Inhibition of Ca-transport ATPase from synaptosomal vesicles by flavonoids

The inhibitory action of the flavonoid quercetin has been examined on the calcium-transport ATPase of synaptosomal vesicles and compared to that of two other flavonoids, morin and rutin. We have found that while quercetin caused a 50% inhibition of calcium transport at a concentration of 15 μM, morin and rutin had similar effects at concentrations of about 200 μM. A similar order of potency was observed also for ATP hydrolysis, though at higher concentrations. Quercetin also strongly inhibited phosphorylation of membrane proteins by ATP in synaptosomal vesicles. Rutin and morin had an almost negligible effect on membrane protein phosphorylation. The order of inhibitory potency of the flavonoids on the Ca²⁺-transport ATPase from synaptosomal vesicles: quercetin > morin > rutin, could be linked to their possible solubility in the membrane lipid phase since: (1) it paralleled their partitioning between a mixture of oil and water; (2) it paralleled their uptake from the reaction mixture by synaptosomal vesicles and phosphatidylcholine liposomes; (3) they had almost equal potency as inhibitors of the water soluble system of histone phosphorylation by protein kinase.     

Inhibition of Ca2+-transport ATPase from synaptosomal vesicles by flavonoids

The inhibitory action of the flavonoid quercetin has been examined on the calcium-transport ATPase of synaptosomal vesicles and compared to that of two other flavonoids, morin and rutin. We have found that while quercetin caused a 50% inhibition of calcium transport at a concentration of 15 μM, morin and rutin had similar effects at concentrations of about 200 μM. A similar order of potency was observed also for ATP hydrolysis, though at higher concentrations. Quercetin also strongly inhibited phosphorylation of membrane proteins by ATP in synaptosomal vesicles. Rutin and morin had an almost negligible effect on membrane protein phosphorylation. The order of inhibitory potency of the flavonoids on the Ca²⁺-transport ATPase from synaptosomal vesicles: quercetin > morin > rutin, could be linked to their possible solubility in the membrane lipid phase since: (1) it paralleled their partitioning between a mixture of oil and water; (2) it paralleled their uptake from the reaction mixture by synaptosomal vesicles and phosphatidylcholine liposomes; (3) they had almost equal potency as inhibitors of the water soluble system of histone phosphorylation by protein kinase.     

Interaction of flavonoid compounds with contractile proteins of skeletal muscle

Flavonoids (quercetin, rutin) influence ATPase activity and actomyosin superprecipitation. Low concentrations (below 20 mumol/l) of flavonoids were found to cause conformational changes in the myosin structure accompanied by an increase in ATPase activity. At higher concentrations an inhibitory action of flavonoids on both ATPase activity and actomyosin superprecipitation occurred. Conformational changes are likely to be due to flavonoids binding to regulatory site near the active centre of the myosin head. The effect of quercetin was stronger than that of rutin.     

Pro-oxidative properties of flavonoids in human lymphocytes

The pro-oxidative properties of the four flavonoids, quercetin, morin, naringenin and hesperetin, in human lymphocyte system were investigated. Naringenin and hesperetin accelerated the oxidation of deoxyribose induced by Fe(3+)/H(2)O(2) in a concentration range of 0-200 microM, but quercetin and morin decreased it when the concentration was greater than 100 microM. The generation of hydrogen peroxide and the superoxide anion and the production of TBARS in lymphocytes were increased with increasing concentration of a flavonoid. Cell membrane protein thiols of the lymphocytes decreased when treated with the four flavonoids. Quercetin and hesperetin had no significant effect (p>0.05) on the activity of glutathione reductase, but morin and naringenin could inhibit the activity of the enzyme at a concentration of 200 microM, when compared to the control group. The glutathione S-transferase activity was slightly decreased by treatment with each of the four flavonoids only at a concentration of 200 microM. Therefore, the DNA damage in lymphocytes induced by the flavonoids in the model system might have been due to their stimulation of oxidative stress in the lymphocytes, which resulted in the decrease of cell membrane protein thiols, increase of lipid peroxidation in cell membrane and in the influence of the antioxidative enzyme activities.     

