Title
Carboxypeptidase A: native, zinc-removed and mercury-replaced forms.
Author
Greenblatt HM; Feinberg H; Tucker PA; Shoham G
Address
Department of Inorganic and Analytical Chemistry, Hebrew University, Jerusalem, Israel, 91904.
Source
Acta Crystallogr D Biol Crystallogr, 1998 May, 54 ( Pt 3):, 289-305
Abstract
The crystal structure of the zinc-containing exopeptidase bovine carboxypeptidase A (CPA) has been refined to high resolution, based on a data set collected from a single crystal, incorporating new sequence information based on cloning of the bovine gene. In addition, new refined structures are available for the zinc-removed form of the enzyme, apo-CPA, as well as the mercury-replaced form, Hg-CPA. The native structure reveals that the zinc-bound water molecule does not appear to more loosely bound than the rest of the zinc ligands, at least when B-factor values are considered. Nor is there any evidence for a secondary location of this water molecule. The apo-enzyme structure does not show any density in the place of the removed zinc ion. The only significant change appears to be a chi2 rotation of one zinc histidine ligand to form an ion-pair interaction with a glutamic acid side chain. The structure of Hg-CPA reveals a solvent Tris molecule bound to the mercury cation, as well as an unidentified cation bound to Glu270. The location of this citation agrees with previous proposals for the binding side of inhibitory zinc. These observations may explain some of the differences in kinetics observed in metal- replaced CPA.

Title
Comparative inhibition of hepatic hydroxymethylbilane synthase by both hard and soft metal cations.
Author
Farmer DJ; Hollebone BR
Source
Can J Biochem Cell Biol, 1984 Jan, 62:1, 49-54
Abstract
The in vitro inhibition of hydroxymethylbilane synthase (EC 4.3.1.8, uroporphyrinogen I synthetase) obtained from livers of Sprague-Dawley rats has been studied with a wide range of di- and tri-valent metal ions. After purification by cell lysis, heat treatment, and centrifugation, the stable, soluble enzyme yielded sigmoidal inhibition curves with increasing concentrations of each of the 16 test ions. Using the negative logarithm of metal concentration for 50% inhibition (the pM50 value), the metal ions could be classified according to their Klopman hardness values. Very soft ions including Hg2+, intermediate ions including Cr3+, and very hard ions including Al3+ all yielded large pM50 values indicating strong inhibition. In comparison to known metal-ion chemical behaviour, these three ions could indicate three different types of inhibitory binding sites at or near the active site: Hg2+ corresponding to sulfur in cysteine, Cr3+ corresponding to nitrogen in histidine, and Al3+ corresponding to oxygen in carboxyl groups. The presence of the first two sites is also indicated by the pH dependence of activity.

Title
Inhibitors of the proton-sucrose symport.
Author
Bush DR
Address
Photosynthesis Research Unit, U.S. Department of Agriculture, Illinois.
Source
Arch Biochem Biophys, 1993 Dec, 307:2, 355-60
Abstract
Sucrose transporters are important components of the assimilate partitioning pathway in many plants. In the results reported here, we examined the effect of several inhibitors on proton-coupled sucrose transport into plasma membrane vesicles isolated from sugar beet leaf tissue. Three compounds that are reversible inhibitors of glucose transporters, phlorizin, cytochalasin B, and forskolin, also inhibited the proton-sucrose symport. Additionally, several reagents that covalently modify specific amino acid residues, including p-chloromercuribenzenesulfonic acid (PCMBS), N-ethylmaleimide (NEM), diethyl pyrocarbonate (DEPC), and Hg2+, were also examined. NEM was not an effective inhibitor of the symport under both energized (pH 6.0) and unenergized (pH 7.7) conditions. In contrast, PCMBS, DEPC, and Hg2+ blocked sucrose transport activity. However, in control experiments it was discovered that Hg2+, but not PCMBS or DEPC, dissipated the proton electrochemical potential difference (delta mu H) that drives sucrose accumulation. It was further demonstrated that Hg2+ dissipated an imposed delta mu+H in protein-free liposomes, thus obscuring its effect on the sucrose symport. In time- and concentration-dependent inactivation experiments, it was shown that DEPC binding was substrate protectable, thereby implicating binding at or near the active site of the carrier. In contrast, PCMBS activity was not linked to substrate binding. DEPC activity was partially reversed with hydroxylamine. This is consistent with specific modification of a histidine residue. Preloading purified vesicles with free histidine did not slow the DEPC-dependent inactivation kinetics. Since these membrane vesicles are predominantly right-side out, the last observation is consistent with a DEPC-sensitive site which is accessible from the outside face of the vesicle. The results with DEPC suggest that a histidine residue is at or near the active site of the sucrose symport and that this amino acid plays a critical role in the reaction mechanism.

