Presence Of Metal Chelators Research Articles

The cdc25 protein contains an intrinsic phosphatase activity

Genetic and biochemical studies have indicated that the cdc25 protein controls the entry into mitosis by triggering tyrosine dephosphorylation of the cdc2 protein kinase. We show that the isolated cdc25 protein can catalyze dephosphorylation of several model phosphatase substrates, including p-nitrophenyl phosphate and two distinct tyrosine-phosphorylated peptides. The cdc25-dependent cleavage reaction closely resembles dephosphorylation by known tyrosine phosphatases: the reaction requires a reducing agent, shows high sensitivity to sodium vanadate, and proceeds efficiently in the presence of metal chelators. Moreover, the phosphatase activity of the cdc25 protein is eliminated by treatment with N-ethylmaleimide or by alteration of a single conserved cysteine residue by site-directed mutagenesis. These observations indicate that the cdc25 protein can function as a tyrosine phosphatase in the absence of any other protein.

Recombinant human tyrosine hydroxylase isozymes

Human tyrosine 3-monooxygenase (tyrosine hydroxylase) exists as four different isozymes (TH1-TH4), generated by alternative splicing of pre-mRNA. Recombinant TH1, TH2 and TH4 were expressed in high yield in Escherichia coli. The purified isozymes revealed high catalytic activity [when reconstituted with Fe(II)] and stability at neutral pH. The isozymes as isolated contained 0.04-0.1 atom iron and 0.02-0.06 atom zinc/enzyme subunit. All three isozymes were rapidly activated (13-40-fold) by incubation with Fe(II) salts (concentration of iron at half-maximal activation = 6-14 microM), and were inhibited by other divalent metal ions, e.g. Zn(II), Co(II) and Ni(II). They all bind stoichiometric amounts of Fe(II) and Zn(II) with high affinity (Kd = 0.2-3 microM at pH 5.4-6.5). Similar time courses were observed for binding of Fe(II) and enzyme activation. In the absence of any free Fe(II) or Zn(II), the metal ions were released from the reconstituted isozymes. The dissociation was favoured by acidic pH, as well as by the presence of metal chelators and dithiothreitol. The potency of metal chelators to remove iron from the hydroxylase correlated with their ability to inhibit the enzyme activity. These studies show that tyrosine hydroxylase binds iron reversibly and that its catalytic activity is strictly dependent on the presence of this metal.

On the regulation of gene expression during Escherichia coli adaptation to hydrogen peroxide

We found previously that 8-hydroxyguanine (oh8Gua) endonuclease in E. coli is induced in response to oxidative stress in a fashion similar to the oxidative response of the Mn-superoxide dismutase (MnSOD). In this study, attempts were made to identify the genes involved in the co-regulation of E. coli endonuclease and MnSOD (sodA). oh8Gua nuclease is induced by molecular oxygen and a superoxide radical generator (paraquat) but not by H2O2, suggesting that the regulation of this endonuclease is dependent on SoxRS but independent of OxyR. This enzyme was induced by paraquat in all of the soxRS mutant strains used (soxR−, soxS− and soxRc), whereas glucose-6-phosphate dehydrogenase (a member of the soxRS regulon) showed the expected responses; therefore, this possibility was excluded. The presence of metal chelators in the growth medium caused the induction of this enzyme, and this induction was suppressed by the addition of Fe++. Consistent with this finding, this enzyme was expressed under anaerobiosis in all of the mutant strains of fnr in particular, as well as fur, arcA, and combinations thereof. These findings suggest that the oxidative regulation of oh8Gua endonuclease is under control of fnr, fur, and arcA, where fnr plays a predominant role. The multiple involvement of regulatory genes as well as co-regulation with antioxidant enzyme will enhance the efficiency of cellular growth and survival in the aerobic environment.

Purification and properties of an extracellular collagenolytic protease produced by the human oral bacterium Bacillus cereus (strain Soc 67).

