Effects of pH and metal ions on antioxidative activities of catechins

The Effects of pH on antioxidative activities of catechol, pyrogallol, and four catechins, and effects of metal ions (Al3+, Ca2+, Cd2+, Co2+, Cr3+, Cu2+, Fe2+, Fe3+, K+, Mg2+, Mn2+, Na+, and Zn2+) on antioxidative activities of (-)-epigallocatechin gallate (EGCG) were studied by an oxygen electrode method. The antioxidative activities of catechins were high and constant at pH 6-12, but decreased in acidic and strong alkaline solutions. Copper(II) ion the most strongly increased the antioxidative activity of EGCG among these metal ions examined, but iron(II) ion largely inhibited the antioxidative activity of EGCG. These effects are discussed considering the formation of metal complexes with catechins and the change in oxidation potentials.     

Characteristics of the vitamin K-dependent carboxylating system in human placenta.

Gamma-carboxyglutamic acid, formed during the post-translational vitamin K-dependent carboxylation of glutamic acid residues in polypeptides has been identified not only in coagulation factors II (prothrombin),, VII, IX and X [1--4], but also in several other plasma proteins [3,5,6] and in protein of bone [7,8] and kidney [9]. In rat liver, carboxylation is mediated through an enzyme system located in the microsomal membrane [10]. The enzyme system requires CO2, O2 and the reduced (hydroquinone) form of the vitamin, as well as a suitable substrate [10,11]. Rat liver microsomes also convert vitamin K1 (phylloquinone) to its stable 2,3-epoxide [12]. Several studies suggest a link between carboxylation and the formation of the epoxide [12--14]. In one of these [14], a survey of rat tissues for vitamin K1 epoxidation revealed that, in addition to liver, this activity was also possessed by kidney, bone, spleen and placenta. In preliminary experiments, vitamin K-dependent carboxylating systems have been found in rat and chick kidney [9], in chick bone [15] and in rat spleen and placenta (unpublished observations). In this communication, we describe some of the basic characteristics of the vitamin K-dependent carboxylating system as found in human placental microsomes.     

Vitamin K-dependent carboxylation of glutamic acid residues to γ-carboxyglutamic acid in lung microsomes

Vitamin K-dependent carboxylation of glutamic acid residues to γ-carboxyglutamic acid was demonstrated in proteins of lung microsomes. The carboxylation was 12% of that in liver microsomes per milligram of mierosomal protein. Carboxylation was very low with microsomes of untreated rats but increased with time up to 42 h after warfarin administration. Carboxylation was highest with microsomes from rats fed a vitamin K-deficient diet. This suggests that a protein(s) accumulates which can be carboxylated in vitro/J. Lung microsomes also catalyzed the vitamin K-dependent carboxylation of the peptide Phe-Leu-Glu-Glu-Leu. The peptide carboxylase activity was 9% of that obtained with liver microsomes. Vitamin K-dependent protein carboxylation required NADH or dithioerythritol, suggesting that vitamin K had to be reduced to the hydroquinone. Accordingly, vitamin K₁ hydroquinone had carboxylating activity without added reducing agents. Menaquinone-3 was considerably more active than phylloquinone. The temperature optimum for carboxylation was around 27 °C.     

Vitamin K-dependent oxygenase/carboxylase; differential inactivation by sulfhydryl reagents

Inhibition of vitamin K-dependent carboxylase and oxygenase by sulfhydryl reagents was compared. Formation of vitamin K epoxide and vitamin K-dependent carboxylation are both strongly (greater than 90%) inhibited by l mM p-hydroxy-mercuribenzoate, and this inhibition is reversed by dithiothreitol. Both activities are also effectively inhibited by N-ethylmaleimide (NEM). Preincubation with vitamin K hydroquinone prevents NEM inhibition of epoxide formation but not of carboxylation. These data argue that separate active sites are required to support vitamin K-dependent epoxide formation and carboxylation and that the binding site vitamin K oxygenase contains an active thiol group.     