Title
Mercury-binding proteins from the marine mussel, Mytilus edulis.
Author
Roesijadi G
Source
Environ Health Perspect, 1986 Mar, 65:, 45-8
Abstract
The marine mussel, Mytilus edulis, possesses low molecular weight, metal-binding proteins which can be induced by and, in turn, bind mercury when individuals are exposed to low, but elevated concentrations of mercury as HgCl2. Induction of the proteins by exposure of mussels to copper, cadmium, or mercury is associated with enhanced tolerance to mercury toxicity. Mercury-binding proteins isolated from gills of mussels occur as two molecular weight variants of about 20-25 and 10-12 kdaltons, respectively, on Sephadex G-75. These have been designated as HgBP20 and HgBP10 following the nomenclature used for cadmium-binding proteins. HgBP20 represents the primary mercury-binding species. These exist as dimers which can be dissociated into subunits by treatment with 1% 2-mercaptoethanol. Further purification of HgBP20 by DEAE-cellulose ion-exchange chromatography resulted in the resolution of three major mercury-binding protein peaks; analysis of two of these showed that both had similar amino acid compositions with 26% half-cystine, 16% glycine, and very low levels of the aromatic amino acids phenylalanine and tyrosine (0.3-0.5%), histidine (0.4%), methionine (about 0.5%), and leucine (about 1%). These are similar to the compositions of proteins reported as mussel thioneins by others. Separation of HgBP20 by anion-exchange high-performance liquid chromatography resulted in the resolution of six peaks, indicating a more complex situation than was evident from DEAE-cellulose separations. Although not completely purified, these also contain cysteine- and glycine-rich proteins.

Title
Degradation of methyl and ethyl mercury by singlet oxygen generated from sea water exposed to sunlight or ultraviolet light.
Author
Suda I; Suda M; Hirayama K
Address
Kyushu National Agricultural Experiment Station, MAFF, Kumamoto, Japan.
Source
Arch Toxicol, 1993, 67:5, 365-8
Abstract
Photodegradation of methyl mercury (MeHg) and ethyl Hg (EtHg) in sea water was studied by sunlight or ultraviolet (UV) light exposure, and by determining inorganic Hg produced by degradation. Sea water containing 1 microM MeHg or EtHg was exposed to sunlight or UV light. N-Acetyl-L-cysteine was added to the solution for preventing Hg loss during the light exposure. MeHg and EtHg in sea water were degraded by sunlight (> 280 nm), UV light A (320-400 nm) and UV light B (280-320 nm), though the amounts of inorganic Hg produced from MeHg were 1/6th to 1/12th those from EtHg. Inorganic Hg production was greater with increasing concentration of sea water. Degradation of MeHg and EtHg by the UV light A exposure was inhibited by singlet oxygen (1O2) trappers such as NaN3, 1,4-diazabicyclo[2,2,2]octane, histidine, methionine and 2,5-dimethylfuran. On the other hand, inhibitors or scavengers of superoxide anion, hydrogen peroxide or hydroxyl radical did not inhibit the photodegradation of alkyl Hg. These results suggested that 1O2 generated from sea water exposed to sunlight, UV light A or UV light B was the reactive oxygen species mainly responsible for the degradation of MeHg and EtHg.

Title
Biosynthesis of porphyrins in Rhodopseudomonas palustris--VI. The effect of metals, thiols and other reagents on the activity of uroporphyrinogen decarboxylase.
Author
Koopmann GE; Batlle AM
Source
Int J Biochem, 1987, 19:4, 373-7
Abstract
The effect of several metals and reagents on the decarboxylation rate of uroporphyrinogen I by using a 16-fold purified preparation of Uroporphyrinogen Decarboxylase from Rhodopseudomonas palustris, was studied. 1 mM Hg2+ and Cu2+ were strong inhibitors, 1 mM Zn2+ and Fe2+ under certain conditions and 1 mM Fe3+ and Cr3+ also inactivated the enzyme, but Pb2+, Cd2+ and Al3+ did not. Metals inhibition was reversed by 1 mM GSH or CySH. 0.1 mM DTNB and PCMB, 1 mM pyridoxal phosphate and 100 mM chloral hydrate, as well as 1 mM 2-methoxy-5-nitrotropone and 0.2 mM diethylpyrocarbonate inhibited Uroporphyrinogen Decarboxylase; while GSH, CySH, N-ethylmaleimide, sodium thioglycolate, 1,4-dithioerythritol, EDTA and O-phenantroline did not modify activity. Data obtained would indicate that one cysteine, one or two histidine residues and probably a lysine group are required for enzyme activity.

