Biphasic kinetics of Zn removal from Zn metallothionein by nitrilotriacetate are associated with differential reactivity of the two metal clusters

We investigated the kinetics of nitrilotriacetate (NTA) extraction of Zn²⁺ from Zn₇-metallothionein (MT) and a metal-hybrid derivative, Zn₄Ag₆MT, in which the Zn²⁺ and Ag⁺ ions occupy sites in the C-terminal and N-terminal β domains of the protein, respectively. Biphasic kinetics were observed for Zn₇MT under pseudo-first-order conditions. Rate constants were (5.2±0.6)×10⁻³ and (1.0±0.3)×10⁻⁴s⁻¹ in 20 mM phosphate, 100 mM KF, pH 7.5 at 23C. In contrast, Zn₄Ag₆MT showed a single kinetic step with a rate constant of (2.9±0.4)×10⁻³s⁻¹. These results indicate that the biphasic reactivity of Zn₇MT stems from differential susceptibility of the metal in the two metal–thiolate clusters to removal by competing ligands, with Zn²⁺ in the more stable -domain cluster reacting faster than that in the less stable β-domain cluster. Such behavior suggests that the structures of the two domains of mammalian MT may have evolved to assure that Cu binding does not compromise the structural characteristics that allow Zn to be rapidly transferred from MT to essential cellular ligands.     

Thionein/metallothionein control Zn(II) availability and the activity of enzymes

Fundamental issues in zinc biology are how proteins control the concentrations of free Zn(II) ions and how tightly they interact with them. Since, basically, the Zn(II) stability constants of only two cytosolic zinc enzymes, carbonic anhydrase and superoxide dismutase, have been reported, the affinity for Zn(II) of another zinc enzyme, sorbitol dehydrogenase (SDH), was determined. Its log K is 11.2 +/- 0.1, which is similar to the log K values of carbonic anhydrase and superoxide dismutase despite considerable differences in the coordination environments of Zn(II) in these enzymes. Protein tyrosine phosphatase 1B (PTP 1B), on the other hand, is not classified as a zinc enzyme but is strongly inhibited by Zn(II), with log K = 7.8 +/- 0.1. In order to test whether or not metallothionein (MT) can serve as a source for Zn(II) ions, it was used to control free Zn(II) ion concentrations. MT makes Zn(II) available for both PTP 1B and the apoform of SDH. However, whether or not Zn(II) ions are indeed available for interaction with these enzymes depends on the thionein (T) to MT ratio and the redox poise. At ratios [T/(MT + T) = 0.08-0.31] prevailing in tissues and cells, picomolar concentrations of free Zn(II) are available from MT for reconstituting apoenzymes with Zn(II). Under conditions of decreased ratios, nanomolar concentrations of free Zn(II) become available and affect enzymes that are not zinc metalloenzymes. The match between the Zn(II) buffering capacity of MT and the Zn(II) affinity of proteins suggests a function of MT in controlling cellular Zn(II) availability.     

Investigation of the effect of metal ions on the reactivity of thiol groups in human 5-aminolaevulinate dehydratase.

The reaction of human 5-aminolaevulinate dehydratase with 5,5'-dithiobis-(2-nitrobenzoic acid) (Nbs2) results in the release of 4 molar equivalents of 5-mercapto-2-nitrobenzoic acid (Nbs) per subunit. Two of the thiol groups reacted very rapidly (groups I and II), and their rate constants were determined by stopped-flow spectrophotometry; the other two thiol groups (groups III and IV) were observed by conventional spectroscopy. Titration of the enzyme with a 1 molar equivalent concentration of Nbs2 resulted in the release of 2 molar equivalents of Nbs and the concomitant formation of an intramolecular disulphide bond between groups I and II. Removal of zinc from the holoenzyme increased the reactivity of groups I and II without significantly affecting the rate of reaction of the other groups. The reactions of the thiol groups in both the holoenzyme and apoenzyme were little affected by the presence of Pb2+ ions at concentrations that strongly inhibit the enzyme, suggesting that Zn2+ and Pb2+ ions may have independent binding sites. Protein fluorescence studies with Pb2+ and Zn2+ have shown that the binding of both metal ions results in perturbation of the protein fluorescence.     