The major collagenolytic proteinase present in the culture filtrate of Bacillus cereus (strain Soc 67, isolated from the human oral cavity) has been purified to homogeneity by a procedure that comprised concentration of ultrafiltered growth medium on a Millipore PTTK00005 membrane, precipitation with ammonium sulfate, gel permeation chromatography, chromatofocusing, fast protein liquid chromatography on an anion-exchange column, and finally fast protein liquid chromatography on a gel column. The enzyme hydrolyzed, with decreasing rates, phenylazobenzyloxy-carbonyl-L-Pro-L-Leu Gly-L-Pro-D-Arg (PZ-PLGPA), furylacrylolyl-L-Leu-Gly-L-Pro-L-Ala, and furylacryloyl-L-Phe-Gly-Gly, while furylacryloyl-Gly-L-Leu-NH2 was not hydrolyzed. The enzyme degraded soluble and insoluble collagens, Azocoll and gelatin. Bradykinin was hydrolyzed at a high rate at the Phe-Ser bond. The enzyme was sensitive to pyrophosphate, L-cysteine, and L-histidine and could be totally inactivated in the presence of metal chelators. The enzyme contains 1 mol of Zn/mol and the hydrolysis of PZ-PLGPA is slightly increased by Ca2+. The enzyme is readily inhibited by heavy metal cations, but Cu2+ and Ni2+ affected the catalysis in opposite ways: increasing levels of Cu2+ decreased the affinity of the enzyme for PZ-PLGPA, whereas Ni2+ had no effect. The effect of Cu2+ also depended on the pH and type of buffer used. Detailed chemical modification experiments suggested that the active site of the enzyme contains at least 1 tyrosyl and 1 lysyl residue, and 1 carboxyl group. The enzyme was not sensitive to sulfhydryl reagents and thiols did not activate the enzyme. The modification studies were unable to reveal active histidyl residues. The ability of the enzyme to hydrolyze PZ-PLGPA, furylacryloyl-L-Leu-Gly-L-Pro-L-Ala, furylacryloyl-L-Phe-Gly-Gly, and various collagenous materials, its inactivity toward furylacryloyl-Gly-L-Leu-NH2, and the results from the chemical modification studies suggest that the B. cereus (Soc 67) collagenolytic enzyme can be regarded as a true collagenase which resembles the Clostridium histolyticum collagenase(s).

Formula omitted] of dopamine and tyramine by mosquito pupae ( Aedes togoi)

The N- acetyltransferase (NAT) activity of mosquito pupae was measured by a radioenzymatic assay, using [ 14C]-, [ 3H]dopamine, [ 14C]tyramine or [ 14C]acetyl-CoA. The pupal extract could also generate acetyl-CoA from ATP, acetate and CoA for this acetylation reaction. Both the dopamine- and tyramine-NAT reactions proceeded linearly up to 20 min at an optimum pH of 8.4. It is possible that the same enzyme is involved in the acetylation of both biogenic amines as shown by the competitive inhibition kinetics obtained, and the similarities of the NAT reaction with both amines, in the presence of metal chelators, metal ions, SH reagents and MAO inhibitors. Mn 2+ stimulated and Zn 2+ inhibited the reaction. The specific activity of NAT in individual pupae measured soon after pupation showed no significant difference between the male and female pupae: the values obtained were, respectively, 893 ± 57 and 861 ± 30 pmol [ 14C]NAcT formed/min per mg protein and 21.9 ± 1.2 and 22.0 ± 1.4 pmol [ 3H]NADA formed/min per mg protein.

Preparation of an enzymatically active cross-linked complex between brain cyclic nucleotide phosphodiesterase and 3-(2-pyridyldithio)propionyl-substituted calmodulin.

Cyclic nucleotide phosphodiesterase (0.07 nM) was activated by near stoichiometric concentrations of [3-(2-pyridyldithio)propionyl]calmodulin (PDP-CaM) after initial incubation of these proteins at 200-fold higher concentrations; activity in assays with EGTA was 80% of that in the presence of Ca2+. The enzyme incubated with native calmodulin under identical conditions required approximately 1 nM for half-maximal activation, and no activation was observed in the absence of calcium. These data suggested formation of a covalent complex between phosphodiesterase and PDP-CaM. On high-performance gel-permeation chromatography in the presence of metal chelators, the complex appeared considerably larger than the native enzyme. Incubation of phosphodiesterase with the thiolated (inactivated) form of PDP-CaM did not change its chromatographic behavior, indicating that reactive sulfhydryl groups were involved in complex formation. Although the total activities recovered from chromatography were not significantly different, maximal activation of PDP-CaM-phosphodiesterase complex was only approximately 20%, whereas the control enzyme was activated 6-8-fold by Ca2+ plus calmodulin. Kinetics of cGMP hydrolysis in the presence of EGTA by the isolated complex differed from those of control enzyme but were indistinguishable from those of control enzyme assayed with saturating Ca2+ and CaM. The calmodulin antagonists W-7 and trifluoperazine had relatively little effect on activity of the PDP-CaM-phosphodiesterase complex. Incubation of the complex with dithiothreitol dramatically increased its Ca2+ and calmodulin responsiveness, suggesting that reduction of the disulfide cross-link released phosphodiesterase from the complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Ascorbate oxidase: Properties of the copper sites and catalytic activity of the native and type 2 Cu depleted enzyme

