Mol Of Zinc Research Articles

Accumulation of Cadmium by Rainbow Trout, Salmo gairdneri, during Extended Exposure

Adult rainbow trout, Salmo gairdneri, were exposed to 3.6 and 6.4 μg Cd/L for 178 d. Cadmium accumulated most rapidly in gill tissue which became saturated at levels 100-fold higher than controls within 24 and 52 d in the high- and low-metal exposures, respectively. Liver cadmium increased 250- to 400-fold over the test period but accumulation exhibited a plateau between 52 and 129 d followed by a rapid rise by 178 d. Kidney cadmium increased consistently throughout the test period to levels approximately 50- to 100-fold higher than control values. Cadmium in the gut and skin increased 10- and 5-fold, respectively, while no increase was recorded in white muscle. A maximum of 2.1% of the cadmium available in a commercial diet (0.2 μg Cd/g dry food} was accumulated in control fish. Although cadmium was not detected in the urine, urinary zinc excretion was elevated in trout exposed to 6.4 μg Cd/L such that 7 mol of zinc was excreted per 1 mol of cadmium accumulated during the initial 24 d of exposure. The whole-body burden of cadmium increased linearly with time in both treatments with a time constant of 0.366 and 0.554%/d for trout exposed to 3.6 and 6.4 μg Cd/L, respectively.

Suppression of gastric ulcer induced by stress and HCL-ethanol by intravenously administered metallothionein-II

A metallothionein isoform metallothionein-II was isolated from the livers of zinc acetate-treated rats. Metallothionein-II, which showed a single band on polyacrylamide gel electrophoresis, was subjected to two kinds of anti-ulcer screening systems. It was shown that intravenously administered metallothionein-II suppressed the formation of rat water-immersion stress- and HCl-ethanol-induced gastric ulcer significantly. The effect may partly be derived from the zinc contained in the metallothionein-II. However, the effect of metallothionein-II was much stronger than that of an equivalent mole of zinc. Apparently, metallothionein-II had an anti-ulcerogenic activity not based on the effect of intrinsic zinc.

Soluble tyrosine hydroxylase (tyrosine 3-monooxygenase) from bovine adrenal medulla: Large-scale purification and physicochemical properties

A new procedure that permits large-scale purification of tyrosine 3-monooxygenase (tyrosine hydroxylase) ( l-tyrosine,tetrahydropteridine: oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2) from the cytosolic fraction of bovine adrenal medulla is described. The homogenous enzyme revealed a subunit M r of 60 000 anda specific activity of 425 nmol · min −1 · mg −1. The N-terminal amino-acid sequence (27 residues) revealed 89% homology with the human pheochromocytoma enzyme as deduced from its cDNA sequence. The pure enzyme contained 0.66 ± 0.09 mol iron, 0.13 mol zinc and 0.62 ± 0.04 mol phosphate per mol subunit of M r = 60 000. A broad light absorption band with its maximum around 700 nm ( ε 700 nm = 1.3 (mM monomer) −1 · cm −1) explains its blue-green color. EPR spectra at 3.6 K revealed high-spin Fe(III) ( S = 5 2 ) in an environment of nearly axial symmetry ( g values at 7.2–6.7, 4.7–5.3 and 1.9–2.0). A close correlation was observed between the absorbance at 700 nm and the intensity of the axial type of EPR spectrum. The absorption peak at 700 nm is compatible with a ligand-to-iron charge-transfer transition as a result of catecholate coordination to the iron. Physicochemical studies suggest that the enzyme does not undergo such major substrate- or cofactor-induced conformational changes as have been reported for the related enzyme, phenylalanine hydroxylase.

Purification and properties of the hydroxylase component of methane monooxygenase.