Conformational studies of A23187 with mono-, di- and trivalent metal ions by circular dichroism spectroscopy

CD studies carried out on A23187 indicate a solvent-dependent conformation for the free acid. Alkali metal ions were found to bind to the ionophore weakly. Divalent metal ions such as Mg2+, Ca2+, Sr2+, Ba2+ and Co2+ and trivalent lanthanide metal ions like La3+ were found to form predominantly 2:1 (ionophore-metal ion) complexes at low concentrations of metal ions, but both 2:1 and 1:1 complexes were formed with increasing salt concentration. Mg2+ and Co2+ exhibit similar CD behaviour that differs from that observed for the other divalent and lanthanide metal ions. The structure of 2:1 complexes involves two ligand molecules coordinated to the metal ion through the carboxylate oxygen, benzoxazole nitrogen and keto-pyrrole oxygen from each ligand molecule along with one or more solvent molecules. Values of the binding constant were determined for 2:1 complexes of the ionophore with divalent and lanthanide metal ions.     

Mimic models of peroxidase--kinetic studies of the catalytic oxidation of hydroquinone by H2O2

The reactions of hydroquinone with hydrogen peroxide catalyzed by transition metal ions Cu2+, Fe2+, Fe3+, Co2+ and Mn2+ were investigated in aqueous solution at 25 degrees C. Two copper (II) complexes (bis(dimethylglyoxime) copper(II) and 5,7,7,12,14,14-hexamethyl-1,4,8,11-tetraazacyclotetradeca-4,11-dienatocopper(II)iodide) were prepared. Their catalytic activities on this oxidation were kinetically investigated in aqueous solution and in cetyltrimethylammonium bromide (CTAB) micellar solution at 25 degrees C. The kinetic equations for micellar catalysis and metallomicellar catalysis were established, respectively. CTAB micelle enhances the reaction rate due to its concentrated and electrostatic effects on substrates and/or intermediate. Metallomicelle exhibits remarkable catalytic activity on this oxidation reaction, which is attributed to the active center and the microenvironment effects. Metallomicelle enhances the rate of reaction by activating hydroquinone anion. The presence of co-ligand of imidazole (or pyridine) remarkably increases the catalytic activity of metal complex in micelle system in contrast to it lowers the activity of the complex in aqueous solution. Metallomicelles could be treated as the mimic models of peroxidase.     

Experimental evidence that flavonoid metal complexes may act as mimics of superoxide dismutase

Radical scavenging activities of flavonoids rutin, taxifolin, (-)-epicatechin, luteolin, and their complexes with transition metal (Fe2+, Fe3+, and Cu2+) towards superoxide were determined using illumination of riboflavin as source and NBT as detector of O*2-. The scavenger potencies of flavonoid metal complexes were significantly higher than those of the parent flavonoids. To elucidate the mechanism of this phenomenon, the rates of superoxide-dependent oxidation of flavonoids and their metal complexes in photochemical system with riboflavin were examined. It was found for the first time that flavonoids bound to metal ions were much less subjected to oxidation compared with those of free compounds. The findings directly demonstrate superoxide scavenging activity of metal ions in complexes with flavonoids and support earlier suggestions that flavonoid metal complexes may exhibit superoxide dismuting activity.     

Metabolism of vitamin K and prothrombin synthesis: anticoagulants and the vitamin K--epoxide cycle.

Vitamin K is primarily located in hepatic microsomes, where the vitamin K-dependent carboxylation in prothrombin synthesis occurs. Recent evidence supports the idea that the carboxylation is linked to the metabolism of the vitamin--specifically the cyclic interconversion of vitamin K and vitamin K epoxide. The primary site of action of coumarin and indandione anticoagulants appears to be an inhibition of the epoxide-to-vitamin K conversion in this cycle. There is a correlation between the inhibition of prothrombin synthesis and the regeneration of vitamin K from the epoxide by anticoagulants. In hamsters and warfarin-resistant rats prothrombin synthesis and the epoxide-K conversion are less sensitive to warfarin than in the normal rat. The epoxide-K conversion is impaired in resistant rats, which may explain their high vitamin K requirement. There is also a correlation between vitamin K epoxidation and vitamin K-dependent carboxylation, but the apparent link may be because vitamin K hydroquinone is an intermediate in the formation of the epoxide and also the active form in carboxylation. The vitamin K-epoxide cycle is found in extrahepatic tissues such as kidney, spleen, and lung and is inhibited by warfarin.     