Title
The 1.7 A refined X-ray structure of the periplasmic glucose/galactose receptor from Salmonella typhimurium.
Author
Zou JY; Flocco MM; Mowbray SL
Address
Department of Molecular Biology, Uppsala University, Sweden.
Source
J Mol Biol, 1993 Oct, 233:4, 739-52
Abstract
The X-ray structure of the periplasmic glucose/galactose receptor (binding protein) of Salmonella typhimurium (GBP-S) has been refined at 1.7 A resolution with an R-factor of 19.0%. The model contains all 309 residues of the amino acid sequence, 153 water molecules, a calcium ion and beta-D-galactose. The protein consists of two very similar structural domains, each of which is composed a core of parallel beta-sheet flanked on both sides by alpha-helices. Three short stretches of amino acid chain (from symmetrically related portions of the structure) link the domains, and presumably act as a hinge to allow their relative movement in functionally important conformational changes. Galactose is bound between the domains, interacting with a number of side-chains from the loops lining the binding cleft. A combination of hydrogen bonding, hydrophobic and steric effects give rise to tight binding (dissociation constant 0.2 microM) and high specificity. Of nine hydrogen bonding groups, three are aspartate, three asparagine, one histidine (unprotonated), one arginine and one water, contributing 13 hydrogen bonds in total. Additional residues pack against (primarily) non-polar faces of the sugar molecule. The precise arrangement of the hydrogen bonding and hydrophobic residues results in an enclosed binding site with a shape that is a composite of those of the allowed sugar molecules. It is presumed that ligands bind to a more open form of the receptor that then closes by rotation in the hinge. Comparison with the GBP-S structure solved earlier in complex with glucose shows no significant changes, even for the aspartate residue most directly involved with the different sugars. Comparison with the galactose/glucose receptor of Escherichia coli indicates that these two proteins are very similar in overall structure, with the main difference being a 2 to 3 degrees rotation in the hinge. This difference appears to be the result of different crystal packing for the two proteins; it is likely that both conformations are normally found in solution.

Title
Pb2+ and Hg2+ binding to alpha-lactalbumin.
Author
Veprintsev DB; Permyakov EA; Kalinichenko LP; Berliner LJ Address
Institute for Biological Instrumentation, Russian Academy of Sciences, Pushchino, Moscow, Russia.
Source
Biochem Mol Biol Int, 1996 Aug, 39:6, 1255-65
Abstract
Interactions of human alpha-lactalbumin with Pb2+ and Hg2+ were studied by intrinsic protein fluorescence. Lead ions bind to the strong Ca2+ binding site of alpha-lactalbumin (association constant Kass approximately 2 x 10(6) M-1) with concomitant spectral changes which are similar to those induced by the binding of Ca2+. Pb2+ also binds to the strong Zn2+ site with Kass approximately 10(5) M-1 and some secondary binding site(s) (which probably contain histidine residues) with apparent Kass approximately 10(4) M-1, causing pronounced aggregation of the protein. Mercury ions bind to alpha-lactalbumin at the primary Zn2+ sites with Kass approximately (1-4) x 10(4) M-1, although the stoichiometry of the binding depends on the conformational state of the protein. Secondary Hg2+ binding sites were suggested to contain histidines, while the strong Hg2+ site contains carboxylates in the coordination sphere and seems to coincide with the strong Zn2+ site. The binding of both Pb2+ and Hg2+ decreases the thermal stability of the Ca(2+)-loaded protein and in some conditions causes pronounced protein aggregation.

Title
Refined 1.8 A X-ray crystal structure of astacin, a zinc-endopeptidase from the crayfish Astacus astacus L. Structure determination, refinement, molecular structure and comparison with thermolysin.
Author
Gomis Rüth FX; Stöcker W; Huber R; Zwilling R; Bode W
Address
Max-Planck-Institut fÂur Biochemie, Martinsried, Germany.
Source
J Mol Biol, 1993 Feb, 229:4, 945-68
Abstract
Astacin, a 200 residue digestive zinc-endopeptidase from the crayfish Astacus astacus L., is the prototype of the "astacin family", which comprises several membrane-bound mammalian endopeptidases and developmentally implicated regulatory proteins. Large trigonal crystals of astacin were grown, and X-ray reflection data to 1.8 A resolution were collected. The astacin structure has been solved by multiple isomorphous replacement using six heavy-atom derivatives, and refined to a crystallographic R-value of 0.158 applying stringent constraints. All 200 residues are clearly defined by electron density; 181 solvent molecules have been localized. Besides the native structure, the structures of Hg-astacin (with a mercury ion replacing the zinc) and of the apoenzyme were also refined. The astacin molecule exhibits a kidney-like shape. It consists of an amino-terminal and a carboxy-terminal domain, with a deep active-site cleft in between. The zinc ion, located at the bottom of this cleft, is co-ordinated in a novel trigonal-bipyramidal geometry by three histidine residues, a tyrosine and by a water molecule, which is also bound to the carboxylate side-chain of Glu93. The amino-terminal domain of astacin consists mainly of two long alpha-helices, one centrally located and one more peripheral, and of a five-stranded pleated beta-sheet. The amino terminus protrudes into an internal, water-filled cavity of the lower domain and forms a buried salt bridge with Glu103; amino-terminally extended pro-forms of astacin are thus not compatible with this structure. The carboxy-terminal domain of astacin is mainly organized in several turns and irregular structures. Because they share sequence identity of about 35%, the structures of the proteolytic domains of the other "astacin" members must be quite similar to astacin. Only a few very short deletions and insertions quite distant from the active-site distinguish their structures from astacin. The five-stranded beta-sheet and the two helices of the amino-terminal domain of astacin are topologically similar to the structure observed in the archetypal zinc-endopeptidase thermolysin; the rest of the structures are, in contrast, completely unrelated in astacin and thermolysin. The zinc ion, the central alpha-helix and the zinc-liganding residues His92, Glu93 and His96 of astacin are nearly superimposable with the respective groups of thermolysin, namely with the zinc ion, the "active-site helix", and His142TL, Glu143TL and His146TL of the zinc-binding consensus motif His-Glu-Xaa-Xaa-His (where Xaa is any amino acid residue).(ABSTRACT TRUNCATED AT 400 WORDS)

