Conformation of [Cd7]-metallothionein-2 from rat liver in aqueous solution determined by nuclear magnetic resonance spectroscopy

The three-dimensional structure of [Cd7]-metallothionein-2 from rat liver was determined in aqueous solution, using nuclear magnetic resonance spectrometry and distance geometry calculations. The experimental data provided proton-proton distance constraints from measurements of nuclear Overhauser effects, constraints on the geometry of the metal-cysteine clusters determined by heteronuclear correlation spectroscopy, and dihedral angle constraints derived from both coupling constants and nuclear Overhauser effects. The structure calculations were performed with the program DISMAN. As in previous studies with rabbit liver metallothionein-2a, the structure calculations were performed separately for the alpha and beta-domains containing the 4 and 3-metal clusters, respectively, since no interdomain constraints were found. For both domains, the global polypeptide fold, the location of polypeptide secondary structure elements, the architecture of the metal-sulfur cluster and the local chirality of the metal co-ordination are very similar to the solution structure of rabbit metallothionein-2a, but show considerable difference relative to the crystal structure of rat metallothionein-2.     

Three-dimensional structure of human [113Cd7]metallothionein-2 in solution determined by nuclear magnetic resonance spectroscopy

The three-dimensional structure of human [113Cd7]metallothionein-2 was determined by nuclear magnetic resonance spectroscopy in solution. Sequence-specific 1H resonance assignments were obtained using the sequential assignment method. The input for the structure calculations consisted of the metal-cysteine co-ordinative bonds identified with heteronuclear correlation spectroscopy, 1H-1H distance constraints from nuclear Overhauser enhancement spectroscopy, and spin-spin coupling constants 3JHN alpha and 3J alpha beta. The molecule consists of two domains, the beta-domain including amino acid residues 1 to 30 and three metal ions, and the alpha-domain including residues 31 to 61 and four metal ions. The nuclear magnetic resonance data present no evidence for a preferred relative orientation of the two domains. The polypeptide-to-metal co-ordinative bonds in human metallothionein-2 are identical to those in the previously determined solution structures of rat metallothionein-2 and rabbit metallothionein-2a, and the polypeptide conformations in the three proteins are also closely similar.     

Three-dimensional solution structure of mouse [Cd7]-metallothionein-1 by homonuclear and heteronuclear NMR spectroscopy

Sequential 1H-NMR assignments of mouse [Cd7]-metallothionein-1 (MT1) have been carried out by standard homonuclear NMR methods and the use of an accordion-heteronuclear multiple quantum correlation (HMQC) experiment for establishing the metal, 113Cd2+, to cysteine connectivities. The three-dimensional structure was then calculated using the distance constraints from two-dimensional nuclear Overhauser effect (NOE) spectroscopy spectra and the Cys-Cd connectivities as input for a distance geometry-dynamical simulated annealing protocol in X-PLOR 3.851. Similar to the mammalian MT2 isoforms, the homologous primary structure of MT1 suggested two separate domains, each containing one metal cluster. Because there were no interdomain constraints, the structure calculation for the N-terminal beta- and the C-terminal alpha-domain were carried out separately. The structures are based on 409 NMR constraints, consisting of 381 NOEs and 28 cysteine-metal connectivities. The only elements of regular secondary structure found were two short stretches of 3(10) helices along with some half-turns in the alpha-domain. Structural comparison with rat liver MT2 showed high similarity, with the beta-domain structure in mouse MT1 showing evidence of increased flexibility compared to the same domain in MT2. The latter was reflected by the presence of fewer interresidue NOEs, no slowly exchanging backbone amide protons, and enhanced cadmium-cadmium exchange rates found in the beta-domain of MT1.     