Soluble enzyme system for vitamin K-dependent carboxylation.

The vitamin K-dependent carboxylating system has been solubilized by Lubrol PX or Triton X-100 treatment of vitamin K-deficient rat liver microsomes. As obtained from vitamin K-deficient rat liver, this soluble preparation is dependent upon the in vitro addition of vitamin K1 for carboxylating activity. The enzyme system is complex and is dependent upon NADH and dithiothreitol for maximum activity. While detergents used to solubilize the enzyme complex do markedly inhibit the activity of the system, the solubilized system is still highly responsive to vitamin K addition and can be used for further study of the carboxylating enzyme system. The requirement for dithiothreitol and the inhibition by p-hydroxymercuribenzoate indicate the involvement of an --SH enzyme in the carboxylating system.     

Oxygen Enhancement of bactericidal activity of rifamycin SV on Escherichia coli and aerobic oxidation of rifamycin SV to rifamycin S catalyzed by manganous ions: the role of superoxide

Oxygen enhanced the bactericidal activity of rifamycin SV to Escherichia coli K12. Anaerobically grown cells, which had a low level of superoxide dismutase, were more susceptible to the bactericidal activity than aerobically grown cells, which contained a high level of superoxide dismutase. Oxygen also enhanced the inhibition of RNA polymerase activity of rifamycin SV, when Mn2+ was used as a cofactor. Rifamycin S was reduced to rifamycin SV by NADPH catalyzed by cell-free extracts of Escherichia coli K12. These results indicate that the inhibition of bacterial growth by rifamycin SV is due to the production of active species of oxygen resulting from the oxidation-reduction cycle of rifamycin SV in the cells. The aerobic oxidation of rifamycin SV to rifamycin S was induced by metal ions, such as Mn2+, Cu2+, and Co2+. The most effective metal ion was Mn2+. In the presence of Mn2+, accompanying the consumption of 1 mol of oxygen and the oxidation of 1 mol of rifamycin SV, 1 mol of hydrogen peroxide and 1 mol of rifamycin S were formed. Superoxide was generated during the autoxidation of rifamycin SV. Superoxide dismutase inhibited the formation of rifamycin S, but scavengers for hydrogen peroxide and the hydroxyl radical did not affect the oxidation. A mechanism of Mn2+-catalyzed oxidation of rifamycin SV is proposed and its relation to bactericidal activity is discussed.     

Probing the functional role of Ca2+ in the oxygen-evolving complex of photosystem II by metal ion inhibition

Photosynthetic oxygen evolution in photosystem II (PSII) takes place in the oxygen-evolving complex (OEC) that is comprised of a tetranuclear manganese cluster (Mn4), a redox-active tyrosine residue (YZ), and Ca2+ and Cl- cofactors. The OEC is successively oxidized by the absorption of 4 quanta of light that results in the oxidation of water and the release of O2. Ca2+ is an essential cofactor in the water-oxidation reaction, as its depletion causes the loss of the oxygen-evolution activity in PSII. In recent X-ray crystal structures, Ca2+ has been revealed to be associated with the Mn4 cluster of PSII. Although several mechanisms have been proposed for the water-oxidation reaction of PSII, the role of Ca2+ in oxygen evolution remains unclear. In this study, we probe the role of Ca2+ in oxygen evolution by monitoring the S1 to S2 state transition in PSII membranes and PSII core complexes upon inhibition of oxygen evolution by Dy3+, Cu2+, and Cd2+ ions. By using a cation-exchange procedure in which Ca2+ is not removed prior to addition of the studied cations, we achieve a high degree of reversible inhibition of PSII membranes and PSII core complexes by Dy3+, Cu2+, and Cd2+ ions. EPR spectroscopy is used to quantitate the number of bound Dy3+ and Cu2+ ions per PSII center and to determine the proximity of Dy3+ to other paramagnetic centers in PSII. We observe, for the first time, the S2 state multiline electron paramagnetic resonance (EPR) signal in Dy3+- and Cd2+-inhibited PSII and conclude that the Ca2+ cofactor is not specifically required for the S1 to S2 state transition of PSII. This observation provides direct support for the proposal that Ca2+ plays a structural role in the early S-state transitions, which can be fulfilled by other cations of similar ionic radius, and that the functional role of Ca2+ to activate water in the O-O bond-forming reaction that occurs in the final step of the S state cycle can only be fulfilled by Ca2+ and Sr2+, which have similar Lewis acidities.     