Ferroxidase kinetics of horse spleen apoferritin.

Protein ferroxidase site(s), which catalyze the reaction between ferrous ion and dioxygen, have long been thought to play a role in core formation in ferritin; however, the mechanism of the reaction has never been studied in detail. In the present work, the enzymatic activity of ferritin was examined using oximetry, the net Fe2+ oxidation reaction being as follows. [formula: see text] The reaction exhibits saturation kinetics with respect to both Fe2+ and O2 (apparent Michaelis constants: Km,Fe = 0.35 +/- 0.01 mM and Km,O2 = 0.14 +/- 0.03 mM). The enzyme has a turnover number kcat = 80 +/- 3 min-1 at 20 degrees C with maximal activity at pH 7. The kinetics are discussed in terms of two mechanisms, one involving monomeric and the other dimeric iron protein complexes. In both instances Fe(II) oxidation occurs in 1-electron steps. Zinc(II) is a competitive inhibitor of iron(II) oxidation at Zn2+/apoprotein ratios > or = 6 (inhibitor constant KI,Zn = 0.067 +/- 0.011 mM) but appears to be a noncompetitive inhibitor at lower ratios (< or = 2), indicating the presence of more than one type of zinc binding site on the protein. At increments of 50 Fe2+/protein or less, all of the iron is oxidized via the protein ferroxidase site(s), independent of the amount of core already present. However, when larger increments are employed, some iron oxidation appears to occur on the surface of the mineral core. The results of these studies emphasize the role of the protein shell in all phases of core growth and confirm the presence of a functionally important catalytic site in ferritin in addition to other binding sites on the protein for iron.     

Indolylacetic acid and N6-(delta 2-isopentenyl) adenine affect NADH binding to yeast alcohol dehydrogenase and inhibit in vitro the enzymatic oxidation of ethanol

Derivative UV spectroscopic data show that the plant growth substances N6-(delta 2-isopentenyl) adenine (i6Ade) and indolylacetic acid (IAA) can bind to the yeast alcohol dehydrogenase (ADH) and affect coenzyme-enzyme binding. This is confirmed by enzyme kinetics studies. At fixed ethanol concentrations (27.8 and 111.1 mM) and varying NAD+ concentrations (0.033-2 mM), as well as at fixed levels of coenzyme (0.67 and 2 mM), and at varying concentrations of ethanol (1.4-111.1 mM), the rate of ethanol oxidation is significantly inhibited by i6Ade and IAA. The kinetics of the ADH reaction is affected by two inhibition constants (KI and K'I) which correspond to the dissociation constants of complexes EI and ESI, respectively. For i6Ade the KI = 0.52 +/- 0.06 mM and K'I = 0.74 +/- 0.07 mM, and for IAA the KI = 0.88 +/- 0.03 mM and K'I = 0.99 +/- 0.02 mM.     

Kinetics of reversible N-ethylmaleimide alkylation of metallothionein and the subsequent metal release