Metal co-ordination in rat liver metallothionein-2 prepared with or without reconstitution of the metal clusters, and comparison with rabbit liver metallothionein-2

Possible origins of the different metal co-ordination topologies in the recently determined structures of rat metallothionein-2 (MT2) in single crystals and rabbit MT2 in solution were investigated. A complete structure determination for rat MT2 in solution by nuclear magnetic resonance (n.m.r.) showed that the differences in the spatial structures cannot be attributed to the different primary structures of the two species. Comparison of [113Cd7]MT2 obtained by reconstitution of the apoprotein in vitro with preparations using a different procedure showed, moreover, that the metal co-ordination observed in solution by n.m.r. is not an artefact of the protein reconstitution. Solutions of high-pressure liquid chromatographically homogeneous biosynthetic preparations of [113Cd, Zn]MT2 were obtained from rat liver following injection of 113Cd into rats in vivo, without further metal exchange after protein isolation. They contain a mixture of several forms of MT2 with different relative metal compositions, giving rise to an increased number of 113Cd resonances. For the components of the four-metal cluster, the major one of these different forms exhibits patterns in the two-dimensional [1H, 113Cd]-correlated spectra that are indistinguishable from those of [113Cd7]MT2, thereby implying identity of cluster coordination and topology. These results are discussed with regard to continued investigations into the differences between the solution structure and crystal structure of MT2.     

Thiolate exchange in [TmR]ZnSR' complexes and relevance to the mechanisms of thiolate alkylation reactions involving zinc enzymes and proteins

The zinc and cadmium thiolate complexes [TmBut]MSCH2C(O)N(H)Ph (M = Zn, Cd) may be obtained via treatment of the respective methyl complex [TmBut]MMe with PhN(H)C(O)CH2SH. The molecular structure of [TmBut]ZnSCH2C(O)N(H)Ph has been determined by X-ray diffraction, thereby demonstrating the presence of an intramolecular N-H S hydrogen bond between the amide N-H group and thiolate sulfur atom. [TmBut]ZnSCH2C(O)N(H)Ph mimics the function of the Ada DNA repair protein by undergoing alkylation with MeI to give [TmBut]ZnI and MeSCH2C(O)N(H)Ph. A series of crossover experiments and 1H NMR magnetization transfer studies establish that thiolate exchange between [TmR]ZnSR' derivatives is facile in this system, an observation that supports the previous suggestion that the alkylation of [TmPh]ZnSCH2C(O)N(H)Ph by MeI may proceed via a sequence that involves dissociation of [PhN(H)C(O)CH2S]-.     

Comparative 113Cd-n.m.r. studies on rabbit 113Cd7-, (Zn1,Cd6)- and partially metal-depleted 113Cd6-metallothionein-2a.

Rabbit 113Cd7-metallothionein-2a (MT) contains two metal-thiolate clusters of three (cluster B) and four (cluster A) metal ions. The 113Cd-n.m.r. spectrum of 113Cd6-MT, isolated from 113Cd7-MT upon treatment with EDTA, is similar to that of 113Cd7-MT, but the cluster B resonances are lower in intensity, suggesting its co-operative metal depletion. (Zn1,113Cd6)-MT, formed upon addition of the Zn(II) ions to 113Cd6-MT, shows 113Cd-n.m.r. features characteristic of cluster B populations containing both Cd(II) and Zn(II) ions. The overall intensity gain of the mixed cluster B resonances per Cd as to those in 113Cd6- and 113Cd7-MT suggests a stabilization effect of the bound Zn(II) ions upon the previously established intramolecular 113Cd exchange within this cluster.     

Two gamma-bends in the backbone conformation of [D-Ala2]-leucine enkephalin in solution