Mechanism of action of t-butyl hydroperoxide in the inhibition of vitamin K-dependent carboxylation

t-Butyl hydroperoxide has been studied as a possible competitive inhibitor of the vitamin K-dependent carboxylation of the pentapeptide PheLeuGluGluIle. Under standard carboxylating conditions the concentrations of reduced phylloquinone and phylloquinone were followed by high-pressure liquid chromatography during 30-min incubations of Triton-solubilized microsomes from rat liver. Under these conditions supporting linear rates of carbon dioxide fixation for 20–30 min, the vitamin KH₂ concentration decreased exponentially to less than 5% of its initial value in 30 min principally due to autooxidation. In the presence of 10 mm t-butyl-OOH, however, the oxidation of vitamin KH₂ was greatly accelerated with none being detected after 7 min. In general, the rate of carboxylation of peptide paralleled the KH₂ concentration. After cessation of carboxylation in the presence of t-butyl-OOH the readdition of KH₂ stimulated additional ¹⁴CO₂ fixation. A known competitive inhibitor of vitamin K, 2-chlorophylloquinone, did not accelerate the oxidation of KH₂ but nonetheless inhibited the vitamin K-dependent carboxylation in a competitive manner. These data have led us to conclude that t-butyl-OOH is not a competitive inhibitor of the vitamin K-dependent carboxylase at the active site of the enzyme but merely acts to promote the oxidation of KH₂.     

Evidence for a common metal ion-dependent transition in the 4-carboxyglutamic acid domains of several vitamin K-dependent proteins

The murine monoclonal antibody H-11 binds a conserved epitope found at the amino terminal of the vitamin K-dependent blood proteins prothrombin, factors VII and X, and protein C. The sequence of polypeptide recognized by antibody H-11 contains 2 residues of gamma-carboxyglutamic acid, and binding of the antibody is inhibited by divalent metal ions. By using a solid-phase immunoassay with 125I-labeled antibody and immobilized vitamin K-dependent protein, binding of the antibody to the vitamin K-dependent proteins was inhibited by increasing concentrations of calcium, manganese, and magnesium ion. The transition midpoints for antibody binding were in the millimolar concentration range and were different for each metal ion. In general, the transition midpoints were lowest for manganese ion, intermediate for calcium ion, and highest for magnesium ion. Antibody H-11 bound specifically to a synthetic peptide corresponding to residues 1-12 of human prothrombin that was synthesized as the gamma-carboxyglutamic acid-containing derivative. Binding of the antibody to the peptide was not inhibited by calcium ion. These data suggest that inhibition of antibody H-11 binding by divalent metal ions is not due simply to neutralization of negative charge by Ca2+. This transition which is conserved in vitamin K-dependent proteins containing the H-11 antigenic site is likely due to a structural transition of the amino-terminal polypeptide possibly from a random (accessible) to ordered (inaccessible) structure.     

DNA structural transitions induced by divalent metal ions in aqueous solutions

Using methods of IR spectroscopy, light scattering, gel-electrophoresis DNA structural transitions are studied under the action of Cu2+, Zn2+, Mn2+, Ca2+ and Mg2+ ions in aqueous solution. Cu2+, Zn2+, Mn2+ and Ca2+ ions bind both to DNA phosphate groups and bases while Mg2+ ions-only to phosphate groups of DNA. Upon interaction with divalent metal ions studied (except for Mg2+ ions) DNA undergoes structural transition into a compact form. DNA compaction is characterized by a drastic decrease in the volume occupied by DNA molecules with reversible formation of DNA dense particles of well-defined finite size and ordered morphology. The DNA secondary structure in condensed particles corresponds to the B-form family. The mechanism of DNA compaction under Mt2+ ion action is not dominated by electrostatics. The effectiveness of the divalent metal ions studied to induce DNA compaction correlates with the affinity of these ions for DNA nucleic bases: Cu2+>Zn2+>Mn2+>Ca2+>Mg2+. Mt2+ ion interaction with DNA bases (or Mt2+ chelation with a base and an oxygen of a phosphate group) may be responsible for DNA compaction. Mt2+ ion interaction with DNA bases can destabilize DNA causing bends and reducing its persistent length that will facilitate DNA compaction.     

