter EAAT4. Administration of LPS resulted in increased oxidative stress, as evidenced by an increase in lipid peroxidation levels, glutathione peroxidase activity, decreased catalase activity and reduced glutathione levels. All changes recorded were attenuated by pretreatment with ouabain. Administration of ouabain plus LPS enhanced the total ganglioside content and EAAT4 levels, but failed to alter the Na,K‐ATPase activity. Our data suggest a neuroprotective effect of ouabain against LPS‐induced oxidative stress by promoting membrane lipid remodeling and increasing the expression of glutamate transporter EAAT4. Our results emphasize that the observed oxidative stress is not correlated with Na,K‐ATPase, but with a possible ouabain‐mediated effect on cellular signaling. The relevance of our results extends beyond LPS‐induced changes in oxidative parameters, as nanomolar doses of ouabain might prove useful in neurodegenerative models. Further study of other cardenolides and related molecules, as well as the development of new molecules derived from ouabain, could also prove useful in the fight against the oxidative and/or general cell stress triggered by neuronal pathologies.

Properties of novel surfactin-derived biosurfactants obtained through solid-phase synthesis

Eight molecules, four peptides (SPs) and four lipopeptides (LPs) derived by rational design from surfactin, a well‐known secreted biosurfactant from Bacillus subtilis, were produced employing Fmoc‐based solid‐phase synthesis. These new peptides were tested to evaluate their potential biosurfactant and biological activities, aiming at possible applications in industrial, biological, pharmaceutical, and medical use. Five molecules (SP1, SP2, SP4, LP5, and LP8) presented potential for medical uses, mainly due to their drug delivery properties as suggested by their synergistic activity with the antibiotic vancomycin against Staphylococcus aureus. All synthetic peptides showed low toxicity against Vero cell cultures, in assays of hemolysis, and in different cytotoxicity assays. In addition, we found that three peptides (SP1, LP6, and LP7) had potential technological and industrial use because of their emulsifying capacity, low toxicity, and ability to physically stabilize solutions. These novel molecules retained some properties of the parental molecule (surfactin, which was originally obtained through nonribosomal synthesis in Bacillus subtilis) but have the advantage of being linear peptides, which can be produced at large scales through the use of conventional heterologous protein expression protocols.

Investigation of Thrombin Activity with PAR 1-based Fluorogenic Peptides

Thrombin, a highly specific protease of blood coagulation, has two exosites that modulate its specificity. We designed two sets of synthetic substrate FRET peptides with 25- or 11- amino acids (aa) each, based on the PAR 1 sequence, to characterize the effect of exosite 1 engagement on substrate catalysis and preference. The 25-aa set encompassed a sequence binding to exosite 1, and structural modeling showed that binding to thrombin did not differ significantly from that of PAR 1 peptide. Modification at the P3´position of the 25 or 11-aa peptides resulted in small effect on kinetic parameters. Ionic strength higher than physiologic depressed thrombin action on the 25-aa peptides. Addition of ligands of the exosite 1 negatively modulated the catalysis of 25-aa substrates. In conclusion, we succeeded to mimic and study in real time, using these synthetic peptides, the influence of ligand binding to exosite 1 on thrombin activity.