Biomedical subjects

J R Meyer-Fernandes

Publications and source records attributed to J R Meyer-Fernandes.

50 records · Page 3Linked to original sources

PubMed1990

Calcium inhibition of the ATP in equilibrium with [32P]Pi exchange and of net ATP synthesis catalyzed by bovine submitochondrial particles.

A previous communication (Fagian, M. M., Pereira da Silva, L. and Vercesi, A. E. (1986) Biochim. Biophys. Acta 852, 262-268) indicated that intramitochondrial calcium inhibits oxidative phosphorylation by decreasing the availability of adenine nucleotides to both the ADP/ATP translocase and the F0F1-ATP synthase complex. In this work we analyzed the interactions of calcium-nucleotide and magnesium-nucleotide complexes with the ATP synthase during catalysis of ATP in equilibrium with [32P]Pi exchange and net synthesis of ATP by submitochondrial particles. Concerning the ATP in equilibrium with [32P]Pi exchange reaction, calcium was ineffective as divalent cation when assayed alone. Furthermore, the addition of calcium increased the magnesium concentration required for half-maximal activation of the exchange, without changing Vmax. With respect to net ATP synthesis, the inhibition by calcium was shown to be due to formation of the CaADP- complex, which competes with MgADP- for the active site of the F0F1-ATP synthase. Moreover, ATP hydrolysis was competitively inhibited by CaATP2-, showing that calcium is able to interact with the enzyme in both forward and backward reactions in the same manner. That high calcium concentrations are required for significant inhibition of ATP synthesis indicates that this inhibition is relevant under conditions in which cytosolic calcium concentrations rise to pathological levels. Therefore, this mechanism may be responsible, in part, for the decrease in cellular ATP content that has been observed to occur when calcium accumulates in the cytosol.

Adenosine Triphosphate↗

PubMed1988

Pyrophosphate formation from acetyl phosphate and orthophosphate: evidence for heterogeneous catalysis.

The formation of [32P]pyrophosphate from acetyl phosphate and [32P]orthophosphate was studied under conditions in which phosphate-metal salts or acetyl phosphate-metal salts precipitate. In the absence of precipitates in purely aqueous media, the initial rate constant of transphosphorylation (kobs) was extremely small and the formation of pyrophosphate was detected only in the presence of calcium. In various combinations, conditions such as high pH, high concentrations of reactants, and the presence of dimethyl sulfoxide caused three types of precipitates to form. In completely aqueous solution with an excess of orthophosphate, the crystals formed at high pH contained 3 mol of calcium for 2 mol of phosphate and they were poorly effective at promoting phosphorolysis. In the presence of dimethyl sulfoxide, the ratio of calcium to phosphate in the sediment was 1:1 and phosphorolysis proceeded at a high rate. In either solvent, an excess of acetyl phosphate caused precipitation of a complex containing 1 mol of acetyl phosphate to 1 mol of calcium. In aqueous media the rate constant of phosphorolysis increased with increasing precipitation of the acetyl phosphate-calcium complex. With destabilization of the anions by dimethyl sulfoxide the increase in kobs for a given amount of acetyl phosphate-calcium precipitated was 200-fold higher. Magnesium did not form precipitates and was ineffective in promoting transphosphorylation in completely aqueous media, either in the presence of excess phosphate or in the presence of excess acetyl phosphate. However, when precipitation of phosphate-magnesium or acetyl phosphate-magnesium was promoted by addition of dimethyl sulfoxide, phosphorolysis was observed with rate constants as high as those found in the presence of calcium. These results indicate that phosphorolysis of acetyl phosphate occurs at higher rates on the surface of solid structures, through highly specific interactions involving acetyl phosphate, orthophosphate, and divalent cations.

Catalysis↗

PubMed1986

Comparison between calcium transport and adenosine triphosphatase activity in membrane vesicles derived from rabbit kidney proximal tubules.

