Differential effects of monovalent, divalent and trivalent metal ions on rat brain hexokinase

The effects of monovalent (Li+, Cs+) divalent (Cu2+, Ca2+, Sr2+, Ba2+, Zn2+, Cd2+, Hg2+, Pb2+, Mn2+, Fe2+, Co2+, Ni2+) and trivalent (Cr3+, Fe3+, Al3+) metals ions on hexokinase activity in rat brain cytosol were compared at 500 microM. The rank order of their potency as inhibitors of brain hexokinase was: Cr3+ (IC50 = 1.3 microM) greater than Hg2+ = Al3+ greater than Cu2+ greater than Pb2+ (IC50 = 80 microM) greater than Fe3+ (IC50 = 250 microM) greater than Cd2+ (IC50 = 540 microM) greater than Zn2+ (IC50 = 560 microM). However, at 500 microM Co2+ slightly stimulated brain hexokinase whereas the other metal ions were without effect. That inhibition of brain glucose metabolism may be an important mechanism in the neurotoxicity of metals is suggested.     

A metal bridge between two enzyme families. 3-deoxy-D-manno-octulosonate-8-phosphate synthase from Aquifex aeolicus requires a divalent metal for activity

The enzymes 3-deoxy-d-manno-octulosonic acid-8-phosphate synthase (KDO8PS) and 3-deoxy-d-arabino-heptulosonic acid-7-phosphate synthase (DAHPS) catalyze analogous condensation reactions between phosphoenolpyruvate and d-arabinose 5-phosphate or d-erythrose 4-phosphate, respectively. While several similarities exist between the two enzymatic reactions, classic studies on the Escherichia coli enzymes have established that DAHPS is a metalloenzyme, whereas KDO8PS has no metal requirement. Here, we demonstrate that KDO8PS from Aquifex aeolicus, representing only the second member of the KDO8PS family to be characterized in detail, is a metalloenzyme. The recombinant KDO8PS, as isolated, displays an absorption band at 505 nm and contains approximately 0.4 and 0.2-0.3 eq of zinc and iron, respectively, per enzyme subunit. EDTA inactivates the enzyme in a time- and concentration-dependent manner and eliminates the absorption at 505 nm. The addition of Cu(2+) to KDO8PS produces an intense absorption at 375 nm, while neither Co(2+) nor Ni(2+) produce such an effect. The EDTA-treated enzyme is reactivated by a wide range of divalent metal ions including Ca(2+), Cd(2+), Co(2+), Cu(2+), Fe(2+), Mg(2+), Mn(2+), Ni(2+), and Zn(2+) and is reversibly inhibited by higher concentrations (>1 mm) of certain metals. Analysis of several metal forms of the enzyme by plasma mass spectrometry suggests that the enzyme preferentially binds one, two, or four metal ions per tetramer. These observations strongly suggest that A. aeolicus KDO8PS is a metalloenzyme in vivo and point to a previously unrecognized relationship between the KDO8PS and DAHPS families.     

The functional role of zinc in angiotensin converting enzyme: implications for the enzyme mechanism

Zinc is essential to the catalytic activity of angiotensin converting enzyme. The enzyme contains one g-atom of zinc per mole of protein. Chelating agents abolish activity by removing the metal ion to yield the inactive, metal-free apoenzyme. Zinc does not stabilize protein structure since the native and apoenzymes are equally susceptible to heat denaturation. Addition of either Zn2+, Co2+, or Mn2+ to the apoenzyme generates an active metalloenzyme; Fe2+, Ni2+, Cu2+, Cd2+, and Hg2+ fail to restore activity. The activities of the metalloenzymes follow the order Zn greater than Co greater than Mn. The protein binds Zn2+ more firmly than it does Co2+ or Mn2+. Hydrolysis of the chromophoric substrate, furanacryloyl-Phe-Gly-Gly, by the active metalloenzymes is subject to chloride activation; the activation constant is not metal dependent. Metal replacement mainly affects Kcat with very little change in Km, indicating that the role of zinc is to catalyze peptide hydrolysis.     