The stability constants of some metal ion complexes of 3',5'-cyclic AMP.

The stability constants of complexes of 3', 5'-cyclic AMP with Mg2+, Ca2+, Mn2+, Ni2+ and Co2+ were estimated at 30 degrees C in solutions of ionic strength about 0.15 containing about 130 mM K+ or tetramethylammonium ions. Values between 13 and 22 M-1 were obtained, indicating that only about 2% of cyclic AMP is present as metal complexes in vivo, but that at commonly used in vitro concentrations of 10 mM bivalent metal ions, 10--20% of cyclic AMP is present as metal complexes. The possible significance of these metal complexes, for example as competitive inhibitors, is discussed.     

Spectral control of metal admixtures in tetracycline]

Analysis of circular dichroism spectra made it possible to offer a method for estimation of tetracycline solutions contamination with metal ions. By its sensitivity the method is much superior to the spectrophotometric one used at present for determination of the antibiotic purity. In the latter method formation of complexes with metals is traced by batochromic displacement of the absorption spectra. The new method is rapid, relatively selective and requires comparatively small quantities of the substance for the analysis, which provides its use under both laboratory and manufacture conditions. The method is based on identification of the circular dichroism spectra of tetracycline complexes with metals in the long wavelength region. The presence of the circular dichroism concervative bands with strictly defined extremums in the spectra of tetracycline low acid solutions contaminated by multiply charged metal ions allowed vs. the circular dichroism spectra of pure tetracycline sample to conclude that the solution contained admixtures and to suggest their nature. It was shown that the charge, ion radius and tetracycline:metal relation were the factors defining the mark and location of the dichroism band extremums. At lambda(extr)-410-415 nm the tetracycline complexes with light metal ions such as Mg2+, Al3+ and Ca2+ were detected by the circular dichroism negative band in the spectra, while the complexes with heavy metal ions such as Sc3+, Sr3+, Cu3+, Cd3+, Ba2+, Y3+ and the cerium subgroup lanthanides were detected by the circular dichroism positive band. The tetracycline complexes with the lanthanides of the second half of the yttrium subgroup (Ho(3+)-Lu3+) were characterized by the presence of the circular dichroism minimum at lambda(min)-425 nm. When the tetracycline concentration was above 1.5 x 10(-3) M, multiligand complexes with circular dichroism negative extremum at lambda(min)-400 nm formed.     

Toxicity evaluation of complex metal mixtures using reduced metal concentrations: application to iron oxidation by Acidithiobacillus ferrooxidans

In this study, we investigated the inhibition effects of single and mixed heavy metal ions (Zn2+, Ni2+, Cu2+, and Cd2+) on iron oxidation by Acidithiobacillus ferrooxidans. Effects of metals on the iron oxidation activity of A. ferrooxidans are categorized into four types of patterns according to its oxidation behavior. The results indicated that the inhibition effects of the metals on the iron oxidation activity were noncompetitive inhibitions. We proposed a reduced inhibition model, along with the reduced inhibition constant (alphai), which was derived from the inhibition constant (KI) of individual metals and represented the tolerance of a given inhibitor relative to that of a reference inhibitor. This model was used to evaluate the toxicity effect (inhibition effect) of metals on the iron oxidation activity of A. ferrooxidans. The model revealed that the iron oxidation behavior of the metals, regardless of metal systems (single, binary, ternary, or quaternary), is closely matched to that of any reference inhibitor at the same reduced inhibition concentration, [I]reduced, which defines the ratio of the inhibitor concentration to the reduced inhibition constant. The model demonstrated that single metal systems and mixed metal systems with the same reduced inhibitor concentrations have similar toxic effects on microbial activity.     