Differential effects of flavonoids on bovine kidney low molecular mass protein tyrosine phosphatase

Among the structurally related flavonoids tested on the bovine kidney low molecular weight protein tyrosine phosphatase (LMrPTP) activity, quercetin activated by about 2.6-fold the p-nitrophenyl-phosphate (p-NPP)-directed reaction, in contrast to morin that acted as a competitive inhibitor, with Ki values of 87, 73 and 50 microM for p-NPP, FMN, and tyrosine-phosphate, respectively. Other related flavonoids, such as rutin, kaempferol, catechin, narigin, phloretin and taxifolin did not significantly affect the LMrPTP activity. The positions of the hydroxyl groups in the structures of the flavonoids were important for their distinct effects on LMrPTP activity. The hydroxyl groups at C3' and C4' and the presence of a double bond at C2 and C3 were essential for the activating effect of quercetin. The absence of the 3'-OH (kaempferol), absence of the double bond (taxifolin) and the presence of the sugar rutinose at the 3-OH (rutin) suppressed the effect of quercetin. The C2'- and C4'-hydroxyl groups, the presence of the double bond, and a C4-ketone group were important requirements for the inhibitory effects of morin.     

The simultaneous separation and determination of six flavonoids and troxerutin in rat urine and chicken plasma by reversed-phase high-performance liquid chromatography with ultraviolet-visible detection

The method of high-performance liquid chromatography (HPLC) with UV-vis detection was used and validated for the simultaneous determination of six flavonoids (puerarin, rutin, morin, luteolin, quercetin, kaempferol) and troxerutin in rat urine and chicken plasma. Chromatographic separation was performed using a VP-ODS column (150 mm x 4.6 mm, 5.0 microm) maintained at 35.0 degrees C. The mobile phase was a mixture of water, methanol and acetic acid (57:43:1, v/v/v, pH 3.0) at the flow rate of 0.8 mL/min. Six flavonoids and troxerutin were analyzed simultaneously with good separation. On optimum conditions, calibration curves were found to be linear with the ranges of 0.10-70.00 microg/mL (puerarin, rutin, morin, luteolin, quercetin, kaempferol) and 0.50-350.00 microg/mL (troxerutin). The detection limits were 0.010-0.050 microg/mL. The method was validated for accuracy and precision, and it was successfully applied to determine drug concentrations in rat urine and chicken plasma samples from rat and chicken that had been orally administered with six flavonoids and troxerutin.     

Rutin-enhanced antibacterial activities of flavonoids against Bacillus cereus and Salmonella enteritidis

The antibacterial activities of flavonoids were found by the paper disk method to be enhanced by combining or mixing them. The combinations of quercetin and quercitrin, quercetin and morin, and quercetin and rutin were much more active than either flavonoid alone. Although rutin did not show activity in itself, the antibacterial activities of quercetin and morin were enhanced in the presence of rutin. The antibacterial activities of flavonoids, in combination with morin and rutin, were evaluated, based on the minimum inhibition concentration (MIC) in a liquid culture, by using Salmonella enteritidis and Bacillus cereus as the test bacteria. The activities of galangin, kaempherol, myricetin and fisetin were each enhanced in the presence of rutin when S. enteritidis was used as the test bacterium. The MIC value for kaempherol was markedly decreased by the addition of rutin. Morin inhibited DNA synthesis, and this effect was promoted by rutin at a concentration of 25 microg/ml.     

Quercetin interaction with the (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum

In sarcoplasmic reticulum vesicles or in the (Ca2+ + Mg2+)-ATPase purified from sarcoplasmic reticulum, quercetin inhibited ATP hydrolysis, Ca2+ uptake, ATP-Pi exchange, ATP synthesis coupled to Ca2+ efflux, ATP-ADP exchange, and steady state phosphorylation of the ATPase by inorganic phosphate. Steady state phosphorylation of the ATPase by ATP was not inhibited. Quercetin also inhibited ATP and ADP binding but not the binding of Ca2+. The inhibition of ATP-dependent Ca2+ transport by quercetin was reversible, and ATP, Ca2+, and dithiothreitol did not affect the inhibitory action of quercetin.     