Intracellular hexose-6-phosphate:phosphohydrolase from Streptococcus lactis: purification, properties, and function.

An intracellular hexose 6-phosphate:phosphohydrolase (EC 3.1.3.2) has been purified from Streptococcus lactis K1. Polyacrylamide disc gel electrophoresis of the purified enzyme revealed one major activity staining protein and one minor inactive band. The Mr determined by gel permeation chromatography was 36,500, but sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single polypeptide of apparent Mr 60,000. The enzyme exhibited a marked preference for hexose 6-phosphates, and the rate of substrate hydrolysis (at 5 mM concentration) decreased in the order, galactose 6-phosphate greater than 2-deoxy-D-glucose 6-phosphate greater than fructose 6-phosphate greater than mannose 6-phosphate greater than glucose 6-phosphate. Hexose 1-phosphates, p-nitrophenylphosphate, pyrophosphate, and nucleotides were not hydrolyzed at a significant rate. In addition, the glycolytic intermediates comprising the intracellular phosphoenolpyruvate potential in the starved cells (phosphoenolpyruvate and 2- and 3-phosphoglyceric acids) were not substrates for the phosphatase. Throughout the isolation, the hexose 6-phosphate:phosphohydrolase was stabilized by Mn2+ ion, and the purified enzyme was dependent upon Mn2+, Mg2+, Fe2+, or Co2+ for activation. Other divalent metal ions including Pb2+, Cu2+, Zn2+, Cd2+, Ca2+, Ba2+, Sr2+, and Ni2+ were unable to activate the enzyme, and the first four cations were potent inhibitors. Enzymatic hydrolysis of 2-deoxy-D-glucose 6-phosphate was inhibited by fluoride when Mg2+ was included in the assay, but only slight inhibition occurred in the presence of Mn2+, Fe2+, or Co2+. The inhibitory effect of Mg2+ plus fluoride was specifically and completely reversed by Fe2+ ion. The hexose 6-phosphate:phosphohydrolase catalyzes the in vivo hydrolysis of 2-deoxy-D-glucose 6-phosphate in stage II of the phosphoenolpyruvate-dependent futile cycle in S. lactis (J. Thompson and B. M. Chassy, J. Bacteriol. 151:1454-1465, 1982).     

Detection of two Zn-finger proteins ofXenopus laevis,TFIIIA, and p43, by probing western blots of ovary cytosol with65Zn2+,63Ni2+, or109Cd2+

Two Zn-finger proteins, TFIIIA (a constituent of 7S RNP particles) and p43 (a constituent of 42S RNP particles), were detected in ovary extracts of juvenile Xenopus laevis females by in vitro binding of radiolabeled divalent metals. Proteins fractionated by SDS-PAGE (sodium dodecylsulfate-polyacrylamide gel electrophoresis) were transferred by Western blotting onto nitrocellulose membranes, probed with 65Zn2+, 63Ni2+, or 109Cd2+, and visualized by autoradiography. Detection limits for TFIIIA were approx 0.07 micrograms/well by 109Cd(2+)-probing, 0.13 micrograms/well by 65Zn(2+)-probing, and 0.26 mu/well by 63Ni(2+)-probing. Protein p43 was more clearly visualized by probing with 63Ni2+ than with 65Zn2+ or 109Cd2+. After purified TFIIIA was cleaved with cyanogen bromide, 65Zn2+, 109Cd2+, and 63Ni2+ distinctly labeled the 22 kDa middle fragment; 65Zn2+ and 109Cd2+ also labeled the 11 kDa N-terminal fragment, but did not label the 13 kDa C-terminal fragment. These results are consistent with the notion that the radioligands were bound to finger-loop domains of TFIIIA, which occur in the middle and N-terminal fragments. Based on the abilities of nonradioactive metal ions to compete with 65Zn2+ for binding to TFIIIA on Western blots, the relative affinities of the metals for TFIIIA were ranked as follows: Zn2+ = Cu2+ greater than or equal to Hg2+ greater than Cd2+ greater than Co2+ greater than or equal to Ni2+. Even at a 1000-fold molar excess, Mn2+ did not compete with 65Zn2+ for binding to TFIIIA. Probing Western blots with the radiolabeled metal ions greatly facilitates the detection, isolation, and quantitation of TFIIIA and p43.     