The solution conformation of [D-Ala2]-leucine enkephalin in its zwitterionic form in DMSO-d6 has been monitored by one- and two-dimensional proton magnetic resonance spectroscopy at 500 MHz. The resonances from the labile amide protons and the nonlabile protons have been assigned from the shift correlated spectroscopy. The chemical shift of the amide and C-alpha protons are found to vary with temperature but in opposite directions, except the C-alpha proton of the terminal tyrosine residue. This behavior has been explained by the shifting of equilibrium between the zwitterionic and neutral forms of the [D-Ala2]-leucine enkephalin and probably conformational changes accompanying temperature variation. The low values of the temperature coefficients of leucine and glycine amide protons indicate that these protons are either intramolecularly hydrogen bonded or solvent shielded. The observation of sequential cross peaks in the nuclear Overhauser effect spectra obtained at various mixing times, tau m (200-900 ms), indicate an extended backbone, which does not corroborate with the presence of a folded structure, i.e., beta-bend type structure. The estimate of interproton distances in conjunction with the low values of temperature coefficients of the leucine and glycine amide protons and vicinal coupling constants 3JHN-C alpha H have been rationalized by the predominance of two gamma-bends in the backbone conformation of [D-Ala2]-leucine enkephalin. The gamma-bend around the D-Ala residue has phi = 80 degrees and psi = 270 degrees, while the one around Phe it has phi = 285 degrees and psi = 90 degrees.     

Amide proton exchange in human metallothionein-2 measured by nuclear magnetic resonance spectroscopy

In human metallothionein-2, the exchange rate constants of ten amide protons were found to range from 1.7 x 10(-4) to 1 x 10(-1) min-1 at pH 6.3 and 8 degrees C. Most of these slowly exchanging protons could be associated with hydrogen bonds in secondary structure elements of the alpha-domain. Amide proton exchange rates thus present an additional criterion for the structural characterization of different metallothioneins, which could be particularly valuable for comparisons of different homologous protein preparations containing nuclear magnetic resonance-inactive metal ions, where the metal-polypeptide co-ordinative bonds cannot be identified directly.     

Inhibition of [3H]platelet activating factor (PAF) binding by Zn2+: a possible explanation for its specific PAF antiaggregating effects in human platelets

Zinc ions in the micromolar range exhibited a strong inhibitory activity toward platelet activating factor (PAF)-induced human washed platelet activation, if added prior to this lipid chemical mediator. The concentration of Zn2+ required for 50% inhibition of aggregation (IC50) was inversely proportional to the concentration of PAF present. The IC50 values (in microM) for Zn2+ were 8.8 +/- 3.9, 27 +/- 5.8, and 34 +/- 1.7 against 2, 5, and 10 nM PAF, respectively (n = 3-6). Zn2+ exhibited comparable inhibitory effects on [3H]serotonin secretion and the IC50 values (in microM) were 10 +/- 1.2, 18 +/- 3.5, and 35 +/- 0.0 against 2, 5, and 10 nM PAF, respectively (n = 3). Under the same experimental conditions, aggregation and serotonin secretion induced by ADP (5 microM), arachidonic acid (3.3 microM), or thrombin (0.05 U/ml) were not inhibited. Introduction of Zn2+ within 0-2 min after PAF addition not only blocked further platelet aggregation and [3H]serotonin secretion but also caused reversal of aggregation. Analysis of [3H]PAF binding to platelets showed that Zn2+ as well as unlabeled PAF prevented the specific binding of [3H]PAF. The inhibition of [3H]PAF specific binding was proportional to the concentration of Zn2+ and the IC50 value was 18 +/- 2 microM against 1 nM [3H]PAF (n = 3). Other cations, such as Cd2+, Cu2+, and La3+, were ineffective as inhibitors of PAF at concentrations where Zn2+ showed its maximal effects. However, Cd2+ and Cu2+ at high concentrations exhibited a significant inhibition of the aggregation induced by 10 nM PAF with IC50 values being five- and sevenfold higher, respectively, than the IC50 for Zn2+, and with the IC50 values for inhibition of binding of 1 nM [3H]PAF being 5 and 19 times higher, respectively, than the IC50 for Zn2+. The specific inhibition of PAF-induced platelet activation and PAF binding to platelets suggested strongly that Zn2+ interacted with the functional receptor site of PAF or at a contiguous site.     