Inhibition of (H+ + K+)-ATPase and H+ accumulation in hog gastric membranes by trifluoperazine, verapamil and 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate

The mechanism of gastric antisecretory action for trifluoperazine, verapamil and 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) has been studied utilizing isolated hog gastric membranes enriched with (H+ + K+)-ATPase. The drugs inhibited the gastric ATPase due to their apparent competition with K+ for the luminal high-affinity K+-site of the ATPase. The dose to inhibit 50% (ID50) of the ATPase in the membranes rendered freely permeable to K+ (20 mM) was 50 microM for trifluoperazine and 1.5 mM for verapamil and TMB-8. In intact hog gastric membranes which develop a pH gradient in the presence of valinomycin, ATP and KCl, however, trifluoperazine at 4 microM, verapamil and TMB-8 at 15 microM inhibited 40 and 30% of the valinomycin-stimulated ATPase activity, respectively, and also blocked the ionophore-dependent intravesicular acidification as measured by aminopyrine accumulation. The enhanced potency of the drugs to inhibit the ATPase in the intact membrane vesicles may be attributed to the accumulation of the drugs as a weak base within the vesicles, where the luminal K+-site of the ATPase is accessible. Calmodulin and Ca2+ had no effect on the extent of H+-accumulation as measured by aminopyrine accumulation in the membrane vesicles which were prepared in the presence of 1 mM EGTA. Since the drugs showed similar potency in interfering with H+ movements either in the membrane vesicles or isolated rabbit gastric glands stimulated by dibutyryl cAMP, it is reasonable to suggest the inhibitory effect of the drugs on (H+ + K+)-ATPase as a primary cause for such interferences in both cases. A trifluoperazine analog and other lipophilic amine drugs similarly inhibited (H+ + K+)-ATPase and H+ accumulation in the membrane vesicles or in the glands. We have concluded that a tertiary amine, the only common functional group among these drugs, is primarily responsible for their ability to interact with the high-affinity K+ site of the gastric ATPase.     

Flavonoids as superoxide scavengers and antioxidants

The superoxide anions scavenging activity and antioxidation of seven flavonoids--quercetin, rutin, morin, acacetin, hispidulin, hesperidin, and naringin--were studied. The superoxide anions were generated in a phenazin methosulphate-NADH system and were assayed by reduction of nitroblue tetrazolium. The scavenging activity ranked: rutin was the strongest, and quercetin and naringin the second, while morin and hispidulin were very weak. The concentration values yielding 50% inhibition of lipid peroxidation in mouse liver homogenate were in order of 10(-6) M for quercetin, rutin, and morin; and of 10(-5) M for acacetin and hispidulin, while naringin and hesperidin had no antioxidative action. In comparison with the antioxidative and scavenging activities of flavonoids, there are no correlations.     

Differential effects of flavonoids on bovine kidney low molecular mass protein tyrosine phosphatase

Among the structurally related flavonoids tested on the bovine kidney low molecular weight protein tyrosine phosphatase (LMrPTP) activity, quercetin activated by about 2.6-fold the p-nitrophenyl-phosphate (p-NPP)-directed reaction, in contrast to morin that acted as a competitive inhibitor, with Ki values of 87, 73 and 50 μM for p-NPP, FMN, and tyrosine-phosphate, respectively. Other related flavonoids, such as rutin, kaempferol, catechin, narigin, phloretin and taxifolin did not significantly affect the LMrPTP activity.

The positions of the hydroxyl groups in the structures of the flavonoids were important for their distinct effects on LMrPTP activity. The hydroxyl groups at C3′ and C4′ and the presence of a double bond at C2 and C3 were essential for the activating effect of quercetin. The absence of the 3′-OH (kaempferol), absence of the double bond (taxifolin) and the presence of the sugar rutinose at the 3-OH (rutin) suppressed the effect of quercetin. The C2′- and C4′-hydroxyl groups, the presence of the double bond, and a C4-ketone group were important requirements for the inhibitory effects of morin.     