Metal-catalyzed oxidation of phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli: inactivation and destabilization by oxidation of active-site cysteines

The in vitro instability of the phenylalanine-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase [DAHPS(Phe)] from Escherichia coli has been found to be due to a metal-catalyzed oxidation mechanism. DAHPS(Phe) is one of three differentially feedback-regulated isoforms of the enzyme which catalyzes the first step of aromatic biosynthesis, the formation of DAHP from phosphoenolpyruvate and D-erythrose-4-phosphate. The activity of the apoenzyme decayed exponentially, with a half-life of about 1 day at room temperature, and the heterotetramer slowly dissociated to the monomeric state. The enzyme was stabilized by the presence of phosphoenolpyruvate or EDTA, indicating that in the absence of substrate, a trace metal(s) was the inactivating agent. Cu2+ and Fe2+, but none of the other divalent metals that activate the enzyme, greatly accelerated the rate of inactivation and subunit dissociation. Both anaerobiosis and the addition of catalase significantly reduced Cu2+-catalyzed inactivation. In the spontaneously inactivated enzyme, there was a net loss of two of the seven thiols per subunit; this value increased with increasing concentrations of added Cu2+. Dithiothreitol completely restored the enzymatic activity and the two lost thiols in the spontaneously inactivated enzyme but was only partially effective in reactivation of the Cu2+-inactivated enzyme. Mutant enzymes with conservative replacements at either of the two active-site cysteines, Cys61 or Cys328, were insensitive to the metal attack. Peptide mapping of the Cu2+-inactivated enzyme revealed a disulfide linkage between these two cysteine residues. All results indicate that DAHPS(Phe) is a metal-catalyzed oxidation system wherein bound substrate protects active-site residues from oxidative attack catalyzed by bound redox metal cofactor. A mechanism of inactivation of DAHPS is proposed that features a metal redox cycle that requires the sequential oxidation of its two active-site cysteines.     

The effect of divalent cations on bovine spermatozoal adenylate cyclase activity.

The effect of divalent cations on bovine sperm adenylate cyclase activity was studied. Mn2+, Co2+, Cd2+, Zn2+, Mg2+ and Ca2+ were found to satisfy the divalent cation requirement for catalysis of the bovine sperm adenylate cyclase. These divalent cations in excess of the amount necessary for the formation of the metal-ATP substrate complex were found to stimulate the enzyme activity to various degrees. The magnitude of stimulation at saturating concentrations of the divalent cations was strikingly greater with M2+ than with either Ca2+, Mg2+, Zn2+, Cd2+ or Co2+. The apparent Km was lowest for Zm2+ (0.1 - 0.2 mM) than for any of the other divalent cations tested (1.2 - 2.3 mM). The enzyme stimulation by Mn2+ was decreased by the simultaneous addition of Co2+, Cd2+, Ni2+ and particularly Zn2+ and Cu2+. The antagonism between Mn2+ and Cu2+ or Zn2+ appeared to have both competitive and non-competitive features. The inhibitory effect of Cu2+ on Mn2+-stimulated adenylate cyclase activity was prevented by 2,3-dimercaptopropanol, but not by dithiothreitol, L-ergothioneine, EDTA, EGTA or D-penicillamine. Ca2+ at concentrations of 1-5 mM was found to act synergistically with Mg2+, Zn2+, Co2+ and Mn2+ in stimulating sperm adenylate cyclase activity. The Ca2+ augmentation of the stimulatory effect of Zn2+, Co2+, Mg2+ and Mn2+ appeared to be specific.     