Title
Structure and function of the Escherichia coli ribonucleotide reductase protein R2.
Author
Nordlund P; Eklund H
Address
Department of Molecular Biology, Swedish University of Agricultural Sciences, Biomedical Center, Uppsala.
Source
J Mol Biol, 1993 Jul, 232:1, 123-64
Abstract
The crystal structure of the ribonucleotide reductase free radical protein R2 from Escherichia coli has been determined by multiple isomorphous replacement and twofold molecular averaging. The structure has been refined at 2.2 A resolution to R = 0.175. The subunit structure of the R2 protein has a novel fold where the basic motif is a bundle of eight long helices. The R2 dimer has two equivalent dinuclear iron centers. Each iron center is well buried in the subunit. The iron atoms have both histidine and carboxyl acid ligands and are bridged by an oxide ion and the carboxylate group of Glu115. One iron atom is octahedrally coordinated with small deviations from ideal values, while the coordination of the other iron ion is more distorted, mainly due to the fact that Asp84 is a bidental ligand to this iron atom. The oxidation of the enzymatically essential tyrosine residue (Tyr122) and the dinuclear iron center by molecular oxygen is suggested to take part in a suitable conserved oxygen-binding pocket between the iron center and the tyrosine zeta-oxygen 5.3 A away from the closest iron ion. The tyrosine proton can be abstracted by the dioxygen and the deprotonated tyrosine residue is then more easily oxidized to a radical species. Tyr122 is buried inside the protein about 10 A from the surface. This has the consequence that the tyrosyl radical cannot participate directly in hydrogen abstraction from the substrate ribose at the active site of the holoenzyme located on the R1 subunit. The radical must then be indirectly involved in the mechanism of the enzyme and an electron transfer reaction between the active site and the tyrosine must take place. Based on the analysis of the available ribonucleotide reductase sequences, the binding surface for the large ribonucleotide reductase protein R1, and a possible route for an electron transport between the buried radical and this surface is described.

Title
Amino acid sequence of the tryptic SH-peptide of thermitase, a thermostable serine proteinase from Thermoactinomyces vulgaris. Relation to the subtilisins.
Author
Baudys M; Kostka V; Hausdorf G; Fittkau S; Höhne WE
Source
Int J Pept Protein Res, 1983 Jul, 22:1, 66-72
Abstract
The following amino acid sequence of the tryptic SH-peptide of thermitase, a thermostable serine proteinase from Thermoactinomyces vulgaris, was determined: Val-Val-Gly-Gly-Trp-Asp-Phe-Val-Asp-Asn-Asp-Ser-Thr- Pro-Gln-Asn-Gly-Asn-Gly-64His-Gly-Thr-His-68Cys-Ala- Gly-Ile-Ala-Ala-Ala-Val-Thr-Asn-Asn-Ser-Thr-Gly-Ile- Ala-Gly-Thr-Ala-Pro-Lys. This sequence shows homology with the highly conservative part of the subtilisin sequences around the active site His-64. The single cysteine residue of thermitase is localized near this histidine residue thus replacing valine in position 68 (according to the numbering of the subtilisins). This becomes evident also from the specific labeling of the active site histidine with a radioactive inhibitor (Z-Ala-Ala-Phe-14CH2-Cl). The tryptic SH-peptide isolated from the modified enzyme contains all the radioactivity and has the same end group and amino acid composition as the tryptic peptide isolated from the tryptic digest of the unlabeled enzyme and subjected to sequential analysis. From sequence homology as well as from secondary structure predictions it may be concluded that the geometry of the active site of thermitase is very similar to that of the subtilisins with the cysteine residue nearby. The inactivation of thermitase by labeling of the SH-group with mercury compounds may then be due to a sterical hindrance or to a more direct interaction of the mercury atom with the charge relay system of the enzyme.