Characteristics of Ca2+ uptake were studied in a vesicular preparation of proximal tubule plasma membranes from rabbit kidney and compared with the properties of both membrane-bound and solubilized Ca2+-ATPase activities. Calcium uptake required both ATP and MgCl2 and revealed two kinetic components with respect to Ca2+ concentration requirements, one with a high affinity for Ca2+ (1.8 microM), operative in the range of cytosolic Ca2+ activity, and one with a low affinity for Ca2+ (250 microM) which may become active only at abnormally high cytosolic Ca2+ concentrations. The high- and low-affinity components were stimulated to similar extents by phosphate, and required similar concentrations of ATP (0.6 mM) for half-maximal activity. The amount of membrane-bound phosphoenzyme formed from ATP in the presence of Ca2+ was the same regardless of whether only one or both sites were saturated, suggesting that occupancy of the second Ca2+ binding site accelerates the enzyme turnover. Inhibition of Ca2+ transport by Na+ was reversed by the addition of ouabain or an ATP-regenerating system, indicating that this inhibitory effect of Na+ on Ca2+ uptake may be due to the accumulation of ADP in the medium as a result of Na+ pump activity. Low concentrations of carbonyl cyanide p-trifluoromethoxyphenylhydrazone and valinomycin (2.5 and 1 microM, respectively) were without effect on Ca2+ uptake in the presence of phosphate, whereas higher concentrations of the ionophores (200 and 100 microM, respectively) reduced uptake by 60% or more. The calmodulin antagonist 48/80 also reduced Ca2+ uptake with half-maximal effectiveness at 100 micrograms/ml. None of these drugs affected either ATPase activity or the EGTA-induced Ca2+ efflux from preloaded vesicles. The Ca2+ dependence of ATP hydrolysis by the membrane-bound enzyme preparation was similar to that observed for Ca2+ uptake by the vesicles. However, with solubilized enzyme, concentrations of Ca2+ similar to that found in the plasma reduced Ca2+-stimulated ATP hydrolysis to one-half of its maximal rate. This indicates that peritubular Ca2+ may play a role in the regulation of Ca2+ transport across the tubular epithelium. ATP could not be replaced by ITP as a substrate for Ca2+ uptake, and the (Ca2+ + Mg2+)ITPase activity of soluble enzyme was 25-fold lower than in the presence of ATP. This is an indication that the active Ca2+ pumping mechanism in proximal tubules is critically dependent on the nucleoside moiety of the substrate.

Adenosine Triphosphatases↗

PubMed1985

Phosphorolysis of acetyl phosphate by orthophosphate with energy conservation in the phosphoanhydride linkage of pyrophosphate.

The formation of pyrophosphate as a result of nucleophilic attack by orthophosphate at the acylphosphate bond of acetyl phosphate was detectable in completely aqueous media, and was enhanced by dimethyl sulfoxide. The reaction had an absolute requirement for divalent cations, the rate constant of phosphorolysis being dependent on the species and concentration of cations as well as on temperature and pH. The amount of pyrophosphate formed depended on both the acetyl phosphate and orthophosphate concentrations. In purely aqueous media, phosphorolysis was barely detectable in the presence of Mg2+, and its rate increased 40-fold when Mg2+ was replaced by Ca2+ or Sr2+. In the presence of Mg2+ the rate of phosphorolysis increased 400-fold when 50 to 80% of the water was replaced by dimethyl sulfoxide. In the latter case, the rate also increased as the pH was raised from 4.0 to 9.0. The entropy of activation was large and negative in the presence of Mg2+ or Ca2+, indicating that the nucleophile is involved in the rate-limiting step of the reaction. Since this thermodynamic parameter became large and positive in the presence of Ca2+ when dimethyl sulfoxide was omitted, it is inferred that the transition state of the same reaction may be changed by the solvent composition and the solvation of reactants.

Cations, Divalent↗

PubMedDate not supplied

Monosaccharides and disaccharides decrease the Km for phosphorylation of a membrane-bound enzyme ATPase.