Methane monooxygenase from Methylobacterium sp. strain CRL-26 which catalyzes the oxygenation of hydrocarbons was resolved into two components, a hydroxylase and a flavoprotein. An anaerobic procedure was developed for the purification of the hydroxylase to homogeneity. The molecular weight of the hydroxylase as determined by gel filtration was 220,000, and that determined by sedimentation equilibrium analysis was about 225,000. The purified hydroxylase contained three nonidentical subunits with molecular weights of about 55,000, 40,000, and 20,000, in equal amounts as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that it is an alpha 2 beta 2 gamma 2 protein. Optical absorption spectra revealed peaks near 408 and 280 nm, and fluorescence spectra revealed emission peaks at 490 and 630 nm. The purified hydroxylase contained 2.8 +/- 0.2 mol of iron and 0.5 +/- 0.1 mol of zinc per mol of protein but negligible amounts of acid-labile sulfide. The antisera prepared against the hydroxylase showed cross-reactivity with hydroxylase components in soluble extracts from other methanotrophs.

Examination of the Zinc Cementation of Cadmium in Aqueous Solutions

The kinetics of the cementation of cadmium by zinc have been studied over a range of solution pH values and initial cadmium concentrations. The kinetics were interpreted in terms of a model postulating a transition from a migration controlled mechanism with consequent half order kinetics to a diffusion controlled regime following first order kinetics. The conditions under which each mechanism was found to apply were consistent with the model in which the immediate reductant was hydride ion generated by the interaction of hydrogen ions with the zinc. The reaction stoichiometry was remarkable in that conditions were observed under which more than the theoretical one mole of cadmium per mole of zinc could be reduced. This was ascribed to the electrolysis of water by the induced electrochemical system. A lag in both cadmium cementation and zinc dissolution was observed in many runs. The zinc dissolution was found to commence about 25% sooner than cadmium cementation, a fact ascribed to the establishment of the reducing electrochemical system.

Characterization of meprin, a membrane-bound metalloendopeptidase from mouse kidney.

Meprin is an intrinsic protein of the brush border, a specialized plasma membrane, of the mouse kidney. It is a metalloendopeptidase that contains 1 mol of zinc and 3 mol of calcium per mol of the 85,000-Mr subunit. The enzyme is isolated, and active, as a tetramer. The behaviour of the enzyme on SDS/polyacrylamide gels in the presence and absence of beta-mercaptoethanol indicates that the subunits are of the same Mr (approx. 85,000) and held together by intersubunit S--S bridges. Eight S-carboxymethyl-L-cysteine residues were detected after reduction of the enzyme with beta-mercaptoethanol and carboxymethylation with iodoacetate. The enzyme is a glycoprotein and contains approx. 18% carbohydrate. Most of the carbohydrate is removed by endoglycosidase F, indicating that the sugar residues are N-linked. The isoelectric point of the enzyme is between pH 4 and 5, and the purified protein yields a pattern of evenly spaced bands in this range on isoelectric focusing. The peptide-bond specificity of the enzyme has been determined by using the oxidized B-chain of insulin as substrate. In all, 15 peptide degradation products were separated by h.p.l.c. and analysed for their amino acid content and N-terminal amino acid residue. The prevalent peptide-bond cleavages were between Gly20 and Glu21, Phe24 and Phe25 and between Phe25 and Tyr26. Other sites of cleavage were Leu6-Cysteic acid7, Ala14-Leu15, His10-Leu11, Leu17-Val18, Gly8-Ser9, Leu15-Tyr16, His5-Leu6. These results indicate that meprin has a preference for peptide bonds that are flanked by hydrophobic or neutral amino acid residues, but hydrolysis is not limited to these bonds. The ability of meprin to hydrolyse peptide bonds between small neutral and negatively charged amino acid residues distinguishes it from several other metalloendopeptidases.

Phosphodiesterase from the venom of crotalus ruber ruber

1. 1. Phosphodiesterase was isolated from the venom of Crotalus ruber ruber from the U.S.A. using the gel filtration on a Sephadex G-75 column, followed by anion or cation exchange chromatography. 2. 2. Phosphodiesterase was homogeneous as established by a single band on acrylamide gel electrophoresis and isolectric focusing electrophoresis. 3. 3. Phosphodiesterase activity was inhibited by ethylenediamine tetraacetic acid (EDTA), o-phenanthroline, thioglycolic acid or p-chloromercuribenzoate (PCMB), but not by soybean trypsin inhibitor (SBTI) or benzamidine. 4. 4. The molecular weight of this enzyme was determined to be approx. 98, 000 and the isoelectric point was found to be pH 10.5 by isoelectric focusing with carrier ampholyte. 5. 5. This enzyme contained 1.04 mol zinc per mol. 6. 6. The Michaelis constants ( K m ) of this enzyme for p-nitrophenyl thymidine-5′-phosphate and inhibition constant ( K i ) for PCMB were found to be 8.3 × 10 −3 and 1.2 × 10 −2 M, respectively.