 The model alkylating agent N-ethylmaleimide (NEM) reacts reversibly at the metal-bound thiolates of Zn₇MT and Cd₇MT. An unprecedented feature of this reaction is that it approaches equilibrium and requires a large excess of NEM (>1 mM for 3 μM protein) to drive it to completion. The complex kinetics of the reaction can be followed by monitoring the release of bound metal ions using the metallochromic dyes Zincon (ZI) for Zn₇MT and pyridylazoresorcinol for Cd₇MT. An initial lag phase is followed by more rapid release of zinc ions. The observed pseudo-first-order rate constants for the two phases are independent of the ZI and Zn₇MT concentrations. The complex NEM concentration dependence of each phase, k _f, obs=k ^f ₁+k ^f ₂ [NEM] and k _s, obs=k ^s ₁+k ^s ₂ [NEM], demonstrates that the forward reactions are second order and the reverse reactions are first order. The alkylation can be reversed using 2-mercaptoethanol to compete for the protein-bound NEM and regenerate the Zn-binding capability of alkylated MT. An explanation of these observations, based on the reversibility of cysteine alkylation by NEM, was developed and tested. The reactions of Cd₇MT are less complete than those of Zn₇MT and occur more slowly. ¹¹¹Cd-NMR studies of the partially alkylated ¹¹¹Cd₇MT reveal that reaction with only four equivalents of NEM completely alters the cluster structure and eliminates the spectral signatures of the α and β clusters, although very little cadmium has been removed from the protein. This finding substantiates the proposed kinetic intermediate, a partially alkylated MT with complete or nearly complete retention of the metal ions, and rules out the possibility of cooperative reactions at either cluster. Received: 5 August 1996 / Accepted: 24 October 1996     

Characterization of the half and overall reactions catalyzed by L-lysine:2-oxoglutarate 6-aminotransferase

Significant differences were found in the reaction rate, and the substrate and reaction specificities between the half reactions and the overall reactions catalyzed by L-lysine: 2-oxoglutarate 6-aminotransferase. The half reactions between an amino donor and the enzyme-bound pyridoxal 5'-phosphate, and also between an amino acceptor and the bound pyridoxamine 5'-phosphate followed first order reaction kinetics. The extrapolated first order rate constants and dissociation constants of the substrates were determined for the half reactions: lysine, 0.87 min-1 and 5.5 mM; glutamate, 1.1 min-1 and 10.5 mM; alanine, 0.66 min-1 and 6.6 mM; 6-aminohexanoate, 0.43 min-1 and 13.3 mM; and 2-oxoglutarate, 0.33 min-1 and 2.5 mM. As compared with the values reported for the overall reactions [Soda, K., Misono, H., & Yamamoto, T. (1968) Biochemistry 7, 4102-4109], the reactivity of the inherent substrates was lower by over 4 orders in the half reaction than that in the overall reaction, and the reactivity of alanine with the bound pyridoxal 5'-phosphate was reduced to 10% of that in the overall reaction. The substrate specificity in the half reaction was much lower than that in the overall reaction, which was re-examined in a reaction system containing the same concentration of the enzyme as that for the half reactions. Lysine 6-aminotransferase catalyzes the transfer of only the terminal amino group of lysine to 2-oxoglutarate in the overall reaction. However, in the half reaction, the 2-amino group as well as the terminal one was transferred to the bound pyridoxal 5'-phosphate. The ratio of reactivity of the 2-amino group to that of the 6-amino group was considerably influenced by the pH of the reaction mixture.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of pH and KCl on aggregation state and sulphydryl groups reactivity of rat skeletal muscle AMP deaminase

The effects of pH and KCl on sedimentation properties and SH groups reactivity of rat skeletal muscle AMP deaminase have been investigated. The values obtained for apparent molecular weight are consistent with an association of AMP deaminase subunits in response to increasing KCl concentration. Increasing pH value from 6.0 to 8.0 causes a reduction in the apparent molecular weight of the enzyme at high KCl concentration, which can be interpreted as due to a deprotonation-induced isomerization process. Removal of Zn2+ from AMP deaminase has effect similar to alkalinization in modifying the sedimentation properties of the enzyme. In the native enzyme at high K+ concentration about 7, 9 and 12 SH groups can be titrated with Nbs2, approximately 1, 2 and 4 SH groups reacting as fast sets, at pH 6.0, 7.0 and 8.0, respectively. Substitution of the 12 SH groups reactive with Nbs2 at pH 8.0 has no effect on the pH-dependent allosteric behaviour of the enzyme. Removal of K+ causes considerable changes in the reactivity of AMP deaminase towards Nbs2, unmasking a class of additional SH groups, so that the total number of titratable SH groups approaches that of 30 determined in denaturing conditions. In the enzyme previously treated with N-ethylmaleimide to alkylate the fast reacting class of SH groups, the class of additional SH groups are substituted by Nbs2 at basic pH, but not at acidic pH, with a concomitant reduction of the enzyme activity.     

