X-ray diffraction analysis of crystals of bovine platelet factor 4

Bovine platelet factor 4 has been crystallized by “vapor dilution” in space group P2₁2₁2₁, $\text{a = 63.7}\text{A}\text{?}\text{, b = 66.7}\text{A}\text{?}\text{, c = 80.5}\text{A}\text{?}$, with four molecules, each 9505 M_r, in the asymmetric unit. The crystals diffract X-rays to better than 2.8 Å resolution.     

Shape,conformation and stability of fibronectin fragments determined by electron microscopy,circular dichroism and ultracentrifugation

Human plasma fibronectin digested with cathepsin D yields a number of fragments: an N-terminal fibrin-binding 30K³ fragment, a gelatin-binding 40K fragment, a 70K fragment containing the 30K and 40K domains, a central 95/105K fragment and a heparin-binding 140K fragment. The 140K fragment is linked by disulphide bonds and forms the C-terminal ends of the fibronectin. Electron microscopy of rotary-shadowed specimens revealed the 40K, 70K and 95/105K fragments as thin rods (diameter about 2.2 nm) with lengths of 13, 25 and 25 nm. respectively. These dimensions agree with the distances between flexible, proteasesusceptible regions in individual fibronectin strands determined previously. The 140K fragment appeared as a V-shaped structure with two arms 21.5 nm long emerging at a preferred angle of about 70 °. The distribution function of this angle was identical to that observed for the angle between the two arms of intact fibronectin. The free energy required for a distortion of this angle by 60 ° was estimated to be 8 kJ/mol. Reduced and alkylated fibronectin was visualized as single strands (about 55 nm long) with kinks of variable angles corresponding to sites of increased flexibility. The hydrodynamic shapes of the fragments in solution were similar to the shapes observed by electron microscopy, indicating that the individual domains of fibronectin maintain their native structure after proteolytic separation. This was also demonstrated by circular dichroism (c.d.) studies. The c.d. spectrum and the thermal melting curve of native fibronectin were well represented by a linear combination of the contributions of the fragments, indicating conformational independence of the domains. The data support earlier findings that fibronectin consists of two thin strands (length about 60 nm), which are composed of several domains separated by flexible and protease-susceptible intervening regions. This very extended structure agrees with the small sedimentation constant found for fibronectin under conditions where electrostatic interactions between domains are repulsive or depressed. Probably because of such interactions the sedimentation constant is larger at neutral pH and low ionic strength, but even under these conditions a very asymmetric shape is still maintained.     

Transition-metal(II) thiocyanate coordination compounds containing 4-allyl-1,2,4-triazole. Structure and magnetic properties.

The synthesis and characterisation of a series of dinuclear and polynuclear coordination compounds with 4-allyl-1,2,4-triazole are described. Dinuclear compounds were obtained for Mn(II) and Fe(II) with composition [M₂(Altrz)₅(NCS)₄], and for Co(II) and Ni(II) with composition [M₂(Altrz)₄(H₂O)(NCS)₄](H₂O)₂. The crystal structure of [Co₂(Altrz)₄(H₂O)(NCS)₄](H₂O)₂ was solved at room temperature. It crystallizes in the monoclinic space group P2₁/n. The lattice constants are a = 18.033(3) Å, b = 13.611(2) Å, c = 15.619(3) Å, β = 92.04(2)° Z = 4. One cobalt ion has an octahedrally arranged donor set of ligands consisting of three vicinal nitrogens of 1,2-bridging triazoles (CoN = 2.14–2.15 Å), one terminal triazole nitrogen (CoN = 2.12 Å) and two N-bonded NCS anions (CON = 2.08 Å). The other Co(II) ion has the same geometry, but the terminal triazole ligand is replaced by H₂O (CoO = 2.15 Å). The crystal structure is stabilised by hydrogen bonding through H₂O molecules, S-atoms of the NCS anions and the lone-pair electron of the monodentate triazole. The magnetic exchange in the Mn, Co and Ni compounds is antiferromagnetic with J-values of ?0.4 cm^?1, ?10.9 cm^?1 and ?8.7 cm^?1 respectively. The Co compound was interpreted in terms of an Ising model. For [Zn₂(Altrz)₅(NCS)₂]∞[Zn(NCS)₄], [Cu₂(Altrz)₃(NCS)₄]∞ and [Cd₂(Altrz)₃(NCS)₄]∞ chain structures are proposed. In the Cu compound thiocyanates appear to be present, bridging via the nitrogen atom, as deduced from the IR spectrum.     