High-level expression of enzymatically active human Cu/Zn superoxide dismutase in Escherichia coli.

Expression of human Cu/Zn superoxide dismutase (SOD) with activity comparable to the human erythrocyte enzyme was achieved in Escherichia coli by using a vector containing a thermoinducible lambda PL promoter and a beta-lactamase-derived ribosomal binding site. The recombinant human SOD was found in the cytosol of disrupted bacteria and represented greater than 10% of the total bacterial protein. The enzyme was purified to homogeneity by salt precipitation, gel filtration chromatography, and ion exchange chromatography. The active enzyme was obtained in high yield only when 1 mol of copper and 1 mol of zinc were incorporated into each mol of subunit during bacterial growth or by reconstitution of the apoenzyme. Human Cu/Zn SOD produced in bacteria has an apparent subunit molecular mass of 19 kDa on NaDodSO4/polyacrylamide gels. The native enzyme behaves as a dimer of 32 kDa as determined by gel filtration. Sequence analysis of the NH2 terminus revealed that the first 14 amino acids corresponded to authentic human SOD except that the NH2-terminal alanine was not acetylated. Thus, the bacterial processing system readily removes the NH2-terminal methionine residue from recombinant human SOD.

Isolation and characterization of a new aminopeptidase from bovine lens.

An aminopeptidase has been purified to homogeneity from bovine lens tissue by gel filtration and DEAE-cellulose chromatography. This enzyme has a molecular weight of 96,000 under both native and denaturing conditions. The purified enzyme hydrolyzed a variety of synthetic substrates as well as di-, tri-, and higher molecular weight peptides. Significantly this enzyme is capable of hydrolyzing arginine, lysine, and proline aminoacyl bonds. The pH optimum for activity and stability was 6.0. Both a reduced sulfhydryl group and a divalent metal ion are essential for activity. The native enzyme contains 1.6 mol of zinc and 1.0 mol of copper/mol of enzyme. No activation was seen upon incubation with either magnesium or manganese; however, heavy metal ions were inhibitory. Bestatin and puromycin were effective inhibitors and no endopeptidase activity could be detected in the purified preparation. This enzyme is clearly distinct from the lens leucine aminopeptidase, but rather, is identical to a cytosolic aminopeptidase III isolated from other tissues. Evidence is presented which argues that this enzyme may be the major lens aminopeptidase under in vivo conditions.

Chemical studies on protease A of Bitis arietans (puff adder) venom

Protease A of Bitis arietans venom is probably a metalloprotease, since it is inhibited by o-phenanthroline and contains 0.77 moles of zinc per mole protein. The enzyme comprises 213 amino acids, including 9 methionine residues and one free sulphydryl group. It contains one polypeptide chain, which is terminated at the carboxyl end by serine. The amino terminal sequence of protease A is: Arg-Ser-Ser-Asp-Pro-Asn- Lys-Tyr-Phe-Asn-Val-Ile-Val-Val-Val-Asp-Asn-Arg-Met-Val-Asn-Tyr-Tyr-Lys-Gly-Glu-Leu- Asn-Lys-Ile-Thr-. Despite difficulties with ‘insoluble peptide core’ formation, a number of peptides were purified from peptic and tryptic digests of S-derivatized protease A.