Properties of the reaction of cis-dichlorodiammineplatinum(II) with metallothionein.

Properties of the reactions of cis-dichlorodiammine Pt(II) and related complexes with zinc metallothionein or apometallothionein have been investigated. During these reactions, platinum binds stoichiometrically to protein sulfhydryl groups and zinc, if present, is displaced. The ammine ligands are also lost in the process, suggesting that Pt(II) has tetrathiolate coordination in metallothionein. This conclusion is supported by extended x-ray absorption-fine structure studies which indicate that there are 4 sulfurs in the first coordination sphere of the platinum centers. The product contains 10 +/- 2 Pt(II) per mol of protein and migrates over Sephadex G-75 as a structure of similar size to zinc metallothionein. The kinetics of reaction are biphasic as monitored by the formation of Pt-thiolate bonds or by the release of zinc from the protein. Both methods yield identical rate laws for the reaction. The first step is independent of Pt(II) concentration but involves the binding of as many as four platinum atoms to the protein with little Pt-sulfhydryl bond formation and without much loss of zinc. The second rate process is first order in both zinc or sulfhydryl binding sites and Pt(II). Neither kinetic step is sensitive to the chloride ion concentration over the range 0-0.5 M. However, the reaction is sensitive to pH between 5.5 and 8.0. trans-Dichlorodiammineplatinum(II) reacts with zinc metallothionein with similar kinetics.     

Protective mechanism of metallothionein against copper-1, 10-phenanthroline induced DNA cleavage

Metallothionein (MT) has been shown to protect DNA against cleavage induced by a variety of mutagenic agents. The mechanism has been attributed to its ability to either chelate transitional metals that participate in the Fenton reaction, or scavenge free radicals by means of the abundant cystenyl residues of the proteins. In the present study, the protective action of MT against DNA cleavage by the copper-1,10-phenanthroline [(OP)(2)Cu(+)] complex was studied in situ. At 0.1 microM, MT inhibited the (OP)(2)Cu(+) induced DNA cleavage by about 50% (IC(50) approximately 0.1 microM). At 2.5 microM, the cleavage activity was completely inhibited. Similar to MT, cysteine can protect against DNA cleavage by (OP)(2)Cu(+) (IC(50) of approximately 3 mM), however, its action was 1500-fold less efficient than MT. The combined action of MT and cysteine was additive. Reduced glutathione (1 and 10 mM) did not protect the (OP)(2)Cu(+) induced DNA cleavage. Sodium azide could inhibit the cleavage only at high concentrations (IC(40) approximately 25 mM). Spectrophotometric analysis showed that MT can inhibit the formation of the DNA[(OP)(2)Cu(+)] complex possibly by chelating Cu. It can also cause a dissociation of the complex after it was formed. In the later case, the mechanism through which MT protects against the DNA cleavage might occur when MT fitted in closely with the complex, competing with the hydroxyl groups of the nucleotides base for Cu, which, in turn, terminate the Fenton-like free radical reaction.     

Expression of human metallothionein III and its metalloabsorption capability in Escherichia coli

Human metallothionein III (MT III) gene was synthesized with Escherichia coli preference codon usage and expressed in E. coli in glutathione-S-transferase (GST) fusion form. The recombinant MT III was released by proteinase Factor Xa digestion and purified with the yield of 2 mg/L culture, and its specific Cd2+ binding capability was confirmed. E. coli strain BL21(DE3), expressing MT III, showed metal tolerance between 0.1 and 0.5 mM Cd2+ and bacterial growth was inhibited at 1 mM Cd2+. MT III expressing E. coli strain showed binding discrimination between different metal ions in combination use, with the preference order of Cd2+ > Cu2+ > Zn2+. It absorbed different metal ions with relatively constant ratio and showed a cumulative absorption capability for mixed heavy metals.     