Kinetic Characterization of Ca2+ Transport in Synaptic Membranes

Lysed synaptosomal membranes were prepared from brain cortices of HA/ICR Swiss mice, and the ATP-stimulated Ca2+ uptake, Ca2+-stimulated Mg2+-dependent ATPase activity, and the Ca2+-stimulated acyl phosphorylation of these membranes were studied. The Km values for free calcium concentrations ([Ca2+]f) for these processes were 0.50 microM, 0.40 microM, and 0.31 microM, respectively. Two kinetically distinct binding sites for ATP were observed for the ATP-stimulated Ca2+ uptake and the Ca2+-stimulated Mg2+-ATPase activity. The high-affinity Km values for ATP for these two processes were 16.3 microM and 28 microM, respectively. These results indicate that the processes studied operate in similar physiological concentration ranges for the substrates [Ca2+]f and ATP under identical assay conditions and, further, that these processes may be functionally coupled in the membrane.     

Interaction of flavonoids with rabbit muscle phosphorylase kinase

We have examined the effect of several flavonoids on the activity of phosphorylase kinase from rabbit skeletal muscle. From 14 flavonoids tested, the flavones quercetin and fisetin were found to be efficient inhibitors of nonactivated phosphorylase kinase when assayed at pH 8.2, causing 50% inhibition at a concentration of about 50 microM, while the flavanone hesperetin stimulated phosphorylase kinase activity about 2-fold when tested at 250 microM. The efficiency of quercetin in inhibiting the kinase is higher when the enzyme is stimulated either by ethanol or by alkaline pH. Both casein and troponin phosphorylation by phosphorylase kinase and the autophosphorylation of the kinase were inhibited by quercetin. In addition, quercetin was found to be a competitive inhibitor of ATP for the phosphorylation of phosphorylase b at pH 8.2. These observations suggest that the inhibitory effect of the flavone is directly on the phosphorylase kinase molecule. Trypsin-activated phosphorylase kinase was inhibited by quercetin and stimulated by hesperetin, as for the native enzyme.     

Oxidative phosphorylation in right-side-out membrane vesicles from Escherichia coli.

Oxidative phosphorylation in Escherichia coli membrane vesicles with a right-side-out orientation and loaded with ADP was investigated. Substrates of the electron transport chain could energize the phosphorylation of ADP, with the order of effectiveness being D-lactate greater than reduced phenazinemethosulfate greater than succinate greater than reduced nicotinamide adenine dinucleotide. Inhibitors of D-lactate oxidation, proton conductors, and inhibitor of the Mg2+ATPase (EC 3.6.1.3) all inhibited oxidative phosphorylation when coupled to D-lactate oxidation. ATP synthesis was absent in membrane vesicles prepared from a mutant strain lacking the Mg2+ATPase. Valinomycin or nigericin partially inhibited oxidative phosphorylation in the presence of potassium. Valinomycin plus nigericin completely inhibited ATP synthesis. The effect of various agents on the respiration-dependent establishment of a transmembrane pH gradient was also examined. NaCN and carbonyl cyanide p-trifluoromethoxyphenylhydrazone inhibited the establishment of a pH gradient while dicyclohexylcarbodiimide had no effect. These results are in good agreement with a chemiosmotic model for oxidative phosphorylation.     

Immunoaffinity purification and characterization of vacuolar H+ATPase from bovine kidney