The role of ATP and divalent cations in the regulation of a cardiac phosphorylase phosphatase (phosphoprotein phosphatase) of Mr = 35,000.

The effects of ATP and divalent cations on a divalent cation-independent phosphorylase phosphatase of Mr = 35,000 (phosphatase S) purified from canine cardiac muscle have been studied. The enzyme can be rapidly inactivated by ATP or other nucleoside di- and triphosphates and PPi, but not by AMP, adenosine, adenine, Pi, EDTA, ethylene glycol bis(beta-aminoethyl ether)N,N' -tetraacetic acid, 1,10-phenanthroline, or 8-hydroxyquinoline. After removing the inactivating agent, such as ATP or PPi, by gel filtraiton followed by exhaustive dialysis, the inactivated enzyme (apophosphatase S) can be reactivated by preincubating with Mn2+ or Co2+, but not with Mg2+, Ca2+, Ni2+, Zn2+, Fe2+, Cu2+, Ba2+, Hg2+, Pb2+, or Cd2+. The Mn2+ -reactivated enzyme, which is less active than the Co2+ -reactivated enzyme, can be again inactivated by preincubating with ATP. The present findings indicate that phosphatase S contains a tightly bound divalent cation, probably Mn2+, in the active site. ATP and PPi, due to their structural similarity to the phosphoprotein substrate and their ability to chelate metal ions, can readily enter the active site to remove the divalent cation(s) essential for the catalytic function. The present findings also indicate that phosphatase S, a common catalytic subunit of several larger molecular forms of nospecific phosphoprotein phosphatase in cardiac muscle, can exist in two interconvertible forms, a metallized form (active) and a demetallized form (inactive). ATP and metal ions may regulate this class of isozymes by mediating the interconversions.     

Thermostable DNA polymerase from Thermus thermophilus B35: influence of divalent metal ions on the interaction with deoxynucleoside triphosphates

The interaction of DNA polymerase from Thermus thermophilus B35 (Tte-pol) with deoxynucleoside triphosphates in the presence of different divalent metal ions has been studied. DNA synthesis and competitive inhibition of the polymerase reaction by non-complementary dNTPs are described with corresponding kinetic schemes. The co-factor properties of some metals (Mg2+, Mn2+, Co2+, Ni2+, Cu2+, Ca2+, Cd2+, and Zn2+) were investigated, and their activating concentration ranges were determined. It was found that kcat values are significantly decreased and Km values slowly decrease when Mn2+ displaces Mg2+. The value of Kd for DNA template-primer is Me2+-independent, whereas Kd values for non-complementary dNTPs decrease in the presence of Mn2+. Tte-pol processivity but not DNA synthesis efficiency is Me2+-type independent.     

Physicochemical characterization of the microbial Fe2+ chelator proferrorosamine from Pseudomonas roseus fluorescens.

The microbial chelating compound proferrorosamine A, produced by Pseudomonas roseus fluorescens, formed a complex with Fe2+ of which the apparent stability constant was found to be 10(23). The following order of increasing stability constants of metal complexes with proferrorosamine was established as: Ba2+, Ca2+, Mg2+, Mn2+ less than Hg2+ less than Zn2+ less than Pb2+ less than Co2+ less than Cu2+ congruent to Fe2+ less than Ni2+. Only Ni(2+)-proferrorosamine had a stability constant which was established as: Ba2+, Ca2+, Mg2+, Mn2+ less than Hg2+ less than Zn2+ less than Pb2+ less than Co2+ less than Cu2+ congruent to Fe2+ less than Ni2+. Only Ni(2+)-proferrorosamine had a stability constant which was ca 32 times higher than Fe(2+)-proferrorosamine. Because of the production of proferrorosamine the growth of Ps. roseus fluorescens was not inhibited in iron limiting media by the addition of 0.15 mmol/l of the weaker chemical Fe2+ chelator 2,2'-dipyridyl. This contrasted with the proferrorosamine-negative mutant K2 and Ps. stutzeri, which only produces Fe(3+)-chelating siderophores. Furthermore, it was found that proferrorosamine was able to dissolve Fe2+ from stainless steel. These results show that proferrorosamine is a strong and selective Fe2+ chelator which could be used as an alternative for the toxic 2,2'-dipyridyl to control lactic acid fermentations.     