Effect of the two conserved prolines of human growth inhibitory factor (metallothionein-3) on its biological activity and structure fluctuation: comparison with a mutant protein

Human neuronal growth inhibitory factor, a metalloprotein classified as metallothionein-3 (MT-3), impairs the survival and the neurite formation of cultured neurons. In these studies the double P7S/P9A mutant (mutMT-3) and single mutants P7S and P9A of human Zn(7)-MT-3 were generated, and their effects on the biological activity and the structure of the protein were examined. The biological results clearly established the necessity of both proline residues for the inhibitory activity, as even single mutants were found to be inactive. Using electronic absorption, circular dichroism (CD), magnetic CD (MCD), and (113)Cd NMR spectroscopy, the structural features of the metal-thiolate clusters in the double mutant Cd(7)-mutMT-3 were investigated and compared with those of wild-type Cd(7)-MT-3 [Faller, P., Hasler, D. W., Zerbe, O., Klauser, S., Winge, D. R., and Vasák, M. (1999) Biochemistry 38, 10158] and the well characterized Cd(7)-MT-2a from rabbit liver. Similarly to (113)Cd(7)-MT-3 the (113)Cd NMR spectrum of (113)Cd(7)-mutMT-3 at 298 K revealed four major and three minor resonances (approximately 20% of the major ones) between 590 and 680 ppm, originating from a Cd(4)S(11) cluster in the alpha-domain and a Cd(3)S(9) cluster in the beta-domain, respectively. Due to the presence of dynamic processes in the structure of MT-3 and mutMT-3, all resonances showed the absence of resolved homonuclear [(113)Cd-(113)Cd] couplings and large apparent line widths (between 140 and 350 Hz). However, whereas in (113)Cd(7)-mutMT-3 the temperature rise to 323 K resulted in a major recovery of the originally NMR nondetectable population of the Cd(3)S(9) cluster resonances, no such temperature effect was observed in (113)Cd(7)-MT-3. To account for the observed NMR features, a dynamic structural model for the beta-domain is proposed, which involves a folded and a partially unfolded state. It is suggested that in the partially unfolded state a slow cis/trans isomerization of Cys-Pro(7) or Cys-Pro(9) amide bonds in (113)Cd(7)-MT-3 takes place and that this process represents a rate-limiting step in a correct domain refolding. In addition, closely similar apparent stability constants of human MT-3, mutMT-3, and rabbit MT-2a with Cd(II) and Zn(II) ions were found. These results suggest that specific structural features dictated by the repetitive (Cys-Pro)(2) sequence in the beta-domain of MT-3 and not its altered metal binding affinity compared to MT-1/MT-2 isoforms are responsible for the biological activity of this protein.     

Computational studies of Cu(II)[peptide] binding motifs: Cu[HGGG] and Cu[HG] as models for Cu(II) binding to the prion protein octarepeat region

The binding of Cu(II) to the prion protein is investigated by computations at the B3LYP level of theory on models of the octarepeat domain of the prion protein. The models incorporate the functionality of the glycine (G) and histidine (H) residues which occur in the octarepeat domain, PHGGGWGQ. The copper complexes are designated Cu[HG] and Cu[HGGG]. Coordination to the metal via the imidazole ring of the histidine, the amide carbonyl groups, and the backbone nitrogen atom of the amide groups were examined, as well as several protonation/deprotonation states of each structure. EPR and CD titration experiments suggest that the octarepeat segments of the unstructured N-terminal domain of prion protein can bind Cu(II) in a 1:1 Cu-to-octarepeat ratio. The results identify the extent to which the Cu(II) facilitates peptide backbone deprotonation, and the propensity of binding in the forward (toward the C-terminus) direction from the anchoring histidine residue. A plausible mechanism is suggested for changing from amide O-atom to deprotonated amide N-atom coordination, and for assembly of the observed species in solutions of Cu[PrP] and truncated models of it. A structure is proposed which has the N2O2 coordination pattern for the minor component observed experimentally by EPR spectroscopy for the Cu[HGGG] model. The most stable neutral Cu[HGGG] structure found, with coordination environment N3O1, corresponds to that observed for Cu[HGGGW] and Cu[HGGG] both in the solid state and as the major component in solution at neutral pH.     

H-7 reduces the nuclear binding of [3H]dexamethasone in rat liver slices but does not affect the phosphorylation of glucocorticoid receptor.