Vacuolar proton-translocating ATPase from bovine kidney was purified in one step by immunoprecipitation and immunoaffinity chromatography using an immobilized anti-H+ATPase monoclonal antibody. The monoclonal antibody affinity matrix coprecipitated polypeptides with Mr of 70,000, a cluster at 56,000, 45,000, 42,000, 38,000, 33,000, 31,000, 15,000, 14,000, and 12,000 from solubilized bovine kidney microsomal membranes, a pattern that was unaffected by different detergent washing conditions. A nearly identical pattern of polypeptides was observed in H+ATPase partially purified by an entirely independent method. The immunoaffinity purified H+ATPase had reconstitutively active ATP-induced acidification and potential generation that was inhibited by N-ethylamaleimide. The purified enzyme had specific activities as high as 3.1 mumol/min/mg protein, dual pH optima at 6.5 and 7.2, and a Km for ATP of 150 microM. The substrate preference was ATP greater than ITP much greater than UTP greater than GTP greater than CTP. The affinity purified H+ATPase was stimulated by phosphatidyl glycerol greater than phosphatidyl inositol much greater than phosphatidyl choline greater than phosphatidyl serine. The immunoaffinity purified enzyme did not require monovalent anions or cations for activity, and the divalent cation preference for activity was Mn, Mg much greater than Ca greater than Co much greater than Sr, Ba. The enzyme was not inhibited by ouabain, azide, or vanadate, but had kappa 1/2 inhibitory concentrations of 22.2 microM for N-ethylmaleimide, 14.9 microM for NBD-Cl, 4.9 microM for N,N'-dicyclohexylcarbodiimide, 13.8 microM for 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, and 315 microM for Zn, values close to those for inhibition of proton transport in the native vesicles. The affinity purified kidney enzyme has similarities to but also significant differences in structural and enzymatic properties from those reported for other vacuolar H+ATPase.     

Relationship between phospholamban and nucleotide activation of cardiac sarcoplasmic reticulum Ca2+ adenosinetriphosphatase

A strong connection with nucleotide activation of Ca2+ATPase and phospholamban inhibition has been found. Phospholamban decreases the number of activatable Ca2+ATPase without affecting substrate affinity or the ability of nucleotide to serve its dual modulatory roles, i.e., catalytic and regulatory. Low concentrations of certain nucleotide mimetics, quercetin, tannin, and ellagic acid, with structural similarity to adenine can unmask phospholamban's inhibitory effect while concurrently acting as competitive inhibitors of nucleotide binding. Micromolar concentrations of tannin (EC50 approximately 0.3 microM) and ellagic acid (EC50 approximately 3 microM) stimulated Ca2+ uptake and calcium-activated ATP hydrolysis at submicromolar Ca2+ in isolated cardiac sarcoplasmic reticulum (SR). Stimulation of Ca2+ATPase was followed by pronounced inhibiton at only slightly higher tannin concentrations (IC50 approximately 3 microM), whereas inhibitory effects by ellagic acid were observed at much greater concentrations (IC50 > 300 microM) than the EC50. A complex relationship between compound, SR protein, and MgATP concentration is a major determining factor in the observed effects. Stimulation was only observed under conditions of phospholamban regulation, while the inhibitory effects were observed in cardiac SR at micromolar Ca2+ and in skeletal muscle SR, which lacks phospholamban. Maximal stimulation of Ca2+ATPase was identical to that observed with the anti-phospholamban monoclonal antibody 1D11. Both compounds appear to relieve the Ca2+ATPase from phospholamban inhibition, thereby increasing the calcium sensitivity of the Ca2+ATPase like that observed with phosphorylation of phospholamban or treatment with monoclonal antibody 1D11. Tannin, even under stimulatory conditions, is a competitive inhibitor of MgATP with a linear Dixon plot. The subsequent inhibitory action of higher tannin concentrations results from competition of tannin with the nucleotide binding site of the Ca2+ATPase. In contrast, ellagic acid produced a curvilinear Dixon plot suggesting partial inhibition of nucleotide activation. The data suggest that nucleotide activation of Ca2+ATPase is functionally coupled to the phospholamban interaction site. These compounds through their interaction with the adenine binding domain of the nucleotide binding site prevent or dissociate phospholamban regulation. Clearly, this portion of Ca2+ATPase needs further study to elucidate its role in phospholamban inhibition.     