Activation of membrane-bound high-affinity calcium ion-sensitive adenosine triphosphatase of human erythrocytes by bivalent metal ions.

The Ca2+-sensitive ATPase (adenosine triphosphatase) of human erythrocyte membranes is activated, not only by Ca2+ ions, but also by a series of other bivalent metal ions including Sr2+, Ba2+, Mn2+, Ni2+, Co2+, Cd2+, Cu2+, Zn2+ and Pb2+. The degree of activation is dependent on the radius of the ion rather than on its nature, in contrast with the dissociation constant of the enzyme--metal ion complex.     

Interaction of metal ions with lupin seed conglutin gamma

Various metal ions were capable of aggregating and precipitating conglutin gamma, an oligomeric glycoprotein purified from Lupinus albus seeds, at neutral pH values. The most effective metal ions, at 60-fold molar excess to the protein, were Zn2+, Hg2+ and Cu2+; a lower influence on the physical status of conglutin gamma was observed with Cr3+, Fe3+, Co2+, Ni2+, Cd2+, Sn2+, and Pb2+, while Mg2+, Ca2+ and Mn2+ had no effect at all. The insolubilisation of the protein with Zn2+, which is fully reversible, strictly depended on both metal concentration and pH. with middle points of the sharp transitions at three-fold molar excess and pH 6.5, respectively. Conglutin gamma is also fully retained on a metal affinity chromatography column at which Zn2+ and Ni2+ were complexed. A drop of pH below 6.0 and the use of chelating agents, such as EDTA and imidazole, fully desorbed the protein. A slightly lower binding to immobilised Cu2+ and Co2+ and no binding with Mg2+, Cd2+ and Mn2+ were observed. The role of the numerous histidine residues of conglutin gamma in the binding of Zn2+ is discussed.     

Petiolar felt-sheath of palm: a new biosorbent for the removal of heavy metals from contaminated water.

Biosorption of heavy metals such as Pb2+, Ni2+, Cd2+, Cu2+, Cr3+ and Zn2+ by petiolar felt-sheath of palm (PFP) from contaminated water was examined. PFP was found to efficiently remove all the toxic metal ions with selectivity order of Pb2+ > Cd2+ > Cu2+ > Zn2+ > Ni2+ > Cr3+. The uptake was rapid, with more than 70% completed within 15 min. The bound metal ions were successfully desorbed and the PFP fibrous-biomass remained effective after several adsorption-desorption cycles.     

Inhibition of glycogen synthase (casein) kinase-1 by divalent metal ions

The specificity of glycogen synthase (casein) kinase-1 (CK-1) for different divalent metal ions was explored in this study. Of nine metal ions (Mg2+, Mn2+, Zn2+, Cu2+, Ca2+, Ba2+, Ni2+, Co2+, Fe2+) tested, only Mg2+ supported significant kinase activity. Several of the other metals, however, inhibited the Mg2+-stimulated kinase activity. Half-maximal inhibitions by Mn2+, Zn2+, Co2+, Fe2+, and Ni2+ were observed at 55, 65, 110, 125, and 284 microM, respectively. Kinetic analyses indicate that the metal ions are acting as competitive inhibitors of CK-1 with respect to the protein substrate (casein) and as noncompetitive inhibitors with respect to the nucleotide substrate (ATP). The inhibition of CK-1 by the different metal ions can be reversed by EGTA.     