Postnatal changes in subcellular distribution of copper,zinc and metallothionein in the liver of bank vole (Clethrionomys glareolus): a possible involvement of metallothionein and copper in cell proliferation

1. Dramatic interdependent changes in the intracellular concentrations of copper (Cu), zinc (Zn) and metallothionein (MT) in the liver of bank voles during the first 30 days of their life were observed.2. The post-mitochondrial Cu, Zn and MT (ZnMT) abruptly decreased between 1 and 3 days following birth but the nuclear MT (CuMT) and Cu increased at the same time, suggesting that Cu displaced Zn already bound to MT in the cytoplasm and subsequently the complex CuMT was translocated to the nuclei.3. The nuclear Cu concentration reached the highest level (62–71% of the total tissue Cu) in the period from day 3 to day 20 post-partum, just prior to and during a rapidly growing liver.4. The data indicate that MT and Cu may be involved in the hepatocyte proliferation.     

Mechanism of phenylalanyl-tRNA synthetase of Escherichia coli K. 10. Modulation of catalytic properties by magnesium.

The association of phenylalanylptRNA and Mg2+ follows a biphasic concentration dependence as indicated by the active site directed fluorescent indicator 2-p-toluidinyl-naphthalene-6-sulfonate. The macroscopic dissociation constants are 0.16 +/- 0.03 and 4.1 +/- mM. The effect of Mg2+ on the association of enzyme and MgATP, on the synergistic binding of MgATP and L-phenylalaninol, and on the pre-steady-state synthesis and pyrophosphorolysis of the enzyme-phenylalanyladenylate complex in the absence and the presence of tRNA Phe has been measured by established equilibrium and stopped-flow techniques using 2-p-toluidinylnaphthalene-6-sulfonate. At 10 mM Mg2+, the association of enzyme and MgATP is biphasic with dissociation constants of 0.25 +/- 0.03 and 9.1 +/- 1.7 mM. At 2 mM Mg2+, a single dissociation constant of 5.0 +/- 0.5 mM is indicated. The coupling constant of the synergistic reaction is 15 at 1 mM Mg2+ and 290 at 10 mM Mg2+. The Hill constant of the sigmoidal dependence is 3.6. The strengthening of the synergism is believed to reflect a Mg2+-dependent coupling of the synergistic reactions at the two active sites of the enzyme, the coupling being negligible at 1 mM and maximal at 10 mM Mg2+. The pre-steady-state rate of adenylate synthesis is accelerated by the presence of Mg2+. The effect is to decrease the value of the Michaelis-Menten constant of MgATP. Another effect is to increase the rate constant when tRNA Phe is present. At subsaturating [MgATP], the [Mg2+] dependence of the observed rate constant is hyperbolical in the absence and sigmoidal (Hill constant, 3.5) in the presence of tRNA Phe. The rate of the pyrophosphorolysis is enhanced by a decrease of the Michaelis-Menten constant of MgPPi. The effects on the thermodynamics and kinetics parallel the occupancy of the low-affinity Mg2+-binding sites of the enzyme.     