Ascorbate oxidase (L-ascorbate: O 2 oxidoreductase, E.C. 1.10.3.3) is the subject of numerous biological investigations. Some of the recent developments in the purification of the enzyme, the number of Cu atoms per active molecule, the stoichiometry of the different Cu sites and the mechanism of reduction by L-ascorbate have been previously reviewed [1]. Ascorbate oxidase from squash Cucurbita p.m. contains 8 Cu atoms/140 000 M r of which 50% are detectable by electron paramagnetic resonance (EPR), i.e. three type-1 Cu, one type-2 Cu and four type-3 Cu [2]. The number and stoichiometry of different Cu sites depend very much on environmental conditions maintained during the process of isolation and purification, such as pH, ionic strength or presence of metal chelators. Thus, dialysis of pure enzyme against acetate buffer, pH 4.5–5.5, may lead to drastic changes in the visible region of the electronic spectra (indicated by a decrease of the purity index A 610/A 330 [1]) and of the EPR spectra (indicated by a decrease of the type-1 signal). In the presence of metal chelators, such as EDTA or dimethylglyoxime [3], the type-2 center is removed selectively (= t2d enzyme) as demonstrated by EPR spectroscopy. Depending on dialysis conditions (pH, concentration of metal chelator, aerobic vs. anaerobic) up to four Cu atoms will be dialysed off. As epxected, the activity of these enzyme samples is lowered to less than 4% of the original activity present. The so-called t2d enzyme (5.8 ± 0.3 Cu/140.000 M r) is still reduced by L-ascorbate in the absence of dioxygen, approx. 5.5 electron equivalents are used for total bleaching of the absorbance at 610 nm. Upon exposure to air, enzyme reoxidation begins as indicated by the appearance of the typical blue color (λ max 610 nm) and the type-1 Cu EPR signal. By comparison with native ascorbate oxidase this seems to be a relatively slow process, complete reoxidation is received after a few minutes at 20 °C, pH 7.0. The time course of the reaction of reduced ascorbate oxidase, or reduced t2d enzyme, with dioxygen was followed by stopped-flow spectrophotometry and rapid-freeze EPR spectroscopy. Prior to reaction with molecular oxygen the protein is reduced by an excess of L-ascorbate (6–8 equivalents of reductant per enzyme), hereafter mixing with O 2/buffer solution is achieved under the strict exclusion of air at temperatures between 5 and 10 °C [1]. In the first rapid phase of the reoxidation of the native enzyme (2–4 msec) the characteristic absorption spectrum and EPR signal of the L-ascorbate radical (λ max 365 nm, g = 2.005) can be detected [1], due to the turnover of the enzyme in the presence of reducing substrate and dioxygen. Total reoxidation of ascorbate oxidase occurs very rapidly, within less than 8 msec at 10 °C as shown optically by the appearance of absorbance at 610 nm and by EPR. By comparison with untreated ascorbate oxidase the reduced t2d enzyme is far less reactive towards dioxygen, the reoxidation process takes several minutes under identical conditions of pH, ionic strength, temperature and oxygen concentration. From the kinetic experiments on the anaerobic reduction of ascorbate oxidase and oxidation of the reduced enzyme conclusions are drawn concerning the modes of internal and external electron transfer. Furthermore, mechanistic aspects of the catalytic function of the multicopper oxidase are discussed.

A neutral metallo endoprotease involved in the processing of an F1-ATPase subunit precursor in mitochondria.