Regulation of the L-lactase dehydrogenase from Lactobacillus casei by fructose-1,6-diphosphate and metal ions.

The lactate dehydrogenase of Lactobacillus casei, like that of streptococci, requires fructose-1,6-diphosphate (FDP) for activity. The L. casei enzyme has a much more acidic pH optimum (pH 5.5) than the streptococcal lactate dehydrogenases. This is apparently due to a marked decrease in the affinity of the enzyme for the activator with increasing pH above 5.5; the concentration of FDP required for half-maximal velocity increase nearly 1,000-fold from 0.002 mM at pH 5.5 to 1.65 mM at 6.6. Manganous ions increase the pH range of activity particularly on the alkaline side of the optimum by increasing the affinity for FDP. This pH dependent metal ion activation is not specific for Mn2+. Other divalent metals, Co2+, Cu2+, Cd2+, Ni2+, Fe2+, Fe2+, and Zn2+ but not Mg2+, will effectively substitute for Mn2+, but the pH dependence of the activation differs with the metal ion used. The enzyme is inhibited by a number of commonly used buffering ions, particularly phosphate, citrate, and tris (hydroxymethyl) aminomethane-maleate buffers, even at low buffer concentrations (0.02 M). These buffers inhibit by affecting the binding of FDP.     

Oxidoreductase activities of polyclonal IgGs from the sera of Wistar rats are better activated by combinations of different metal ions

It was shown that IgGs purified from the sera of healthy Wistar rats contain several different bound Me2+ ions and oxidize 3,3'-diaminobenzidine through a H2O2-dependent peroxidase and H2O2-independent oxidoreductase activity. IgGs have lost these activities after removing the internal metal ions by dialysis against EDTA. External Cu2+ or Fe2+ activated significantly both activities of non-dialysed IgGs containing different internal metals (Fe > or = Pb > or = Zn > or = Cu > or = Al > or = Ca > or = Ni > or = Mn > Co > or = Mg) showing pronounced biphasic dependencies corresponding to approximately 0.1-2 and approximately 2-5 mM of Me2+, while the curves for Mn2+ were nearly linear. Cu2+ alone significantly stimulated both the peroxidase and oxidoreductase activities of dialysed IgGs only at high concentration (> or = 2 mM), while Mn2+ weakly activated peroxidase activity at concentration >3 mM but was active in the oxidoreductase oxidation at a low concentration (<1 mM). Fe2+-dependent peroxidase activity of dialysed IgGs was observed at 0.1-5 mM, but Fe2+ was completely inactive in the oxidoreductase reaction. Mg2+, Ca2+, Zn2+, Al2+ and especially Co2+ and Ni2+ were not able to activate dialysed IgGs, but slightly activated non-dialysed IgGs. The use of the combinations of Cu2+ + Mn2+, Cu2+ + Zn2+, Fe2+ + Mn2+, Fe2+ + Zn2+ led to a conversion of the biphasic curves to hyperbolic ones and in parallel to a significant increase in the activity as compared with Cu2+, Fe2+ or Mn2+ ions taken separately; the rates of the oxidation reactions, catalysed by non-dialysed and dialysed IgGs, became comparable. Mg2+, Co2+ and Ni2+ markedly activated the Cu2+-dependent oxidation reactions catalysed by dialysed IgGs, while Ca2+ inhibited these reactions. A possible role of the second metal in the oxidation reactions is discussed.     