The disaccharides trehalose and sucrose, and to a lesser extent the monosaccharides glucose and fructose, decrease the apparent Km of the Ca2+, Mg(2+)-ATPase of sarcoplasmic reticulum for Pi. This effect is more pronounced at pH 7.4 than at pH 6.2. The enzyme is not phosphorylated by Pi when the temperature of the medium is decreased to 0 degree C, but when 1.5 M trehalose or sucrose is present phosphoenzyme formation increases to 0.5 mumol E-P/g protein.

Animals↗

PubMedDate not supplied

Uncoupling by trehalose of Ca2+ transport and ATP hydrolysis by the plasma membrane (Ca2+ + Mg2+) ATPase of kidney tubules.

Trehalose, the disaccharide of glucose, inhibits both initial rate and maximal capacity of ATP-dependent Ca2+ transport in inside-out vesicles of basolateral membrane from kidney proximal tubules. This inhibition (I0.5 = 60 mM) cannot be attributed to an increase in Ca2+ permeability, since the rate of EGTA-stimulated Ca2+ efflux from preloaded vesicles is not modified by trehalose. In the presence of 600 mM trehalose, Ca2+ uptake was almost completely inhibited, but the Ca(2+)-stimulated ATPase activity was unaffected; thus trehalose uncouples the Ca2+ transport from the ATPase activity. The Ca2+ transport inhibition by trehalose is reversible, since the inhibition disappeared when the vesicles were pre-incubated with 600 mM trehalose and then diluted in reaction medium to measure Ca2+ accumulation. Other mono- and disaccharides such as glucose, fructose, galactose, sucrose, maltose and lactose were tested but were not so effective as trehalose. The uncoupling of Ca2+ transport from hydrolysis can be explained by an interaction of trehalose with the phospholipid environment of the enzyme that induces conformational changes in specific domains of the enzyme so as to impair the coupling process.

Adenosine Triphosphate↗

PubMedDate not supplied

Polyols that accumulate in renal tissue uncouple the plasma membrane calcium pump and counteract the inhibition by urea and guanidine hydrochloride.

Sorbitol and mannitol, two stereoisomeric osmolytes, inhibit the ATP-dependent Ca2+ transport in inside-out vesicles derived from basolateral membranes from kidney proximal tubules. This inhibition (I0.5 = 400 and 390 mM respectively) cannot be attributed to an increase in Ca2+ permeability, since the rate of EGTA-stimulated Ca2+ efflux from preloaded vesicles is not modified by these osmolytes. In the presence of 1 M sorbitol or mannitol, Ca2+ uptake is inhibited by 70 and 75%, respectively. Since the Ca(2+)-stimulated ATPase activity is unaffected, sorbitol and mannitol uncouple the Ca2+ transport from the ATPase activity. The inhibition of Ca2+ transport by these osmolytes is reversible, since the inhibition disappears when the vesicles are preincubated with 1 M sorbitol or mannitol and then diluted 25-fold in reaction medium to measure Ca2+ accumulation. On the other hand, these osmolytes protect the (Ca2+ + Mg2+) ATPase from the inhibition of Ca2+ transport and ATPase activity by urea and guanidinium. These data suggest that the high concentrations of polyols that renal cells accumulate during antidiuresis, may regulate Ca2+ transport across the plasma membrane. In addition, polyols may protect the (Ca2+ + Mg2+) ATPase from the deleterious structural effects of urea, a compound that also accumulates during antidiuresis.

Animals↗

PubMedDate not supplied

Trehalose protects yeast pyrophosphatase against structural and functional damage induced by guanidinium chloride.