Isolation and biochemical characterization of hemorrhagic toxin f from the venom of Crotalus atrox (Western Diamondback Rattlesnake)

Hemorrhagic toxin f (HT- f) was isolated from Crotalus atrox (Western Diamondback Rattlesnake) venom by a five-step purification procedure. Homogeneity was established by the formation of a single band in acrylamide gel electrophoresis, isoelectric focusing, and sodium dodecyl sulfate (SDS)-electrophoresis. HT- f has a molecular weight of 64,000 and contains 572 amino acid residues. It contains 1 mol of zinc per mol of protein. Zinc is essential for both hemorrhagic and proteolytic activities. HT- fpossesses proteolytic activity hydrolyzing the Val-Asn, Gln-His, Leu-Cys, His-Leu, Ala-Leu, and Tyr-Leu bonds of oxidized insulin B chain. HT- f did not coagulate fibrinogen to fibrin, yet it did hydrolyze the γ chain of fibrinogen without affecting either the Aα or Bβ chains. This is the first time that a hemorrhagic toxin was shown to have fibrinogenase activity. HT- f was shown to differ immunologically from other hemorrhagic toxins such as HT- a and HT- c. HT- f also possesses lethal toxicity. When zinc was removed the apo-HT- f lost its lethal toxicity. HT- f produced not only local hemorrhage in the skin and muscle, but also produced systemic hemorrhage in internal organs such as the intestine, kidney, lung, heart, and liver.

Phospholipase C from Clostridium perfringens: Preparation and characterization of homogeneous enzyme

A new procedure for the purification of phospholipase C from Clostridium perfringens has been devised that results in essentially pure enzyme. The procedure consists of ammonium sulfate fractionation, ion-exchange chromatography on QAE-Sephadex, and affinity chromatography on phosphatidylcholine linked to Sepharose. The molecular weight of the enzyme, determined by sodium dodecyl sulfate-gel electrophoresis, amino acid analysis, and gel filtration, is 43,000; and the isoelectric point is pH 5.4. The enzyme was optimally active with phosphatidylcholine dispersed in sodium deoxycholate, although appreciable activity was observed with either phosphatidylcholine or sphingomyelin dispersed with ethanol. The requirement for metal ions in the assay could be met by a number of different ions. The pure enzyme was found to contain 2 mol zinc per mol enzyme, thus implicating it as a zinc metalloenzyme.

Anticoagulant proteases from western diamondback rattlesnake (Crotalus atrox) venom.

Crotalus atrox venom contains agents that render human fibrinogen and plasma incoagulable by thrombin. To elucidate the mechanism of alteration of fibrinogen clotting function by the venom, four immunochemically different proteases, I, II, III, and IV, were purified from the venom by anion-exchange chromatography and column gel filtration. All four proteases had anticoagulant activity rendering purified fibrinogen incoagulable. Proteases I and IV do not affect fibrinogen in plasma but in purified fibrinogen cleave the A alpha chain first and then the B beta and gamma chains. Both enzymes are metalloproteases containing a single polypeptide chain with 1 mol of zinc, are inhibited by (ethylenedinitrilo)tetraacetate and human alpha 2-macroglobulin, and have an optimal temperature of 37 degrees C and an optimal pH of 7. Protease I has a molecular weight (Mr) of 20 000 and is the most cationic. Protease IV has an Mr of 46 000 and is the most anionic glycoprotein with one free sulfhydryl group. Proteases II and III degrade both purified fibrinogen and fibrinogen in plasma, cleaving only the B beta chain and leaving the A alpha and gamma chains intact. Both enzymes are alkaline serine proteases, cleave chromogenic substrates at the COOH terminal of arginine or lysine, are inhibited by diisopropyl fluorophosphate and phenylmethanesulfonyl fluoride, and have an optimal temperature of 50-65 degrees C. Protease II is a single polypeptide chain glycoprotein with an Mr of 31 000. Protease III is a two polypeptide chain protein with an Mr of 24 000, each of the two chains having an Mr of 13 000; its activity is not affected by major protease inhibitors of human plasma. Proteases II and III are enzymes with unique and limited substrate specificity by cleaving only the B beta chain, releasing a peptide of Mr 5000 and generating a fibrinogen derivative of Mr 325 000, with intact A alpha and gamma chains and poor coagulability. Since the two enzymes are active in human plasma and serum, it is postulated that proteases II and III can mediate anticoagulant effects in vivo after envenomation.