Preparation and properties of metal ion derivatives of the lentil and pea lectins

Lentil lectin (LcH) and pea lectin (PSA) belong to the class of D-glucose/D-mannose binding lectins and resemble concanavalin A (Con A) closely in physicochemical, structural, and biological properties. LcH and PSA, like Con A, are Ca2+-Mn2+ metalloproteins that require the metal ions for their saccharide binding and biological activities. Studies of the relationship between the metal ions binding and saccharide binding activity in LcH and PSA have been difficult due to the problem of metal ion replacement in these proteins. We now report a method of metal ion replacement in both lectins that allows substitution of the Mn2+ in the native proteins with a variety of transition metal ions, as well as substitution of the Ca2+ with Cd2+ in a particular complex. The following metal ion derivatives of both LcH and PSA have been prepared: Ca2+-Zn2+, Ca2+-Co2+, Ca2+-Ni2+, and Cd2+-Cd2+. All of these derivatives are as active as the native lectins, as demonstrated by precipitation with specific polysaccharides, saccharide inhibition of precipitation, and hemagglutination assays. The yields of these derivatives are good (generally greater than 70%), and the degree of metal ion incorporation is high (generally greater than 90%). The method of preparation is quite different from that for metal ion substitution in Con A, which proceeds via the apoprotein. In contrast, the apoproteins of LcH and PSA are unstable, aggregate above pH 4.0, and cannot be remetallized once formed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of Vitamin K-dependent Protein Precursor Propeptide,Vitamin K Hydroquinone,and Glutamate Substrate Binding on the Structure and Function of γ-Glutamyl Carboxylase

The γ-glutamyl carboxylase utilizes four substrates to catalyze carboxylation of certain glutamic acid residues in vitamin K-dependent proteins. How the enzyme brings the substrates together to promote catalysis is an important question in understanding the structure and function of this enzyme. The propeptide is the primary binding site of the vitamin K-dependent proteins to carboxylase. It is also an effector of carboxylase activity. We tested the hypothesis that binding of substrates causes changes to the carboxylase and in turn to the substrate-enzyme interactions. In addition we investigated how the sequences of the propeptides affected the substrate-enzyme interaction. To study these questions we employed fluorescently labeled propeptides to measure affinity for the carboxylase. We also measured the ability of several propeptides to increase carboxylase catalytic activity. Finally we determined the effect of substrates: vitamin K hydroquinone, the pentapeptide FLEEL, and NaHCO₃, on the stability of the propeptide-carboxylase complexes. We found a wide variation in the propeptide affinities for carboxylase. In contrast, the propeptides tested had similar effects on carboxylase catalytic activity. FLEEL and vitamin K hydroquinone both stabilized the propeptide-carboxylase complex. The two together had a greater effect than either alone. We conclude that the effect of propeptide and substrates on carboxylase controls the order of substrate binding in such a way as to ensure efficient, specific carboxylation.     

Steady-state kinetic studies of the metal ion-dependent decarboxylation of oxalacetate catalyzed by pyruvate kinase

Steady-state kinetic studies with differing divalent metals ions have been carried out on the pyruvate kinase-catalyzed, divalent cation-dependent decarboxylation of oxalacetate to probe the role of the divalent metal ion in this reaction. With either Mn2+ or Co2+, initial velocity patterns show that the divalent metal ion is bound to the enzyme in a rapid equilibrium prior to the addition of oxalacetate. Further, there is no change in the initial velocity patterns or the kinetic parameters in the presence or absence of K+, indicating that K+ is not required for oxalacetate decarboxylation. Dead-end inhibition of the decarboxylation reaction by the physiological substrate phosphoenolpyruvate indicates that phosphoenolpyruvate binds only to the enzyme-metal ion complex and not to free enzyme. The pKi values for both Mn2+ and Co2+ decrease below a pK of 7.0, and increase above a pK of 8.9. Since these pK values are the same for both ions, both of the observed pK values must be attributable to enzymatic residues. The pK of 7.0 is presumably that of a ligand to the metal ion, while the pK of 8.9 is probably that of the lysine involved in enolization of pyruvate in the normal physiological reaction. However, with Co2+ as divalent cation, the V for oxalacetate decreases above a pK of 8.0, the V/K decreases above two pK values averaging 7.8, and the pKi for oxalate decreases above a single pK of 7.3. These data indicate that metal-coordinated water is displaced during the binding of substrates or inhibitors and the other pK value observed in both V and V/K pH profiles (pK of 8.3 with Co2+ and 9.2 with Mg2+) is an enzymatic residue whose deprotonation disrupts the charge distribution in the active site and decreases activity.