The kinetics and divalent cation inhibition of plasma membrane ATPase in the yeast Candida albicans

The kinetics of ATP hydrolysis and cation effects on ATPase activity in plasma membrane from Candida albicans ATCC 10261 yeast cells were investigated. The ATPase showed classical Michaelis-Menten kinetics for the hydrolysis of Mg X ATP, with Km = 4.8 mM Mg X ATP. Na+ and K+ stimulated the ATPase slightly (9% at 20 mM). Divalent cations in combination with ATP gave lower ATPase activity than Mg X ATP (Mg greater than Mn greater than Co greater than Zn greater than Ni greater than Ca). Divalent cations inhibited the Mg X ATPase (Zn greater than Ni greater than Co greater than Ca greater than Mn). Free Mg2+ inhibited Mg X ATPase weakly (20% inhibition at 10 mM). Computed analyses of substrate concentrations showed that free Zn2+ inhibited Zn X ATPase, mixed (Zn2+ + Mg2+) X ATPase, and Mg X ATPase activities. Zn X ATP showed high affinity for ATPase (Km = 1.0 mM Zn X ATP) but lower turnover (52%) relative to Mg X ATP. Inhibition of Mg X ATPase by (free) Zn2+ was noncompetitive, Ki = 90 microM Zn2+. The existence of a divalent cation inhibitory site on the plasma membrane Mg X ATPase is proposed.     

Three adenine nucleotide binding sites in F1-F0 mitochondrial ATPase as revealed by presteady-state and steady-state kinetics of ATP hydrolysis. Evidence for two inhibitory ADP-specific noncatalytic sites

Preincubation of submitochondrial particles with ADP in the presence of Mg2+ results in the complete inhibition of ATPase which is slowly reactivated in the assay mixture containing ATP and the ATP regenerating system. Significantly, the rate of activation increases as the concentration of ADP in the preincubation mixture rises from 1 microM to 20 microM and reaches a constant value at higher ADP concentrations. The first-order rate constant for the activation process in the assay mixture is ATP-dependent at any level of inhibitory ADP. The data obtained strongly suggest that two ADP-specific inhibitory sites and one ATP-specific hydrolytic site are present in F1-F0 ATPase. Taking into account the (3 alpha.3 beta).gamma.delta.epsilon structure of F1, it is concluded that the synchronous discharge of ADP from two inhibitory sites during the activation occurs after ATP binds to the ATPase catalytic site.     

Membrane-bound adenosine triphosphatase of Escherichia coli. III. Effects of sodium azide on the enzyme functions.

1) Sodium azide and diphenyl phosphorazidate (DPPA) inhibited purified membrane-bound ATPase [coupling factor of oxidative phosphorylation; EC 3.6.1.3] of Escherichia coli non-competitively with Ki values of 39 and 51 micrometer, respectively. 2) Sodium azide and DPPA inhibited the activity of ATPase bound to the membrane as effectively as that of the purified enzyme. 3) The effects of sodium azide on succinate-dependent ATP synthesis, Pi-ATP exchange, and ATP hydrolysis reactions by the membrane vesicles were compared under the same conditions. At concentrations below 1.0 mM, sodium azide inhibited ATP hydrolysis, but Pi-ATP exchange and ATP synthesis were almost unaffected. At 10 mM sodium azide, both Pi-ATP exchange and ATP synthesis reactions were completely inhibited, probably because at this concentration, sodium azide acted as a proton-conducting uncoupler.     

A study of cyclic AMP-dependent protein phosphorylation in Schistocerca gregaria CNS: a comparison to that in mammalian CNS

1. The effect of cyclic AMP (10 microM) on the incorporation of 32P into protein was studied in cell-free preparations of Schistocerca gregaria cerebral ganglia. 2. Cyclic AMP-dependent phosphorylation of total protein was maximal after 60 sec, had a pH optimum of 7 to 8, was not affected by temperature (22-37 degrees C) and had a Km of 77 microM ATP. 3. Cyclic AMP increased the phosphorylation of total and specific protein in soluble fractions greater than synaptosomal greater than microsomal greater than crude membrane fractions. 4. In a direct comparison of locust brain to rat cerebral cortex, cyclic AMP stimulated the increased phosphorylation of only three protein bands, whereas in identical fractions of locust brain the phosphorylation of at least 12 protein bands was observed.