Structure of the cytochrome c oxidase dimer electron microscopy of two-dimensional crystals

We have studied the structure of beef heart mitochondrial cytochrome c oxidase dimers by image-processing of electron micrographs of the vesicle crystal form. Specimens were prepared by different procedures, which contrast different features of the crystals. Heavy-atom shadowing of freeze-dried crystals contrasts the exterior or M-side surface (mitochondrial matrix-side) and reveals a 100 Å long ellipsoidal dimer oriented with its long axis in the (?1, 1) direction of the 95 Å × 125 Å rectangular unit cell. The M-side surface structure correlates well with the intra-bilayer structure revealed by contrast matching extra-bilayer protein with glucose. Frozen suspensions of vesicle crystals fracture predominantly along hydrophilic surfaces revealing the interior C-side (mitochondrial cytoplasm-facing surface) of vesicle crystals. The C-side surface revealed in shadowed replicas of fracture surfaces shows the ends of the dimers furthest from the bilayer surface; they consist of two structural domains separated by 70 to 80 Å. We present a new interpretation of the structure of the cytochrome oxidase dimer based on these data and on the y-shaped monomer structure described by Fuller et al. (1979). A cytochrome oxidase dimer is formed from two y-shaped monomers joined along one set of identical M-domain arms with the other arms approximately 70 Å apart along a unit cell diagonal in the (?1, 1) direction. The arms of the monomers lie within and perpendicular to the phospholipid bilayer, and they protrude approximately 25 Å beyond the bilayer surface on the M-side. The y tails represent the C-side domains, which are closely apposed across the dimer 2-fold axis near the C-side bilayer surface. Further away from the bilayer surface, C-side domains split away from one another forming a large cleft.     

Metal—phenoxyalkanoic acid interactions. Part 14. The crystal and molecular structures of diaquabis(4-fluorophenoxyacetato)copper(II) bis(4-fluorophenoxyacetic acid)dihydrate and tetra-μ-(4-fluorophenoxyacetato-0,0′)-bis[(2-aminopyrimidine)copper(II)]

The crystal structures of two copper(II) complexes of 4-fluorophenoxyacetic acid (4-FPAH) have been determined by X-ray diffraction. [Cu(4-FPA)₂(H₂O)₂]·2(4-FPAH)·2H₂O (1) is triclinic, space group P1 with Z = 1 in a cell of dimensions a = 14.808(2), b = 9.832(2), c = 6.847(2) Å, α = 87.77(2), β = 98.41(2), γ = 112.33(2)° and was refined to a residual of 0.038 for 1697 ‘observed’ reflections. The coordination sphere in this complex is tetragonally distorted octahedral comprising two waters [CuO, 1.940(3) Å], two unidentate carboxylate oxygens [CuO, 1.942(2) Å] and two ether oxygens [CuO, 2.471(2) Å]. Two adducted [4-FPAH] acid molecules are linked to the un-coordinated oxygens of the acid ligands by hydrogen bonds [2.547(4) Å]. [Cu₂(4-FPA)₄(2-aminopyrimidine)₂] (2) is triclinic, space group P1 with Z = 1 in a cell of dimensions a = 12.688(2), b = 11.422(2), c = 7.951(1) Å, α = 78.74(1), β = 107.51(1), γ = 75.78(1)°, and was refined to a residual of 0.042 for 2683 ‘observed’ reflections. (2) is a centrosymmetric tetracarboxylate bridged dimer with four similar CuO (equatorial) distances [1.967–1.987 Å; 1.977(3) Å mean] and the axial position occupied by the hetero nitrogen of the 2-aminopyrimidine ligand [CuN, 2.176(3) Å]. The Cu---Cu separation is 2.710(1) Å. Crystal data are also presented which confirm the isostructurality of complex (2) with [Cu₂(phenoxyacetate)₄(2-aminopyrimidine)₂], the Co^II, Mg^II and Mn^II4-fluorophenoxyacetate complexes with their phenoxyacetic and 4-chlorophenoxyacetic acid analogues, and of Cd^II4-fluorophenoxyacetate with Cd^II and Zn^II phenoxyacetates.     