Oxidation reactivity of zinc–cysteine clusters in metallothionein

Evaluating the reactivity of the metal–thiolate clusters in metallothionein (MT) is a key step in understanding the biological functions of this protein. The effects of the metal clustering and protein environment on the thiolate reactivity with hydrogen peroxide (H₂O₂) were investigated by performing quantum theory calculations with chemical accuracy at two levels of complexity. At the first level, the reactivity with H₂O₂ of a model system ([(Zn)₃(MeS)₉]^3?, MeS is methanethiolate) of the β domain cluster of MT was evaluated using density functional theory (DFT) with the mPW1PW91 functional. At the second level of complexity, the protein environment was included in the reactant system and the calculations were performed with the hybrid ONIOM method combining the DFT–mPW1PW91 and the semiempirical PM6 levels of theory. In these conditions, the energy barrier for the oxidation of the most reactive terminal thiolate was 21.5 kcal mol^?1. This is 3 kcal mol^?1 higher than that calculated for the terminal thiolate in the model system [(Zn)₃(MeS)₉]^3? and about 7 kcal mol^?1 higher than that obtained for the free thiolate. In spite of this rise of the energy barrier induced by the protein environment, the thiolate oxidation by H₂O₂ is confirmed as a possible way for metal release from MT. On the other hand, the results suggest that the antioxidant role of MT in the living cell cannot be as important as that of glutathione (which bears a free thiol).     

Apo-metallothionein emerging as a major player in the cellular activities of metallothionein

Observations of apo-metallothionein (apo-MT) have been made under a variety of physiologic circumstances, including zinc deficiency in cell culture and in rodents, cellular induction of MT by dexamethasone with concurrent Zn deficiency, a variety of tumors under normal Zn conditions, MT induction by Zn and Bi citrate, induction of hepatic MT after tumor cell injection into nude mice, and overexpression of cardiac MT in MT transgenic mice. Experiments demonstrating both the lability of Zn and Cu bound to MT and the cellular stability of apo-MT are described to help rationalize the widespread presence of this metal-depleted species. Finally, comparative in vitro and cellular experiments examined the relative reactivity of Zn- and apo-MT with nitric oxide species, showing that apo-MT is much more reactive chemically and that in cells it may be a principal reactive species within the MT pool.     

Inhibitory kinetics of beta-N-acetyl-D-glucosaminidase from prawn (Litopenaeus vannamei) by zinc ion

Prawn (Litopenaeus vannamei) beta-N-acetyl-D-glucosaminidase (NAGase, EC 3.2.1.52) is involved in the digestion and molting processes. Zinc is one of the most important metals often found in the pollutant. In this article, the effects of Zn(2+) on prawn NAGase activity for the hydrolysis of pNP-NAG have been investigated. The results showed that Zn(2+) could reversibly and noncompetitively inhibit the enzyme activity at appropriate concentrations and its IC(50) value was estimated to be 6.00 +/- 0.25 mM. The inhibition model was set up, and the inhibition kinetics of the enzyme by Zn(2+) has been studied using the kinetic method of the substrate reaction. The inhibition constant was determined to be 11.96 mM and the microscopic rate constants were also determined for inactivation and reactivation. The rate constant of the inactivation (k(+0)) is much larger than that of the reactivation (k(-0)). Therefore, when the Zn(2+) concentration is sufficiently large, the enzyme is completely inactivated. On increasing the concentration of Zn(2+), the fluorescence emission peak and the UV absorbance peak are not position shifted, but the intensity decreased, indicating that the conformation of Zn(2+)-bound inactive NAGase is stable and different from that of native NAGase. We presumed that Zn(2+) made changes in the activity and conformation of prawn NAGase by binding with the histidine or cysteine residues of the enzyme.     

Metal binding properties and structure of a type III metallothionein from the metal hyperaccumulator plant Noccaea caerulescens

Metallothioneins (MT) are low molecular weight proteins with cysteine-rich sequences that bind heavy metals with remarkably high affinities. Plant MTs differ from animal ones by a peculiar amino acid sequence organization consisting of two short Cys-rich terminal domains (containing from 4 to 8 Cys each) linked by a Cys free region of about 30 residues. In contrast with the current knowledge on the 3D structure of animal MTs, there is a striking lack of structural data on plant MTs. We have expressed and purified a type III MT from Noccaea caerulescens (previously Thlaspi caerulescens). This protein is able to bind a variety of cations including Cd(2+), Cu(2+), Zn(2+) and Pb(2+), with different stoichiometries as shown by mass spectrometry. The protein displays a complete absence of periodic secondary structures as measured by far-UV circular dichroism, infrared spectroscopy and hydrogen/deuterium exchange kinetics. When attached onto a BIA-ATR biosensor, no significant structural change was observed upon removing the metal ions.     