Trehalose is accumulated at very high concentrations in yeasts when this organism is submitted to a stress condition. This report approaches the question on the protective effect of trehalose and its degradation product, glucose, against structural and functional damage promoted by guanidinium on yeast cytosolic pyrophosphatase. Here it is shown that both, 1 M trehalose or 2 M glucose, are able to attenuate at almost the same extent the conformational changes promoted by guanidinium chloride on the pyrophosphatase structure. On the other hand while 1 M trehalose increases 3.8 times the Ki (from 0.15 to 0.57 M) for guanidinium chloride inhibition of pyrophosphatase activity, 2 M glucose did not even duplicate this parameter (from 0.15 to 0.25 M). These data support evidences for a functional reason for the accumulation by yeasts of trehalose, and not other compound, during stress conditions.

Cytosol↗

PubMedDate not supplied

Inhibition of succinic dehydrogenase and F0F1-ATP synthase by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS).

This study shows that incubation of rat liver mitochondria in the presence of the thiol/ amino reagent 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) is followed by inhibition of both succinate supported respiration and oxidative phosphorylation. Half-maximal inhibition of succinic dehydrogenase activity and succinate oxidation by mitochondria was attained at 55.3 and 60.8 microM DIDS, respectively. DIDS did inhibit the net ATP synthesis and ATP<=>[32P]Pi exchange reaction catalyzed by submitochondrial particles in a dose-dependent manner (Ki = 31.7 microM and Ki = 32.7 microM), respectively. The hydrolytic activities of uncoupled heart submitochondrial particles and purified F1-ATPase were also inhibited 50% by 31.9 and 20.9 microM DIDS, respectively.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMedDate not supplied

Trypanosoma brucei: ecto-phosphatase activity present on the surface of intact procyclic forms.

The results presented in this paper indicate that procyclic forms of Trypanosoma brucei possess a phosphatase activity detected in the external cell surface able to hydrolyze about 0.7 nmol.mg-1.min-1 p-nitrophenylphosphate. A faster rate of hydrolysis was observed when membrane-enriched fractions were used. This activity is weakly sensitive to 1 mM NaF, 10 mM tartrate and 10 mM levamizole but strongly inhibited by 0.1 mM vanadate. Inhibition by both NaF and vanadate have a competitive character. This phosphatase activity decreases by increasing the pH from 6.8 to 8.4, a pH range in which cell viability was maintained during at least 1 hour. In the membrane-enriched fractions this phosphatase activity showed to be an acid phosphatase. In addition, intact cells could catalyze the dephosphorylation of [32P]phosphocasein phosphorylated at serine and threonine residues.

4-Nitrophenylphosphatase↗

PubMedDate not supplied

A contribution of the mitochondrial adenosinetriphosphatase inhibitor protein to the thermal stability of the F0F1-ATPase complex.

A complete inactivation is observed after a 3 min pre-incubation at 70 degrees C with mitochondrial F0F1-ATPase complex depleted of the ATPase natural inhibitor protein (ammonium-Sephadex submitochondrial particles) and activated MgATP-submitochondrial particles (particles that after a 4 h-pre-incubation at 42 degrees C released the endogenous inhibitor protein). However, latent MgATP-submitochondrial particles (particles containing the inhibitor protein) pre-incubated under the same conditions are totally inactivated only after 15 min of pre-incubation. When ammonium-Sephadex particles are reconstituted with 20 micrograms/ml of purified ATPase inhibitor protein there is an increase of 15-fold in the half-time for thermal inactivation (t0.5), showing that the inhibitor protein protects the mitochondrial F0F1-ATPase complex against thermal inactivation.

Adenosine Triphosphate↗

PubMedDate not supplied

Effects of trehalose and ethanol on yeast cytosolic pyrophosphatase.