The effect of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), an inhibitor of protein kinase C, on the nuclear binding of [3H]dexamethasone and on the phosphorylation of glucocorticoid receptor was studied in rat liver slices to ascertain the role of protein kinase C in the expression of glucocorticoid action. H-7 reduces the nuclear binding of [3H]dexamethasone in rat liver slices. It does not affect the extent of phosphorylation of glucocorticoid receptor both in the absence or in the presence of glucocorticoid. These findings indicate that protein kinase C may be involved in the nuclear binding of glucocorticoid receptor but does not directly influence the receptor phosphorylation.     

Determination of N-7-[2H3]methyl guanine in rat urine by gas chromatography-mass spectrometry following administration of trideuteromethylating agents or precursors

A gas chromatographic-mass spectrometric method has been developed for the determination of N-7-[2H3]methyl guanine in urine in the presence of large natural levels of N-7-methyl guanine. Urine is fractionated on heptanesulfonic acid-treated C-18 Sep-pak cartridges, followed by derivatization to give a volatile N-heptafluorobutyryl-O6-2,3,4,5, 6-pentafluorobenzyl derivative which is separated on an SE52 fused silica capillary column. Using N-7-ethyl guanine as an internal standard, the total amount of N-7-methyl guanine is determined by gas chromatography-flame ionization detection. The percentage of N-7-[2H3]methyl guanine is then measured by gas chromatography-mass spectrometry, enabling the amount of deuterated base to be determined. Preliminary experiments with [2H3]methyl methanesulfonate in rats showed measurable excretion of N-7-[2H3]methyl guanine. 4-(Di[2H3]methylamino)antipyrine alone gave no detectable amount of alkylated base, but coadministration of nitrite resulted in excretion of deuterated N-7-methyl guanine.     

Mechanism of benzo[a]pyrene-induced Cyp1a-1 gene expression in mouse Hepa 1c1c7 cells: role of the nuclear 6 s and 4 s proteins.

Treatment of wild-type (wt) aryl hydrocarbon (Ah)-responsive mouse Hepa 1c1c7 cells with benzo[a]pyrene (B[a]P) caused a concentration-dependent induction of ethoxyresorufin O-deethylase (EROD) activity. In contrast, B[a]P was inactive as an inducer in Ah nonresponsive class 1 and class 2 mutant cell lines. In parallel experiments, the nuclear fractions from wt cells treated with 10(-7) M [3H]B[a]P contained both the 4 s carcinogen binding protein and the 6 s (Ah receptor) complexes, whereas only the 4 s complex was present in the nuclear fraction of the class 2 mutant cells. The results obtained from cotreatment of wt Hepa 1c1c7 cells with 10(-6) or 10(-7) M B[a]P and 5 x 10(-7) or 10(-7) M 6-methyl-1,3,8-trichlorodibenzofuran (MCDF) showed that MCDF inhibited the induction of EROD activity and Cyp1a-1 mRNA levels by B[a]P. Moreover, using 10(-7) M [3H]B[a]P and unlabeled MCDF, it was shown that MCDF not only inhibited the induction response but also caused a concentration-dependent decrease in levels of the nuclear 6 s complex but not the 4 s complex. In contrast, in situ competition studies with unlabeled 10(-6) M benzo[ghi]-perylene (B[ghi]P) resulted in the elimination of the nuclear [3H]B[a]P 4 s complex (but not the 6 s complex); however, the EROD activity and Cyp1a-1 mRNA levels in cells treated with 10(-7) M B[a]P in the presence or absence of 10(-6) M B[ghi]P were not significantly different. These results indicate that the 4 s binding protein is not required for the induction of Cyp1a-1 gene expression in Hepa 1c1c7 cells and suggest that B[a]P and 2,3,7,8-tetrachlorodibenzo-p-dioxin induce Cyp1a-1 gene expression via a common mechanism which involves binding to the Ah receptor.     