A new insight into the Ag+ and Cu+ binding sites in the metallothionein beta domain

The copper(I) and silver(I) binding properties of the beta fragment of recombinant mouse metallothionein I have been studied by electronic absorption and circular dichroism spectroscopy. When possible, the stoichiometry of the species formed was confirmed by electrospray mass spectrometry. The behaviour observed differs from that reported for the native protein. Titration of either Zn3-beta MT at pH 7 or apo-beta MT at pH 3 with Cu+ leads to the formation of species having the same stoichiometry and structure: Cu6-beta MT, Cu7-beta MT and Cu10-beta MT. In the first stage of the titration of Zn3-beta MT with Cu+ at pH 7 one additional species of formula Cu4Zn1-beta MT was detected. In contrast, the titration of Zn3-beta MT at pH 7.5 and of apo-beta MT at pH 2.5 with Ag+ proceeds through different reaction pathways, affording ZnxAg3-beta MT, Ag6-beta MT and Ag9-beta MT or Ag3-beta MT, Ag6-beta MT and Ag9-beta MT, respectively. The CD envelope corresponding to species with the same stoichiometric ratio, Ag6-beta MT and Ag9-beta MT, indicates that they have a different structure at each pH value. On the basis of the differences observed, the postulated similarity between copper and silver binding to metallothionein may be questioned.     

Heavy metal intracellular balance and relationship with metallothionein induction in the gills of carp

Determination of metal levels (Cu, Zn, Cd, Ag, Hg) in soluble and insoluble fractions of gill homogenates has been performed after 7 d exposure of carp (Cyprinus carpio) to moderate concentrations of Cd, Ag, and Hg in water. Metallothionein levels have been quantified by polarographic method before and after contamination and a subsequent decontamination phase (7 d). The influence of pretreatment by zinc (7 d) has also been evaluated. Metallothionein level variations have been interpreted as having regard to interrelated flows of metal between subcellular fractions. Special interest has been focused on heat-stable compound (HSC)-bound heavy metal flows within the cytosol, taking in account that MT is the major component of these ligands. Our data showed differences between the ability of metals to bind cytosolic ligands and HSCs, and their respective potency for MT induction in gill. Regardless of pretreatment, mercury gave the highest increase of gill MT, and after the decontamination MT level remained high compared to control. Cadmium and silver gave similar increases, but a significant difference with control appeared only after the decontamination step with silver, whereas 1 week of contamination was enough for cadmium. Our experimental conditions gave the following order of potency for MT induction in gill: Hg≫Cd>Ag>Zn.     

Oxygen and carbon monoxide kinetics of Glycera dibranchiata monomeric hemoglobin.

Oxygen and carbon monoxide kinetics of Glycera dibranchiata monomeric hemoglobin have been studied using laser photolysis, air flash, and stopped flow techniques. The reactions of this hemoglobin with both ligands were found to be more rapid than the corresponding reactions involving myoglobin and were also biphasic in nature, the rate constants being approximately an order of magnitude different for the fast and slow phases in each case. No pH or hemoglobin concentration dependence of the pseudo-first order rate constants was apparent between pH 6 and 9 and in the concentration range of 1.25 to 40 muM heme. Both fast and slow pseudo-first order oxygen combination rate constants varied linearly with oxygen concentration between 16 and 1300 muM. A first order slow relaxation was also noted which was linearly dependent on heme concentration and inversely dependent on oxygen concentration. This reaction has been shown to be due to a replacement of oxygen by carbon monoxide. The presence of this reaction is a result of the high affinity of Glycera monomer for carbon monoxide as shown by the partition coefficient Mr = approximately 20,000 ana an equilibrium dissociation constant of the order L = 1.1 X 10(-9) M.