Trehalose has been described to protect several enzymes against destabilizing conditions. This sugar is naturally accumulated by yeast as a stress protectant. A common stress condition that yeast is normally submitted is the presence of ethanol, the by-product of fermentation process of several yeast. In this paper we show the effects of trehalose and ethanol, alone or together, on yeast pyrophosphatase, and the effects of these compounds on inhibition and unfolding of pyrophosphatase promoted by urea. We show that both trehalose and ethanol inhibit pyrophosphatase in a dose-dependent manner, and that the presence of ethanol does not modify the inhibition promoted by trehalose as well as the presence of trehalose does not modify the inhibition promoted by ethanol. The effects of trehalose on pyrophosphatase are completely reversible, but the inhibition caused by ethanol was only partially reversible. Incubation of pyrophosphatase with 10% (v/v) ethanol promoted an inhibition of 15%, and the control activity was completely recovered after removal of ethanol. On the other hand, when pyrophosphatase was incubated with 20% (v/v) ethanol an inhibition of 40% of the control activity was observed which persisted after removal of ethanol. Ethanol also potentiates the inhibition of pyrophosphatase promoted by urea, and contributes for an irreversible inactivation and unfolding of pyrophosphatase in the presence of urea. Trehalose, that protects this enzyme against the inhibition and unfolding promoted by the chaotropic compound urea, was inefficient to protect against the effects of ethanol. Trehalose was also efficient to prevent an irreversible inactivation induced by urea.

Ethanol↗

PubMedDate not supplied

Effects of naturally occurring polyols and urea on mitochondrial F0F1ATPase.

We show that urea inhibits the ATPase activity of MgATP submitochondrial particles (MgATP-SMP) with Ki = 0.7 M, probably as a result of direct interaction with the structure of F0F1-ATPase. Counteracting compounds (sorbitol, mannitol or inositol), despite slightly (10-20%) inhibiting the ATPase activity, also protect the F0F1-ATPase against denaturation by urea. However, this protection was only observed at low urea concentrations (less than 1.5 M), and in the presence of three polyols, the Ki for urea shift from 0.7 M to 1.2 M. Urea also increases the initial activation rate of latent MgATP-SMP in a dose-dependent-manner. However, when the particles (0.5 mg/ml) were preincubated in the presence of 1 M, 2 M or 3 M urea, a decrease in the activation level occurred after 1 h, 30 and 10 min, respectively. At high MgATP-SMP concentration (3 mg/ml) a decrease in activation was observed after 2 h, 1 h and 20 min, respectively. These data indicate that the effect of urea on the activation of MgATP-SMP depends on time, urea and protein concentrations. It was also observed that polyols suppress the activation of latent MgATP-SMP in a dose-dependent manner, and protect the particles against urea denaturation during activation. We suppose that a decrease in membrane mobility promoted by interactions of polyols with phospholipids around the F0F1-ATPase may also increase the compactation of protein structure, explaining the inhibition of natural inhibitor protein of ATPase (IF1) release and the activation of the enzyme.

Adenosine Triphosphate↗

PubMedDate not supplied

An ecto-ATPase activity present in Leishmania tropica stimulated by dextran sulfate.

In this study we report the effects of sulfated polysaccharides on the ecto-ATPase activity of intact cells of Leishmania tropica. Increasing concentrations of dextran sulfate stimulated progressively the ecto-ATPase activity, but did not modify other ecto-enzymes present on the surface of this parasite, such as 5'nucleotidase, 3'nucleotidase and a membrane-bound acid phosphatase activity. This stimulation was not observed when other sulfated polysaccharides such as chondroitin sulfates and heparin were tested. It depends on size and charge of the dextran sulfated molecule. When the cells were incubated in the presence of dextran sulfate Mr 8,000; 40,000 and 500,000 the stimulation of the ecto-ATPase activity was 11%; 23%; and 63%, respectively, and the stimulation was not observed when desulfated dextran (Mr 40,000) was used. The effects of dextran sulfate also depend on pH of the medium. At pH 7.5, the stimulation was over 60%, whereas at pH 8.5 only 25%. The effects of dextran sulfate 500,000 on the ecto-ATPase activity was totally abolished by spermidine and partially by putrescine, two polyamines synthesized and released by Leishmania.

4-Nitrophenylphosphatase↗