A general approach to the synthesis of neutral and cationic binuclear trithiocarbonato-bridged complexes of palladium(II) or of palladium(II)—platinum(II)

Addition (1:2) of Tl₂CS₃ to solutions of perchloratocomplexes of palladium(II) Pd(OClO₃)(C₆F₅)(PR₃) leads to neutral binuclear derivatives of the type (PR₃)(C₆F₅)Pd(μ-S₂CS)Pd(C₆F₅)(PR₃)₂, whilst the reaction of perchloratocomplexes of palladium(II) or platinum(II) with the neutral Pd(η²-CS₃)(PR₃)₂ affords cationic complexes of the type [L₂Pd(μ-S₂CS)M(C₆F₅)L₂]ClO₄ (M = Pd or Pt). Spectral data (IR and ³¹P, NMR) permit the inequivocal structural characterization of both the neutral and the cationic complexes.     

Binding of metal ions to polysaccharides. VI. Spectroscopic and potentiometric studies of the binding of copper(II) and nickel(II) to some monosaccharide units in acidic,neutral and alkaline aqueous media

Binding of Cu²⁺ and Ni²⁺ to glucosamine, N-acetyl- glucosamine and other derivatives of glucose was investigated in acidic, neutral and alkaline aqueous media using H⁺ and Cu²⁺ potentiometry and ligand- field and ESR spectroscopy. In neutral medium, site binding with copper(II) and nickel(II) occurs when the monosaccharide possesses a potentially coordinating amine or charged group not attached to C-1. At high pH, a coordination entity is only formed if the C-1 hydroxyl group can be deprotonated and other stabilizing groups are present. The role of groups attached to C-1 reflects the different behaviour of monosaccharides compared with polysaccharides.     

Lysine to 13C-labeled homoarginine conversion in microbial cytochromes c. Electron transport rates and 13C nuclear magnetic resonance spectroscopy

Seven microbial and one mammalian species of cytochrome c have been reacted with O-methylisourea to convert lysine residues to homoarginines containing enriched ¹³C. This set of guanidinated cytochromes has been assayed for electron transport reactivity and the nuclear magnetic resonance spectra of incorporated label have been obtained. The set consisted of c-type cytochromes from horse, Saccharomyces cerevisiae, Candida krusei, Paracoccus denitrificans, Pseudomonas aeruginosa, Rhodospirillum rubrum, Rhodopseudomonas capsulata, and Rhodopseudomanas spheroides. All derivatives demonstrated high electron transport reactivity with cytochrome oxidases; at some concentrations this rate was 100% or higher compared to corresponding native rates. All labeled ferricytochrome spectra followed a common pattern giving about five resolved or partially resolved resonance peaks. Two of these, at approximately 158.1 and 157.3 parts per million, correspond to single carbon sites. They have been assigned to labeled lysine 27 and lysine 79 (horse numbering), respectively, on the basis of sequence comparisons and an approximate chemical shift calculation. Labeled ferrocytochrome spectra were obtained and shown to be more diverse than the set of ferric spectra. Poly-[¹³C]homoarginine was prepared and shown to be an inhibitor of the horse cytochrome c-cytochrome oxidase reaction but an activator for the reactions of Paracoccus cytochrome c550. Relaxation measurements indicated that polyhomoarginine forms a complex with both cytochromes c.     

Synthesis and x-ray structure of a Hg(II) complex with adenine N(1)-oxide. A model for mercury binding to DNA

The synthesis and crystal structure of the adenine N(1)-oxide complex with mercury(II) chloride, (C₅H₅N₅O)HgCl₂ are reported. Crystals of the coordination compound belong to the monoclinic system, space group P2₁/n with the following primary crystallographic data: a = 6.685(1) Å, b = 11.798(2) Å, c = 10.155(1) Å, β = 100.22(1)°, V = 906.04 ų, Z = 4. The structure was elucidated by conventional Patterson and Fourier methods and refined by the full matrix least-squares technique on the basis of 1977 observed reflections to an R value of 0.074. The basic unit of the structure is a dimer, with a centre of symmetry, consisting of two HgCl₂ moieties and two adenine N(1)-oxide ligands. A polymeric structure results from the bridging interactions of chloride ions. Adenine N(1)-oxide acts as a bidentate bridging ligand, coordinating through N(7) and O(1). The coordination geometry around the mercury ion is a distorted square pyramid with N(7) and three chlorines (two of which are centro-symmetrically related) forming the square plane and O(1) occupying the axial position. Hg also interacts indirectly with N(6) through a Cl

HN hydrogen bond. Principal intracomplex geometrical parameters are as follows: HgN(7) = 2.61(1) Å, HgO(1) = 2.55(1) Å, HgCl(1) = 2.330(3) Å, HgCl(2) = 2.318(3) Å, HgCl(2′) = 3.347(3) Å. The cis angles range from 77.5° to 107.9° and the two trans angles are 155.5° and 163.1°. The centro-symmetrically related bases overlap partially and pack at a distance of 3.2 Å. The glide-related bases are linked by a hydrogen bond, N(9)H

O(1) and are inclined to one another by 109.7°. The results are compared with those derived from spectroscopic and other physicochemical studies on metal interaction with adenine N(1)-oxide. Based on the present structural observations and earlier experimental results a possible mechanism is proposed for mercury interaction with DNA.     