A post-translational processing assay of the precursor to the yeast F1-ATPase subunit has been utilized to examine a mitochondrial endoprotease which cleaves this subunit precursor to the size of a mature subunit. The endoprotease is extracted from purified mitochondria as a soluble complex of Mr = 115,000 which is composed of subunits of lower molecular weight when examined on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It exhibits a pH optimum of between pH 7 and 8 and is inactive at pH 6.5 and below. The mitochondrial endoprotease is insensitive to serine esterase inhibitors, but is inhibited by EDTA and o-phenanthroline. Restoration of precursor subunit processing activity in the presence of metal chelators is strictly dependent on excess Co2+ and Mn2+ over other heavy metals examined. These and additional data indicate that this soluble metallo endoprotease is involved in the processing of other cytoplasmically synthesized precursor subunits of the ATPase complex in addition to the subunit 2 precursor. The role of this processing enzyme in the assembly of mitochondrial inner membrane complexes is discussed in light of the current model of mitochondrial biogenesis.

Stability of lyophobic colloids in the presence of metal chelates and chelating agents

Coagulation, electrophoresis and adsorption data of various metal chelates and chelating agents on silver halide sols, PVC latex, and montmorillonite clay have been shown. The effects are most pronounced with the silver halide sols which are believed to be due to the coordination of the chelates and chelating agents with the crystal lattice ions of the colloidal particles. The PVC latex stability in the presence of the same solute species depends strongly on the concentration of the stabilizing surface active agent (dodecyl sulfate ions).

Reactions of Diazoketones in the Presence of Metal Chelates. III. Reactions with Carbonyl Compounds

Abstract The reaction of α-diazoacetophenone with carbonyl compounds such as benzaldehyde, acetophenone and p-phenylacetophenone, has been studied in the presence of bis(acetylacetonato) copper (II). The reaction gave the corresponding tetrahydrofurans and dioxolanes accompanied by some by-products derived from benzoylcarbene. The mechanism of the reaction has been discussed in terms of an intermediate with a structure of betaine type, produced by the reaction of a ketocarbene-metal chelate complex with carbonyl compounds.

Doxynucleotide-polymerizing enzymes of calf thymus gland. IV. Inhibition of terminal deoxynucleotidyl transferase by metal ligands.

The polymerization of deoxynucleoside triphosphates, catalyzed by terminal deoxynucleotidyl transferase from calf thymus gland, is strongly inhibited by various metal chelators. The reaction appears to be first order with respect to enzyme, deoxynucleoside triphosphate, and initiator, indicating that there is only one catalytic center for each enzyme molecule. The presence of metal chelator does not affect the molecular state of the enzyme or the order of reaction. An analysis of the kinetics of o-phenanthroline inhibition demonstrates that the ligand is noncompetitive with nucleoside triphosphate substrate and strictly competitive with oligodeoxynucleotide initiator. These results, obtained in the presence of millimolar Mg(++), suggest that the initiator binds to the enzyme through a transition metal atom and ligand inhibition occurs at that site. The precise spatial arrangement of the coordination orbitals around a metal atom provides a model to visualize the specificity of the enzyme for the 3'-OH end of the growing polynucleotide chain.

Reactions of Diazoketones in the Presence of Metal Chelates. II. Reactions with Olefinic Compounds

Abstract The reaction of α-diazoacetophenone with olefinic compounds, such as vinyl acetate, cis- and trans-stilbenes and cyclohexene, has been studied in the presence of bis(acetylacetonato)copper-(II). The reaction gave the corresponding cyclopropane derivatives, the addition products of benzoylcarbene to the double bond of olefinic compounds in good yields, accompanied by cis-and trans-dibenzoylethylenes, dibenzoylethane or dilactone. Addition of carbene was found to be stereospecific in the cases of cis- and trans-stilbenes. A mechanism has been advanced in which a ketocarbene-metal chelate complex is included as an intermediate of the reaction.

Reactions of Diazoketones in the Presence of Metal Chelates. I. Reactions in Alcohols

Abstract The reaction of diazoacetophenone with alcohols has been studied in the presence of metal chelates. Metal chelates, such as bis(acetylacetonato)copper(II), bis(acetylacetonato)nickel(II), and bis(acetylacetonato)lead(II), were found to be active catalysts for this reaction; they modified the reaction to give alkoxyacetophenones instead of alkyl phenylacetates. When tertiary amines, such as triethylamine, pyridine, and quinoline, were added to the reaction mixture, the formation of alkoxyacetophenones was suppressed and that of alkyl phenylacetates was promoted in proportion to the increase in the amount of amines added. The mechanism of the reaction can reasonably be explained by assuming the intermediacy of a ketocarbene-metal chelate complex.