Cadmium evokes inositol polyphosphate formation and calcium mobilization. Evidence for a cell surface receptor that cadmium stimulates and zinc antagonizes

Cd2+ and other divalent metals mobilized cell Ca2+ in human skin fibroblasts. The divalent metals produced a large spike in cytosolic free Ca2+ and strikingly increased net Ca2+ efflux similarly to bradykinin. One-tenth microM Cd2+ half-maximally increased 45Ca2+ efflux. The potency order of the Ca2+ mobilizing metals was: Cd2+ greater than Co2+ greater than Ni2+ greater than Fe2+ greater than Mn2+. Cd2+ probably acts at an extracellular site because loading the cells with a heavy metal chelator only slightly inhibited Cd2+-evoked 45Ca2+ efflux. Cd2+ increased [3H]inositol polyphosphates; [3H]inositol trisphosphate increased 4-fold in 15 s. Zn2+ reversibly blocked 45Ca2+ efflux evoked by Cd2+ but not that produced by bradykinin. Zn2+ competitively (Ki = approximately 0.4 microM) inhibited net Ca2+ efflux produced by Cd2+. Cd2+ also evoked Ca2+ mobilization in umbilical artery muscle, endothelial, and neuroblastoma cells, and the divalent cation agonist and antagonist specificities were similar to those in the fibroblasts. The divalent metals appear to trigger Ca2+ mobilization via a reversible interaction with an external site on the cell surface, which may be considered a "Cd2+ receptor."     

Probing the role of metal ions in the catalysis of Helicobacter pylori 3-deoxy-D-manno-octulosonate-8-phosphate synthase using a transient kinetic analysis

3-Deoxy-d-manno-2-octulosonate-8-phosphate (KDO8P) synthase catalyzes the net condensation of phosphoenolpyruvate and d-arabinose 5-phosphate to form KDO8P and inorganic phosphate (Pi). Two classes of KDO8P synthases have been identified. The Class I KDO8P synthases (e.g. Escherchia coli KDO8P synthase) catalyze the condensation reaction in a metal-independent fashion, whereas the Class II enzymes (e.g. Aquifex aeolicus) require metal ions for catalysis. Helicobacter pylori (H. pylori) KDO8P synthase, a Zn2+-dependent metalloenzyme, has recently been found to be a Class II enzyme and has a high degree of clinical significance since it is an attractive molecular target for the design of novel antibiotic therapy. Although the presence of a divalent metal ion in Class II KDO8P synthases is essential for catalysis, there is a paucity of mechanistic information on the role of the metal ions and functional differences as compared with Class I enzymes. Using H. pylori KDO8P synthase as a prototypical Class II enzyme, a steady-state and transient kinetic approach was undertaken to understand the role of the metal ion in catalysis and define the kinetic reaction pathway. Metal reconstitution experiments examining the reaction kinetics using Zn2+, Cd2+, Cu2+, Co2+, Mn2+, and Ni2+ yielded surprising results in that the Cd2+ enzyme has the greatest activity. Unlike Class-I KDO8P synthases, the Class II metallo-KDO8P synthases containing Zn2+, Cd2+, Cu2+, and Co2+ show cooperativity. This study presents the first detailed kinetic characterization of a metal-dependent Class II KDO8P synthase and offers mechanistic insight for how the divalent metal ions modulate catalysis through effects on chemistry as well as quaternary protein structure.     

Comparative Evaluation of Trace Metal Distribution and Correlation in Human Malignant and Benign Breast Tissues