Five-coordinated metal complexes of bis(2-hydroxy-1-naphthylideneimine-3-propyl)amine and their reactivity towards dioxygen. Part I. An electrochemical investigation on manganese(II), iron(II), cobalt(II), nickel(II) and copper(II) complexes

The electrochemical behaviour in aprotic solvent of the complexes {M[bis-(2-hydroxy-l-naphthylideneimine-3-propyl)amine]}, where M = Mn(II), Co(II), Fe(II), Ni(II) and Cu(II) is reported. The complexes were prepared and characterized by elemental analysis, infrared and visible spectroscopy and magnetic susceptibility measurements. In addition the reactivity towards dioxygen of the Mn(II), Fe(II) and Co(II) derivatives was investigated, mainly by cyclic voltammetry and gas-volumetric uptake measurements. The results indicate that the Co(II) complexes are able to add dioxygen reversibly, while Mn(II) and Fe(II) compounds undergo an irreversible oxygenation process. The pathway of the dioxygenation processes is tentatively interpreted on the basis of the electrochemical responses. The results confirm that the location of the oxidation potential allows one to predict whether a compound is able to react with dioxygen, but it is not sufficient to predict whether the dioxygenation reaction proceeds reversibly.     

Chromium(III) complexes and their relationship to the glucose tolerance factor. Part II. Structure and biological activity of amino acid complexes

A number of octahedral chromium complexes with amino acids are ligands have been prepared and their structures assigned on the basis of their chromatographic and spectral properties. These include complexes with the general structure Cr(AA)₂(H₂O)₂ where the amino acids glycine, glutamic acid and glutamine act as bidentate ligands. The analogous compound with cysteine as ligand is stable at low pH, but at high pH a terdentate cysteine complex, Cr(cysteine)₂^?, is formed. These complexes, as well as a solution of monodentate glycine aquo complexes, and Cr-nicotinic acid-glycine and Cr-nicotinic acid-cysteine complexes of undetermined structure, have been assayed for glucose tolerance factor activity using a yeast assay. Only Cr(glutamine)₂- (H₂O)₂⁺, Cr-nicotinic acid-glycine and the mixture of complexes Cr(glycine)_n(H₂O)_6-n⁺³ showed significant activity. It is proposed that a trans arrangement of the non-coordinated nitrogen atoms in the ligands of these complexes can mimic the structural features of the glucose tolerance factor which are essential for biological activity.     

Complexes of dibenzoyldisulphide with metal halides. Part III. Complexes of dibenzoyldisulphide with cobalt(II), nickel(II), manganese(II), copper(II), iron(III) and chromium(III) halides

Adducts (1:1) of halides of cobalt(II), nickel(II), manganese(II), copper(II), iron(III) and chromium(III) with dibenzoyldisulphide have been isolated and characterized on the basis of elemental analysis, molar conductance, magnetic susceptibility, infrared spectra, molecular weight and thermogravimetric analysis data.     

Cell-mediated immune response to syngeneic ultraviolet-induced tumors. II. The properties and antigenic specificities of cytotoxic T lymphocytes generated in vitro following removal from syngeneic tumor-immunized mice.

The immune responses of C3Hf mice to syngeneic fibrosarcomas induced with either ultraviolet light or methychlolanthrene (MCA) were measured in vitro by the ability of cytotoxic lymphocytes (CTL) from immunized animals to kill ⁵¹Cr-labeled tumor targets in a 6-hr assay. The CTL were generated by the in vitro culturing of draining popliteal lymph node (DLN) cells derived from animals that were footpad immunized 8 days previously. It was determined that CTL activity could be generated using DLN from both normal (uv tumorresistant) and uv-exposed (uv tumor-susceptible) C3H mice. The kinetics of CTL generation between these two groups, however, was different in that the lymphocytes from normal animals appeared to differentiate into CTL faster than the lymphocytes from the uv-irradiated mice. The in vitro generation of CTL activity was found to be extremely radiosensitive and was also inhibited by the presence of viable tumor cells within the cell culture. Once generated, it was observed that the CTL were extremely insensitive to the effects of gamma irradiation. It was also established that the CTL is a T lymphocyte that appears to be Ia^?. The CTL derived from mice immunized to syngeneic uv- or MCA-induced tumors were capable of expressing cross-reactive non-MHC-restricted killing of multiple tumor targets. Cold cell inhibition experiments confirmed the presence of cross-reactive determinants on various tumors and also established the presence within a single CTL preparation of effector cells with specificity for both the unique tumor specific transplantation antigens as well as the common (cross-reactive) tumor-associated antigens.     