Local Tissue Oamaging (Hemorrhage and Myonecrosis) Toxins from Rattlcsnake and Other Pit Viper Venoms

AbstractSix hemorrhagic toxins have been isolated from Crotalus atrox (western diamondback rattlesnake) venom. All of them contain one mole of zinc per mole of protein, although the molecular weights vary from 24,000 to 68,000. All have proteolytic activity when tested on dimethyl-casein. Removal of zinc eliminates the hemorrhagic and proteolytic activities. Proteolytic specificity of hemorrhagic toxin a is highly specific and hydrolyzes the X-Leu bond. Hemorrhagic toxin b is less specific and hydrolyzes the X-Phe, X-Ser, and X-Leu bonds.There are several types of myonecrotic [muscle damaging) toxins, and of those, myotoxin a from C. viridis viridis (prairie rattlesnake) venom is the most extensively investigated. Myotoxin a consists of 42 amino acid residues with 3 disulfide bonds. There is no similarity in amino acid sequence and disulfide bond positions when compared to well-known post-synaptic neurotoxins (both Type I and II) of snake venoms. It is rather curious and interesting to note that there is ...

A fully active DNA polymerase I from Escherichia coli lacking stoichiometric zinc

DNA polymerase I produced by an Escherichia coli strain lysogenized with a λ polA phage has been purified using a modification of the method of Kelley and Stump (Kelley, W.S. and Stump, K.H. (1979) J. Biol. Chem. 254, 3206–3210). This modification involves an additional phosphocellulose column and a hydroxylapatite column and results in a preparation of DNA polymerase I which is at least 97% pure. The metal content of the purified enzyme was analyzed and only 0.13 moles of zinc were detected per mole of DNA polymerase I. Since the enzyme still retained its full activity, this result did not support previous investigations which stated that zinc bound to the enzyme in stoichiometric amounts was necessary for polymerase activity. These findings suggest that the mechanism of DNA polymerase I with respect to the role of zinc should be reevaluated.

Formamide-induced dissociation and inactivation of Escherichia coli alkaline phosphatase. Metal-dependent reassociation and restoration of activity from isolated subunits.

Alkaline phosphatase from Escherichia coli has been reversibly dissociated by treatment with low concentrations of formamide. The monomer retains the capacity to bind metals and to regenerate catalytically active dimer that is identical with the native dimeric enzyme. The rate and extent of dissociation of dimer to monomer depend upon pH, ionic strength, temperature, formamide concentration, and enzyme-bound metal. Under appropriate experimental conditions, reassociation can be greatly slowed, allowing the properties of the monomer to be examined in solution. The formamide-induced apo monomer has a conformation distinct from that of the dimer and zinc- or cobalt-containing monomers. The monomer tightly binds 1 mol of zinc or cobalt in a metal-binding site altered from those of the dimer but is catalytically inactive. pH, ionic strength, and formamide concentration all influence reassociation. Hydrophobic forces are implicated as important in subunit interactions. The effect of metal content on the dissociation--reassociation process underscores the essential role that metals play in maintaining enzyme tertiary structure and reveals a new role in stabilizing the quaternary structure.

Purification and characterization of nucleoside diphosphatase from rat-liver microsomes. Evidence for metalloenzyme and glycoprotein.

Nucleoside diphosphatase was purified from rat liver microsomes more than 3000-fold with a 16% yield using a procedure including concanavalin-A--Sepharose and phenyl-Sepharose column chromatography. The purified enzyme had a specific activity of 2500 units/mg protein and appeared homogeneous by gel electrophoresis. The enzyme had a sedimentation coefficient of 6.5 S by sucrose-density gradient centrifugation. The enzyme had a sedimentation coefficient of 6.5 S by sucrose-density gradient centrifugation, and a Stokes' radius of 4.8 nm was estimated by the gel filtration technique. Its molecular weight is 130,000, but only one single band of Mr 65,000 was detected after sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The native enzyme seems thus to be composed of two identical subunits. The purified enzyme was confirmed to be a glycoprotein containing approximately 9% carbohydrates. The enzyme had a pH optimum of 7.5, an isoelectric point of 4.85 and a Km of 2.5 mM for UDP. On the basis of direct measurement of metal content in the native enzyme, the rat liver nucleoside diphosphatase was found to be a metalloenzyme containing 0.9 mol zinc and 0.1 mol manganese/mol 65,000-Mr subunit. Metal-free nucleoside diphosphatase has been prepared. The activity of the metal-free enzyme was restored by the addition of several divalent cations, zinc being the most effective.