Selected trace metals were analyzed in human malignant and nonmalignant (benign) breast tissue samples by the flame atomic absorption spectrophotometric method. In malignant tissues, dominant mean concentrations were revealed by Na, K, Ca, Mg, Fe, Zn, and Al at 927, 552, 231, 61.7, 36.5, 18.3, and 8.94 microg/g, respectively, while the mean metal levels in benign tissues were 903, 435, 183, 63.3, 24.7, 14.5, and 10.1 microg/g, respectively. Average concentrations of Cd, Co, Cr, Cu, Fe, Mn, K, Ca, and Zn were noted to be significantly higher in the malignant tissues compared with the benign tissues. Significantly strong correlations (r > 0.50) in malignant tissues were observed between Mn and Co, Mn and Cd, Cd and Cr, Fe and Mn, Cd and Co, Fe and Co, Mg and Pb, Cd and Fe, Mg and Ni, Pb and Ni, Ni and Sr, and Fe and Pb, whereas, Cd and Co, Cd and Mn, Co and Mg, Co and Mn, Cu and Mn, Co and Ni, Mg and Ni, Cd and Cu, Cd and Ni, Ca and Mg, Mn and Pb, Cu and Ni, Fe and Ni, Cd and Mg, Co and Cu, Cr and Na, and Cd and Cr revealed strong and significant relationships in benign tissues at p < 0.001. Principal component analysis of the metals data yielded six principal components for malignant tissues and five principal components for benign tissues, with considerably different loadings, duly supported by cluster analysis. The study revealed a considerably different pattern of distribution and mutual correlations of trace metals in the breast tissues of benign and cancerous patients.     

Hemolysis of human red blood cells induced by the combination of diethyldithiocarbamate (DDC) and divalent metals: modulation by anaerobiosis, certain antioxidants and oxidants.

The objective of the present communication is to describe the role played by combinations between diethydithiocarbamate (DDC) and divalent metals in hemolysis of human RBC. RBC which had been treated with DDC (10-50 microM) were moderately hemolyzed (about 50%) upon the addition of subtoxic amounts of Cu2+ (50 microM). However, a much stronger and a faster hemolysis occurred either if mixtures of RBC-DDC were immediately treated either by Co2+ (50 microM) or by a premixture of Cu2+ and Co2+ (Cu:Co) (50 microM). While Fe2+ and Ni2+, at 50 microM, initiated 30-50% hemolysis when combined with DDC (50 microM), on a molar basis, Cd2+ was at least 50 fold more efficient than any of the other metals in the initiation of hemolysis by DDC. On the other hand, neither Mn2+ nor Zn2+, had any hemolysis-initiating effects. Co2+ was the only metal which totally blocked hemolysis if added to DDC prior to the addition of the other metals. Hemolysis by mixtures of DDC + (Cu:Co) was strongly inhibited by anaerobiosis (flushing with nitrogen gas), by the reducing agents glutathione, N-acetyl cysteine, mercaptosuccinate, ascorbate, TEMPO, and alpha-tocopherol, by the PLA2 inhibitorbromophenacylbromide (BrPACBr), by tetracycline as well as by phosphatidyl choline, cholesterol and by trypan blue. However, TEMPO, BrPACBr and PC were the only agents which inhibited hemolysis induced by DDC: Cd2+ complexes. On the other hand, none of the classical scavengers of reactive oxygen species (ROS) employed e.g dimethylthiourea, catalase, histidine, mannitol, sodium benzoate, nor the metal chelators desferal and phenanthroline, had any appreciable inhibitory effects on hemolysis induced by DDC + (Cu:Co). DDC oxidized by H2O2 lost its capacity to act in concert either with Cu2+ or with Cd2+ to hemolyze RBC. While either heating RBC to temperatures greater than 37 degrees C or exposure of the cells to glucose-oxidase-generated peroxide diminished their susceptibility to hemolysis, exposure to the peroxyl radical from AAPH, enhanced hemolysis by DDC + (Cu:Co). The cyclovoltammetry patterns of DDC were drastically changed either by Cu2+, Co2+ or by Cd2+ suggesting a strong interaction of the metals with DDC. Also, while the absorbance spectrum of DDC at 280 nm was decreased by 50% either by Co2+, Cd2+ or by H2O2, a 90% reduction in absorbance occurred if DDC + H2O2 mixtures were treated either by Cu2+ or by Co2+, but not by Cd2+. Taken together, it is suggested that DDC-metal chelates can induce hemolysis by affecting the stability and the integrity of the RBC membrane, and possibly also of the cytoskeleton and the role played by reducing agents as inhibitors might be related to their ability to deplete oxygen which is also supported by the inhibitory effects of anaeobiosis.