Copper(II) halide complexes of 2-aminobenzophenone. Crystal and molecular structure of bis(2-aminobenzophenone)dichlorocopper(II)

The complexes CuX₂L₂ (X = Cl, Br; L = 2-aminobenzophenone) were prepared and characterized by means of magnetic and spectroscopic measurements. For the Cl compound the crystal structure was also determined. Crystals are triclinic, space group P$\text{1}$, with a = 13.397(3), b = 10.752(2), c = 9.205(2) Å, α = 72.26(1)°, β = 91.58(1)°, γ = 106.86(1)°, and Z = 2. The structure was solved by the heavy-atom method and refined by least-squares calculations to R = 0.034 for 2581 counter data. It consists of discrete CuX₂L₂ monomers showing distorted trigonal bipyramidal coordination geometry about the copper ion. The amino nitrogens are axial ligands, with the equatorial positions occupied by two chlorine atoms and a carbonyl oxygen from one L molecule acting as a bidentate ligand. Infrared and ligand field spectroscopies and magnetic measurements, interpreted on the basis of the known crystal structure, also suggest a similar structure for the related Br compound.     

Biologically important coordination compounds of 4-acetyl-2-(acetylamino)-5-dimethyl-δ2-1,3,4-thiadiazole with manganese(II), iron(II), cobalt(II) and nickel(II)

The complexes of 4-Acetyl-2-(acetylamino)-5- dimethyl-δ²-1,3,4-thiadiazole (AAT) with Mn(II), Fe(II), Co(Il) and Ni(II) have been prepared and characterized on the basis of elemental analyses, molar conductance, magnetic moments, electronic and infrared spectral studies. The most probable structures of the complexes have been proposed on the basis of their physicochemical properties. The fungitoxicity of AAT and its complexes has been evaluated on pathogenic fungi.     

Detection of aldolase activity on polyacrylamide gels: application to 2-keto-4-hydroxyglutarate aldolase.

A method is described for the detection of 2-keto-4-hydroxyglutarate aldolase activity after electrophoresis of the enzyme on polyacrylamide gels. When gels are incubated with substrate (2-keto-4-hydroxyglutarate), activity is seen as a yellow-colored band due to interaction of the product )glyoxylate) with ortho-aminobenzaldehyde and glycine. Positive results have been obtained using either crude cell-free preparations or homogeneous enzyme from Escherichia coli as well as with highly purified samples of aldolase from bovine liver or kidney extracts. The method is potentially applicable to other aldolases that liberate an aliphatic aldehyde as a product; modifications and limitations of the procedure for detecting fructose 1,6-diphosphate aldolase, 2-keto-3-deoxy-6-phosphogluconate aldolase, and 2-deoxyribose-5-phosphate aldolase activities have been explored.     

Ligand and carbon monoxide affinities of iron(II) ‘C4-capped’ porphyrins

Ligand and CO binding constants are reported for Fe(C₄-Cap) [1]. The results show that the CO affinities are Fe(C₂-Cap)(B) ?Fe(C₃-Cap)(B) > Fe(C₄-Cap)(B). Also the CO affinities are Fe(Cap)(1,5-DCIm) > Fe(Cap) (1,2-Me₂Im). These results are explained in terms of the possible steric factors involved.     

Cardiac tropomyosin crystals and their interactions with troponin subunits

Crystals and paracrystals of bovine cardiac tropomyosin and their mixtures with different combinations of troponin subunits were examined in the electron microscope after negative staining. Although the cardiac proteins gave most of the same crystalline and paracrystalline patterns as observed previously with skeletal muscle tropomyosin and troponin, two important differences were noted. Cardiac troponin T was incapable of forming hexagonal networks with either skeletal or cardiac muscle tropomyosins, while skeletal troponin T readily associated in this manner with tropomyosins from either tissue source. Also the characteristic paracrystalline pattern seen with skeletal muscle tropomyosin, troponin T and troponin C only in the presence of calcium was consistently obtained with mixtures of the corresponding cardiac components when calcium was absent.