The effect of cadmium ions and cadmium-metallothionein on the activities of phospholipid-synthesizing enzymes of rat liver microsomes in vitro

The effects of cadmium ions or cadmium-metallothionein on the activities of acyl-CoA:1acyl- sn-glycerol 3-phosphoric acid or 1-acyl- sn-glycero 3-phosphocholine acyltransferase of rat liver microsomes have been studied, in vitro. Cadmium ions were found to cause a noncompetitive type inhibition of these two acyltransferases. The K i values were calculated, and found to be smallest (1.7 × 10 −5 m) for palmitoyl-CoA and greatest (1.0 × 10 −4 m) for linoleoyl-CoA, among the several fatty acyl-CoA's tested on the 1-acyl- sn-glycerol 3-phosphoric acid acyltransferases. With the 1-acyl- sn-glycero 3-phosphocholine acyltransferase, the K i values were found to be smallest for the plamitoyl-CoA acyltransferase (3.8 × 10 −5 m) and largest for thearachidonoyl-CoA acyltransferase (1.1 × 10 −4 m). In contrast, mouse liver cadmium-metallothionein, including 4 mol of cadmium and 2 mol of zinc in one molecule of metallothionein, was not found to be inhibitory or rather stimulative on the above two acyltransferases at the same concentration of cadmium tested in the cadmium ion inhibitor experiments. The above results demonstrate that there is a strong and irreversible inhibition by cadmium ions on acyl-CoA acyltransferases, but that when cadmium acts on the enzyme in the form of a cadmium-metallothionein complex, the inhibition effect does not occur. These findings may reflect differing degrees of toxicity of these two types of cadmium compounds in mammalian tissues.

Binding of inhibitory metals to yeast enolase

Certain divalent cations can inhibit yeast enolase by binding at sites that are distinct from those metal binding sites normally associated with catalytic activity, i.e., the conformational and catalytic binding sites. By using a buffer that does not compete with metal ions (tetrapropylammonium borate) Zn, Co, Mn, Cu, Cd, and Ni are found to exhibit similar inhibitory characteristics. Inhibition by those metals is alleviated by the addition of imidazole or tris buffer and, for zinc, by a metal chelating agent (Calcein). Inhibition by zinc was examined in detail through binding studies and enzymatic activity measurement. In tetrapropylammonium buffers at pH 8.0, enolase binds up to four moles of zinc per mole of enzyme (two moles per subunit). An imidazole concentration of 0.05 M reduces the binding: in the absence of substrate, just two moles of zinc per enzyme are bound. The enzyme will bind two additional moles of zinc upon the addition of substrate in either buffer, but the enzyme in tetrapropylammonium buffer is nearly inactive. Inhibition is, therefore, correlated with the binding of two moles of zinc per mole of enzyme. Some additional metal ions, Ca, Tb, Hg, and Ag also caused inhibition of yeast enolase but not by binding to the inhibitory site described.

Interaction of Streptonigrin with Metals and with DNA

The antitumor antibiotic, streptonigrin, interacted with zinc, copper, and manganese but not with calcium or magnesium, as indicated by spectral shifts and difference spectra. The titration data showed the formation of 1:1 complexes, and further titration continued to show spectral shifts until a molar ratio for zinc to streptonigrin of 5-10 to 1 was reached. Streptonigrin interacted with DNA only in the presence of a metal ion such as zinc. Streptonigrin titration with DNA at varying zinc molar equivalents revealed that one antibiotic molecule required 5-7 moles of zinc and 20-25 moles of DNA-phosphorus for complexation. Similar values were obtained from gel permeation chromatography.