Title
Enzymatic properties of the sweet-tasting proteins thaumatin and monellin after partial reduction.
Author
Van Der Wel H; Bel WJ
Source
Eur J Biochem, 1980 Mar, 104:2, 413-8
Abstract
After partial reduction of disulfide bonds in the thaumatins, the sweet-tasting proteins from the fruits of Thaumatococcus danielii Benth, a rapid autodigestion was demonstrated. In the presence of suitable substrates, the reduced thaumatins showed protease, amidase and esterase activity. Thiol-blocking reagents like mercury(II) chloride inhibited the enzymatic activity. Of the thaumatins b, c, I, II and III (with increasing isoelectric points), thaumatin I showed the lowest enzymatic activity. In this series, the enzymatic activity increased from thaumatin I to thaumatin III as well as from thaumatin I to thaumatin b. Acetylation of the epsilon-amino group of lysine residues in the thaumatins by acetic anhydride, causing a decrease in basicity, led to an increase in enzymatic activity, which is correlated with the number of acetyl groups introduced. Comparison of the amino acid sequence of thaumatin I with that of cysteine proteases of plant origin showed no similarities. Moreover, the thaumatins lack histidine, one of the amino acids in the active site of the cysteine proteases. Monellin, the sweet-tasting protein from the fruits of Dioscoreophyllum cumminsii Diels, is not enzymatically active. However, when monellin with acetylated epsilon-amino groups of lysine residues was brought into a reducing environment it appeared to be enzymatically active. The similarities in properties of the thaumatins and monellin suggest a structural relationship between these proteins.

Title
Suppression of aminoacyladenylate synthesis by methyl mercury in vitro and in vivo.
Author
Kuznetsov DA; Zavijalov NV; Govorkov AV; Richter V
Source
Toxicol Lett, 1987 Apr, 36:2, 161-5
Abstract
Methyl mercury (MeHg) at a concentration of 20 microM significantly inhibits the synthesis of aminoacyladenylates (AAA) in vitro from serine and histidine, and fails to inhibit AAA synthesis from phenylalanine, leucine, arginine and aspartate. In vivo administration of MeHg (single i.p. injection of 50 nmol/g body weight) leads to 75-80% suppression of AAA synthesis from serine, histidine, phenylalanine, leucine, arginine and aspartate in rat brain tissue.

Title
Crystallographic studies of inhibitor binding sites in human carbonic anhydrase II: a pentacoordinated binding of the SCN- ion to the zinc at high pH.
Author
Eriksson AE; Kylsten PM; Jones TA; Liljas A
Address
Department of Molecular Biology, Biomedical Center, Uppsala, Sweden.
Source
Proteins, 1988, 4:4, 283-93
Abstract
The binding of four inhibitors--mercuric ion, 3-acetoxymercuri-4-aminobenzenesulfonamide (AMS), acetazolamide (Diamox), and thiocyanate ion--to human carbonic anhydrase II (HCA II) has been studied with X-ray crystallography. The binding of mercury to HCA II at pH 7.0 has been investigated at 3.1 A resolution. Mercuric ions are observed at both nitrogens in the His-64 ring. One of these sites is pointing toward the zinc ion. The only other binding site for mercury is at Cys-206. The binding of the two sulfonamide inhibitors AMS and Diamox, has been reinvestigated at 2.0 and 3.0 A, respectively. Only the nitrogen of the sulfonamide group binds to the zinc ion replacing the hydroxyl ion. The sulfonamide oxygen closest to the zinc ion is 3.1 A away. Thus the tetrahedral geometry of the zinc is retained, refuting earlier models of a pentacoordinated zinc. The structure of the thiocyanate complex has been investigated at pH 8.5 and the structure has been refined at 1.9 A resolution using the least-squares refinement program PROLSQ. The crystallographic R factor is 17.6%. The zinc ion is pentacoordinated with the anion as well as a water molecule bound in addition to the three histidine residues. The nitrogen atom of the SCN- ion is 1.9 A from the zinc ion but shifted 1.3 A with respect to the hydroxyl ion in the native structure and at van der Waals' distance from the O gamma l atom of Thr-199. This is due to the inability of the O gamma l atom of Thr-199 to serve as a hydrogen bond donor, thus repelling the nonprotonated nitrogen. The SCN- molecule reaches into the deep end of the active site cavity where the sulfur atom has displaced the so-called "deep" water molecule of the native enzyme. The zinc-bound water molecule is 2.2 A from the zinc ion and 2.4 A from the SCN- nitrogen. In addition, this water is hydrogen bonded to the O gamma l atom of Thr-199 and to another water molecule. We have observed that solvent and inhibitor molecules have three possible binding sites on the zinc ion and their significance for the catalysis and inhibition of HCA II will be discussed. All available crystallographic data are consistent with a proposed catalytic mechanism in which both the OH moiety and one oxygen of the substrate HCO3- ion are ligated to the zinc ion.