Photoaffinity labeling of the nuclear Ah receptor from mouse Hepa 1c1c7 cells using 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin

The photoinduced formation of the covalently labeled cytosolic and nuclear aryl hydrocarbon (Ah) receptors was studied using 2,3,7,8-[3H]tetrachlorodibenzo-p-dioxin (TCDD) as the photoaffinity label. Irradiation of TCDD alone at wavelengths of greater than 300 nm resulted in rapid degradation of this compound (t 1/2 = 8 min). In a separate experiment, the unliganded cytosolic Ah receptor was only slowly inactivated (t 1/2 = 48 min) using the greater than 300 nm light source. Preliminary experiments with rat hepatic cytosol did not result in significant formation of specifically bound [3H]TCDD-protein covalent adducts which could be visualized by autoradiography. Irradiation of [3H]TCDD-nuclear Ah receptor complexes isolated from mouse Hepa 1c1c7 cells for 15 min gave approximately a 40% overall yield of the radiolabeled Ah receptor protein adduct. Denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the [3H]TCDD-nuclear Ah receptor photoadduct gave a single major radiolabeled protein with an apparent molecular size of 91 kDa. The chromatographic properties of the control (dark) and photolabeled nuclear Ah receptor complexes were comparable using Sephacryl S-300 and DNA-Sepharose columns. Velocity sedimentation of both the control (dark) and irradiated nuclear Ah receptor complexes gave specifically bound peaks which sedimented at 6.5 S. However, the trichloroacetic acid-precipitable (buffer-reconstituted) [3H]TCDD-nuclear Ah receptor photo-covalent adduct was eluted from the Sephacryl S-300 column in the void volume and did not exhibit a specifically bound peak after velocity sedimentation analysis due to protein aggregate formation. In contrast, the elution profile of the aggregate on a DNA-Sepharose column was similar to that observed for the control (dark) and photolabeled complexes, which were eluted from the column with salt concentrations between 0.24 and 0.28 M. These photolabeling studies show that [3H] TCDD can act as a photoaffinity label for the Ah receptor and can be utilized as photoligand to probe further the structure and function of this protein.     

Cations decrease specific [3H]-spiroperidol binding in human prefrontal cortex

Ligand binding at many physiologically relevant receptors is regulated by divalent cations. To determine whether [3H]-spiroperidol binding sites in prefrontal cortex might be physiologically relevant receptors, we examined the effect of ions on the binding of this ligand in postmortem human prefrontal cortex. Our results indicate that several cations decreased [3H]-spiroperidol binding in a dose-dependent fashion. Of these, Cd++ and Zn++ were the most able to decrease [3H]-spiroperidol binding with IC50 of 5.5 +/- 2.4 X 10(-6)M and 5.6 +/- 1.1 X 10(-5)M respectively. These findings indicate that [3H]-spiroperidol may bind at physiologically relevant receptors in human prefrontal cortex.     

Divalent cation competition with [3H]saxitoxin binding to tetrodotoxin- resistant and -sensitive sodium channels. A two-site structural model of ion/toxin interaction