Title
Metal binding properties of a monoclonal antibody directed toward metal-chelate complexes.
Author
Blake DA; Chakrabarti P; Khosraviani M; Hatcher FM; Westhoff CM; Goebel P; Wylie DE; Blake RC 2nd
Address
Department of Ophthalmology, Tulane University School of Medicine, New Orleans, Louisiana 70112, USA. dblake@tmcpop.tmc.tulane.edu
Source
J Biol Chem, 1996 Nov, 271:44, 27677-85
Abstract
A monoclonal antibody that recognizes cadmium-EDTA complexes has been produced by the injection of BALB/c mice with a metal-chelate complex covalently coupled to a carrier protein. The ability of purified antibody to recognize 16 different metal-EDTA complexes was assessed by measuring equilibrium binding constants using a KinExATM immunoassay instrument. The antibody bound to cadmium- and mercury-EDTA complexes with equilibrium dissociation constants of 21 and 26 nM, respectively. All other metal-EDTA complexes tested, including those of Mn(II), In(III), Ni(II), Zn(II), Co(II), Cu(II), Ag(I), Fe(III), Pb(II), Au(III), Tb(III), Ga(III), Mg(II), and Al(III) bound with affinities from 20- to 40,000-fold less than that determined for the cadmium-EDTA complex. With the exception of mercury and magnesium, the binding of divalent metal-chelate complexes was well-correlated with the size of the metal ion. The amino acid sequences of the heavy and light chain variable regions were deduced from polymerase chain reaction-amplified regions of the corresponding genes and subsequently used to construct molecular models of the antigen binding region. The key residue for cadmium binding in the model for 2A81G5 appeared to be histidine 96 in the heavy chain.

Title
Certain photooxidized derivatives of tryptophan bind with very high affinity to the Ah receptor and are likely to be endogenous signal substances.
Author
Rannug A; Rannug U; Rosenkranz HS; Winqvist L; Westerholm R; Agurell E; Grafström AK
Address
Research Department, National Board of Occupational Safety and Health, Solna, Sweden.
Source
J Biol Chem, 1987 Nov, 262:32, 15422-7
Abstract
The purpose of the present study was to determine whether ultraviolet light (UV) irradiation of amino acids produces compounds with affinity for the Ah receptor. Aqueous solutions of L-tryptophan were exposed to radiation from an unfiltered high-pressure mercury lamp. The photoproducts formed were solvent-extracted or concentrated on Sep-Pak C18 cartridges. The concentrated extracts or eluants were treated for their ability to compete with 3H-labeled 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Binding was assayed in liver cytosolic preparations from Sprague-Dawley rats using a technique based on hydroxylapatite separation. Photoproducts with receptor affinity were formed in a time-dependent manner. Histidine and tryptamine also gave products upon UV irradiation that competed with TCDD. Commercial tryptophan, at least aged, contained trace amounts of impurities with receptor affinity. Analysis by TLC and high-pressure liquid chromatography of the photo-products of tryptophan showed a minimum of three different binding compounds. Two of the products were studied in greater detail. One of them, showing UV absorbance and yellow fluorescence, gave a molecular ion (M+) of 284 and the other gave M+ 312 but showed little UV absorption and fluorescence. The concentration, based on mass spectrometry quantifications, of the two compounds that displaced more than 50% of TCDD was found to be extremely low, giving Kd values of 0.44 nM (M+ 312) and 0.07 nM (M+ 284). The existence of high affinity receptors for oxidized amino acids is postulated and their possible role in the proliferative cellular responses to TCDD and tryptophan is discussed briefly.

Title
Possible role of a copper complex in the pathogenesis of rheumatoid arthritis.
Author
Gerber DA
Source
Agents Actions Suppl, 1981, 8:, 165-84
Abstract
Clinical and experimental evidence suggest that rheumatoid arthritis may be due to a deficiency in joint fluid of an inhibitor of protein denaturation. This inhibitor may be a histidine-cystine-copper complex. A deficiency of this inhibitor may permit the nonimmunological alteration of joint fluid IgG to a pseudoimmune complex which could initiate the inflammation and immunological abnormalities of rheumatoid arthritis.

Title
Growth characteristics in laboratory animals fed zinc-deficient, copper-deficient, of histidine-supplemented diets.
Author
Van Wouwe JP; Veldhuizen M
Address
Department of Pediatrics, Drechtsteden Hospital Jacobus, Zwijndrecht, The Netherlands.
Source
Biol Trace Elem Res, 1996 Oct, 55:1-2, 71-7
Abstract
The effects of growth in male Wistar rats and female Swiss Random mice were studied during dietary zinc (Zn) deficiency, copper (Cu) deficiency, and during the feeding of a histidine (His) supplement Growth was analyzed by comparing the characteristics of the decreasing exponential growth curve plotted for the experimental period. When the animals were pair-fed the experimental diets, the growth pattern in the animals remained unaltered. The growth rate decreased during Zn deficiency by a factor of 0.64 over a period of 10 d (male young adult rats) and by a factor of 0.76 over a period of 28 d (female weaning mice). On the other hand, a supplement of His increased the growth rate by a factor of 1.11 (in the mice). The effect of Cu deficiency on the growth rate was not statistically significant (in the rats). However, Cu deficiency causes effects in the Zn status that may over-compensate minor growth retardation during Cu deficiency. The effect of the His supplement is explained by its having an effect on the Zn-absorption (His enhancing Zn transport over the gut) and by a stimulating effect of this amino acid on the thickness of the growth plate in bone.

Title
The core metal-recognition domain of MerR.
Author
Zeng Q; StÁlhandske C; Anderson MC; Scott RA; Summers AO
Address
The Department of Microbiology, University of Georgia, Athens 30602, USA.
Source
Biochemistry, 1998 Nov, 37:45, 15885-95
Abstract
MerR, the metalloregulatory protein of the mercury-resistance operon (mer) has unusually high affinity and specificity for ionic mercury, Hg(II). Prior genetic and biochemical evidence suggested that the protein has a structure consisting of an N-terminal DNA binding domain, a C-terminal Hg(II)-binding domain, and an intervening region involved with communication between these two domains. We have characterized a series of MerR deletion mutants and found that as little as 30% of the protein (residues 80-128) forms a stable dimer and retains high affinity for Hg(II). Biophysical measures indicate that this minimal Hg(II)-binding domain assumes the structural characteristics of the wild-type full-length protein both in the Hg(II) center itself and in an immediately adjacent helical protein domain. Our observations are consistent with the core Hg(II)-binding domain of the MerR dimer being constituted by a pair of antiparallel helices (possibly in a coiled-coil conformation) comprised of residues cysteine 82 through cysteine 117 from each monomer followed by a flexible loop through residue cysteine 126. These antiparallel helices would have a potential Hg(II)-binding site at each end. However, just as in the full-length protein, only one of these potential binding sites in the deleted proteins actually binds Hg(II). Title
A carbon-13 NMR comparative study of metal ion substitutions in human carbonic anhydrase I carboxymethylated at active-site histidine-200.
Author
Khalifah RG; Morley PJ
Source
Arch Biochem Biophys, 1984 Aug, 232:2, 632-9
Abstract
Human carbonic anhydrase I (EC 4.2.1.1), the low-activity isozyme, has a reactive active-site Histidine-200 that is known to be specifically modified at N tau with haloacetates. Using [1-13C]bromoacetate, we previously introduced a highly sensitive 13C NMR probe into the active site of the enzyme and studied the interaction of the carboxymethyl carboxylate with the active-site zinc, as well as the ionization properties of the carboxymethylated histidine-200 side chain. In the present work, these studies have been extended to metalloderivatives of the enzyme in which the intrinsic zinc has been replaced by Cd2+, Hg2+, and Co2+. In the former two metals, spin-1/2 isotopes (113Cd and 199Hg) in the absence of inhibitory halides were utilized to search for two-bond spin-spin couplings in the spectrum of the 13C-enriched carboxymethyl carboxylate under conditions where coordination exists in the Zn and Co derivatives. The absence of splittings and titration studies of the chemical shift of the resonance both established the absence of coordination. The pH dependence of the carboxylate, which reflects the ionization of the CmHis-200 ring, was observed in the presence and absence of bound inhibitors. Marked differences were seen among the four metalloderivatives in all these properties, suggesting great sensitivity of the active site to the nature of the metal inserted. The data suggest extreme caution in extrapolating results from metal ion substitution studies to the native zinc enzyme, and may reflect functional significance of this sensitivity in the catalysis.

Title
Biotoxicity of mercury to Chlorella vulgaris as influenced by amino acids.
Author
Mohapatra DK; Mohanty L; Mohanty RC; Mohapatra PK
Address
Department of Botany, Uktal University, Bhubaneswar, India.
Source
Acta Biol Hung, 1997, 48:4, 497-504
Abstract
The toxicity of mercury ion, on Chlorella vulgaris, is largely influenced by amino acids. Five amino acids, namely alanine, asparagine, glutamic acid, cysteine and histidine, were added separately to the medium containing static dose of mercury. Survival (%) of the alga was reduced with the increasing concentrations of mercury. Of these five amino acids, cysteine was found to be the most effective while alanine and glutamic acid were the least effective on reducing the toxic effect of mercury on the alga measured in terms of growth, chlorophyll and protein content. The order of detoxification was Alanine < Glutamate < Asparargine < Histidine < Cysteine. Amino acids from ligands with Hg2+ making it less toxic to the alga and produce an additional source of energy for growth and development.