Monovalent and divalent cations competitively displace tetrodotoxin and saxitoxin (STX) from their binding sites on nerve and skeletal muscle Na channels. Recent studies of cloned cardiac (toxin-resistant) and brain (toxin-sensitive) Na channels suggest important structural differences in their toxin and divalent cation binding sites. We used a partially purified preparation of sheep cardiac Na channels to compare monovalent and divalent cation competition and pH dependence of binding of [3H]STX between these toxin-resistant channels and toxin-sensitive channels in membranes prepared from rat brain. The effects of several chemical modifiers of amino acid groups were also compared. Toxin competition curves for Na+ in heart and Cd2+ in brain yielded similar KD values to measurements of equilibrium binding curves. The monovalent cation sequence for effectiveness of [3H]STX competition is the same for cardiac and brain Na channels, with similar KI values for each ion and slopes of -1. The effectiveness sequence corresponds to unhydrated ion radii. For seven divalent cations tested (Ca2+, Mg2+, Mn2+, Co2+, Ni2+, Cd2+, and Zn2+) the sequence for [3H]STX competition was also similar. However, whereas all ions displaced [3H]STX from cardiac Na channels at lower concentrations, Cd2+ and Zn2+ did so at much lower concentrations. In addition, and by way of explication, the divalent ion competition curves for both brain and cardiac channels (except for Cd2+ and Zn2+ in heart and Zn2+ in brain) had slopes of less than -1, consistent with more than one interaction site. Two-site curves had statistically better fits than one-site curves. The derived values of KI for the higher affinity sites were similar between the channel types, but the lower affinity KI's were larger for heart. On the other hand, the slopes of competition curves for Cd2+ and Zn2+ were close to - 1, as if the cardiac Na channel had one dominant site of interaction or more than one site with similar values for KI. pH titration of [3H]STX binding to cardiac channels showed a pKa of 5.5 and a slope of 0.6-0.9, compared with a pKa of 5.1 and slope of 1 for brain channels. Tetramethyloxonium (TMO) treatment abolished [3H]STX binding to cardiac and brain channels and STX protected channels, but the TMO effect was less dramatic for cardiac channels. Trinitrobenzene sulfonate preferentially abolished [3H]STX binding to brain channels by action at an STX protected site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Polypeptide fold in the two metal clusters of metallothionein-2 by nuclear magnetic resonance in solution

The solution conformation of rabbit liver Cd27+-metallothionein-2 was determined by nuclear magnetic resonance (n.m.r.) and distance geometry. The n.m.r. data are based on complete sequence-specific resonance assignments for the polypeptide chain. This letter describes the global arrangement of the polypeptide chain, which forms two distinct domains containing metal clusters of three and four Cd ions, respectively.     

(+)-3-[2-(Benzo[b]thiophen-2-yl)-2-oxoethyl]-1-azabicyclo[2.2.2]octane as potent agonists for the alpha7 nicotinic acetylcholine receptor

A series of 3-substituted 1-azabicyclo[2.2.2]octanes was discovered as the alpha7 nicotinic acetylcholine (alpha7) receptor agonists. It was found that (+)-3-[2-(benzo[b]thiophen-2-yl)-2-oxoethyl]-1-azabicyclo[2.2.2]octane (+)-15b has potent agonistic activity for the alpha7 receptor.     

Synthesis of 1 alpha-hydroxy[7-3H]cholecalciferol and its metabolism in the chick.

1. 1 alpha-Hydroxy[7-3H]cholecalciferol (specific radioactivity of 2-Ci/mmol) was synthesized, and its metabolism in chicks studied. 2. 1 alpha-Hydroxy[7-3H]cholecalciferol was metabolized very rapidly in the chick to 1 alpha,25-dihydroxy[7-3H]cholecalciferol and to a metabolite less polar than 1 alpha-hydroxycholecalciferol. Intestine exhibited highest accumulation of 1 alpha-25-dihydroxy[7-3H]cholecalciferol, and liver exhibited highest accumulation of the non-polar metabolite. 3. Tissue uptake of 1 alpha-hydroxy[7-3H]cholecalciferol and its metabolites in chicks that were dosed continuously for 16 days with 1 alpha-hydroxy[7-3H]cholecalciferol did not exceed by very much that observed in tissues obtained from chicks that were dosed with a single injection of 1 alpha-hydroxy[7-3H]cholecalciferol 24 h before killing, except for liver and kidney. 4. Lowest accumulation of metabolites was noted in muscle and bone, and for the latter, highest uptake of 1 alpha,25-dihydroxy[7-3H]cholecalciferol was noted in the epiphysial periosteum and the metaphysis. 5. Formation of 1 alpha,24,25-trihydroxy[7-3H]cholecalciferol was not observed in the chicks that were dosed continuously with 1 alpha-hydroxy[7-3H]cholecalciferol, despite the fact that plasma calcium and phosphorus were normal and despite the presence of renal 24-hydroxylase activity. 6. The vitamin D status of the chicks did not appear to affect the metabolic profile of the administered 1 alpha-hydroxy[7-3H]cholecalciferol.