Title
Biotoxicity of mercury as influenced by mercury(II) speciation.
Author
Farrell RE; Germida JJ; Huang PM
Address
Department of Soil Science, University of Saskatchewan, Saskatoon, Canada.
Source
Appl Environ Microbiol, 1990 Oct, 56:10, 3006-16
Abstract
Integration of physicochemical procedures for studying mercury(II) speciation with microbiological procedures for studying the effects of mercury on bacterial growth allows evaluation of ionic factors (e.g., pH and ligand species and concentration) which affect biotoxicity. A Pseudomonas fluorescens strain capable of methylating inorganic Hg(II) was isolated from sediment samples collected at Buffalo Pound Lake in Saskatchewan, Canada. The effect of pH and ligand species on the toxic response (i.e., 50% inhibitory concentration [IC50]) of the P. fluorescens isolated to mercury were determined and related to the aqueous speciation of Hg(II). It was determined that the toxicities of different mercury salts were influenced by the nature of the co-ion. At a given pH level, mercuric acetate and mercuric nitrate yielded essentially the same IC50s; mercuric chloride, on the other hand, always produced lower IC50s. For each Hg salt, toxicity was greatest at pH 6.0 and decreased significantly (P = 0.05) at pH 7.0. Increasing the pH to 8.0 had no effect on the toxicity of mercuric acetate or mercuric nitrate but significantly (P = 0.05) reduced the toxicity of mercuric chloride. The aqueous speciation of Hg(II) in the synthetic growth medium M-IIY (a minimal salts medium amended to contain 0.1% yeast extract and 0.1% glycerol) was calculated by using the computer program GEOCHEM-PC with a modified data base. Results of the speciation calculations indicated that complexes of Hg(II) with histidine [Hg(H-HIS)HIS+ and Hg(H-HIS)2(2+)], chloride (HgCl+, HgCl2(0), HgClOH0, and HgCl3-), phosphate (HgHPO4(0), ammonia (HgNH3(2+), glycine [Hg(GLY)+], alanine [Hg(ALA)+], and hydroxyl ion (HgOH+) were the Hg species primarily responsible for toxicity in the M-IIY medium. The toxicity of mercuric nitrate at pH 8.0 was unaffected by the addition of citrate, enhanced by the addition of chloride, and reduced by the addition of cysteine. In the chloride-amended system, HgCl+, HgCl2(0), and HgClOH0 were the species primarily responsible for observed increases in toxicity. In the cysteine-amended system, formation of Hg(CYS)2(2-) was responsible for detoxification effects that were observed. The formation of Hg-citrate complexes was insignificant and had no effect on Hg toxicity.

Title
Modulation of mu, delta and kappa opioid receptors in rat brain by metal ions and histidine.
Author
Tejwani GA; Hanissian SH
Address
Department of Pharmacology, College of Medicine, Ohio State University, Columbus 43210-1239.
Source
Neuropharmacology, 1990 May, 29:5, 445-52
Abstract
The effect of zinc (Zn2+) and several other trace elements was studied on the binding of the opioid receptor agonists [3H] DAGO [( ([Tyr-D-Ala-Gly-Methyl-Phe-Glyol]-enkephalin)a, [3H] DSTLE ([Tyr-D-Ser-Gly-Phe-Leu-Thr]-enkephalin) and [3H] EKC (ethylketocyclazocine), which are specific for the mu, delta and kappa opioid receptors, respectively, in the cerebral cortex of the rat. Physiological concentrations of zinc were inhibitory to mu receptor binding, whereas the delta and kappa receptors were relatively insensitive to this inhibition. Scatchard analysis, using these opioid agonists, revealed curvilinear plots; concentrations of zinc equal to or less than the IC50 (the concentration of cation which caused 50% inhibition of the binding of opioid ligand to its receptor), increased the KD (the dissociation constant) of all three subtypes of receptor, with no effect on the Bmax (the maximum number of binding sites) and abolished the high affinity sites of the delta and kappa receptors. Copper, cadmium and mercury also inhibited the binding of these ligands to their receptors. Histidine was most effective in preventing the inhibitory effects of zinc and copper, whereas it was less effective on cadmium and without any effect on the inhibition caused by mercury. Magnesium and manganese were stimulatory to opioid receptor binding, whereas cobalt and nickel had dual (stimulatory and inhibitory) effects. Non-inhibitory concentrations of zinc significantly decreased the stimulatory effects of magnesium and manganese on the mu and delta receptors, suggesting that part of the effect of zinc was through prevention of the actions of stimulatory cations.(ABSTRACT TRUNCATED AT 250 WORDS)