Structure of Cu2(indomethacin)4 and the reaction with superoxide in aprotic systems

The copper complex of indomethacin (1-(p-chlorobenzoyl)-5-methoxy-2-methyl-indole acetate), a common anti-inflammatory drug, was prepared and characterized. Crystal structure determination revealed the dimeric form of the 1:2 complex, namely Cu₂(indomethacin)₄ · L₂, in the unit cell. Suprisingly, the copper-copper distance (263 pm) was very close to metallic copper (256 pm). The two coordination sites in the copper-copper axis can be readily replaced by superoxide. An intriguing similarity to Cu₂(acetate)₄ was seen.Due to the lipophilic nature of the indomethacin ligand, this copper complex reacted with superoxide in aprotic solvents. The superoxide dismutating activity was successfully demonstrated in Me₂SO/water and acetonitrile/water mixtures using the nitro-blue tetrazolium assay and pulse radiolysis. The second-order rate constant of 6 · 10⁹ M^?1 · s^?1 in strictly aqueous systems dropped only slightly to 1.1 · 10⁹ M^?1 · s^?1 when aprotic solvents were used. This is the fastest rate constant ever observed for a copper-dependent dismutation of superoxide. The KO₂-induced lipid peroxidation in both erythrocytes and liver microsomes was suppressed by 70% in the presence of 1 · 10^?10 mol · ml^?1 of Cu₂(indomethacin)₄. The inhibitory action dropped to 25% when Cu₂Zn₂superoxide dismutase was employed. The formation of copper · indomethacin in rat serum after administration of indomethacin was shown in vitro and in vivo.     

The binding of pentaammineruthenium (III) to RNase B and RNase A+d(pA)4 in the crystalline state

The binding of pentaammineruthenium (III) to ribonuclease A and B both free and complexed with d(pA)₄ has been examined in the crystalline state through the application of X-ray diffraction and difference Fourier techniques. In crystals of native RNase B, the reagent was observed to have many binding sites, some entirely electrostatic in nature and others consistent with coordination to histidine residues. The primary histidine in the latter case was 105 with 119 also partially substituted. In crystals of RNase A+d(pA)₄ complex only a single, extremely strong site of substitution was observed, and this was 2.4 Å from the native position of the imidazole ring of histidine 105. Thus, the results of these X-ray diffraction studies appear to be quite consistent with the findings of earlier NMR studies and with the results obtained in crystals of the gene 5 DNA binding protein.     

X-ray absorption spectroscopy of selenium-containing amino acids

Received: 2 April 1999 / Accepted: 17 September 1999     

Synthesis of alkali metal hexahydroaluminate complexes using dimethyl ether as a reaction medium

A novel method of preparation of hexahydroaluminate complexes M₃AlH₆ (M = Li, Na or K) from the corresponding alkali metal hydride and tetrahydroaluminate has been explored, using dimethyl ether (Me₂O) as a solvent at near-ambient temperatures. The results are compared with those obtained using a recently established mechanochemical approach. Characterization of the products by powder X-ray diffraction revealed M₃AlH₆ to be formed in high yield for M = Li and Na, but not for M = K. The attempted preparation of Li₂NaAlH₆ and Li₂KAlH₆ was unsuccessful.     

The Cap-Snatching SFTSV Endonuclease Domain Is an Antiviral Target

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Production, crystallization, and preliminary X-ray analysis of rabbit skeletal muscle troponin complex consisting of troponin C and fragment (1-47) of troponin I.

Troponin is a ternary protein complex consisting of subunits TnC. TnI, and TnT, and plays a key role in calcium regulation of the skeletal and cardiac muscle contraction. In the present study, a partial complex (CI47) was prepared from Escherichia coli-expressed rabbit skeletal muscle TnC and fragment 1-47 of TnI, which is obtained by chemical cleavage of an E. coli-expressed mutant of rabbit skeletal muscle TnI. Within the ternary troponin complex, CI47 is thought to form a core that is resistant to proteolytic digestion, and the interaction within CI47 likely maintains the integrity of the troponin complex. Complex CI47 was crystallized in the presence of sodium citrate. The addition of trehalose improved the diffraction pattern of the crystals substantially. The crystal lattice belongs to the space group P3(1)(2)21, with unit cell dimensions a = b = 48.2 A, c = 162 A. The asymmetric unit presumably contains one CI47 complex. Soaking with p-chloromercuribenzenesulfonate (PCMBS) resulted in loss of isomorphism, but enhanced the quality of the crystals. The crystals diffracted up to 2.3 A resolution, with completeness of 91% and R(merge) = 6.4%. The crystals of PCMBS-derivative should be suitable for X-ray studies using the multiple-wavelength anomalous diffraction technique. This is the first step for elucidating the structure of the full troponin complex.     

Structural characterization of KKT4, an unconventional microtubule-binding kinetochore protein

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Chondroitin sulphate at the endothelial lumen of pig aorta: Assay by radiolabelling and by X-ray microanalysis

Trypsin-releasable glycosaminoglycans from the luminal surface of intact pig aorta were measured following metabolic labelling with³⁵S]sulphate. Chondroitin sulphate was found to be present at a surface density equal to that already established for heparan sulphate (5×10¹¹ chains per cm²). This result was confirmed by X-ray microanalysis of the luminal sulphur content before and after treatment with specific glycosaminoglycan-degrading enzymes. This result implies that approximately half of the luminal surface is occupied by sulphated glycosaminoglycans.     

Application of PIXE analysis to the study of the regional distribution of trace elements in normal human brain

A particle-induced X-ray emission (PIXE) analysis method is presented, which allows measurement of eight elements (i.e., K, Ca, Mn, Fe, Cu, Zn, Se, and Rb) in human brain samples of only a few mg dry weight. The precision and accuracy of the method were investigated by analyzing animal brain matter with both PIXE and instrumental neutron activation analysis (INAA). The method was applied to measure the 8 elements in 46 different regions of 3 human brains. The sections analyzed originated from either the left or the right cerebral hemisphere, brain stem, and cerebellum. For one of the brains, sections were also analyzed from 26 corresponding regions of both hemispheres. For all elements, similar concentrations were found in the corresponding areas of the left and right sides of the brain. The concentrations (in μg/g dry weight) of the elements K, Fe, Cu, Zn, Se, and Rb were consistently higher in cortical structures than in white matter. Deep nuclei and brain stem, which have a mixed composition, showed intermediate values for K, Zn, Se, and Rb. A hierarchical cluster analysis indicated that the various brain regions clustered into two large groups, one comprising gray and mixed matter regions and the other, white and mixed matter brain areas.     

Structural and functional domains of cellobiohydrolase I from trichoderma reesei

Limited proteolysis (papain) of the cellobiohydrolase I (CBH I, 65 kDa) from Trichoderma reesei led to the seperation of two functional domains: a core protein (55 kDa) containing the active site, and a C-terminal glycopeptide (10 kDa) implicated in binding to the insoluble matrix (cellulose). The quaternary structures of the intact CBH I and its core in solution are now compared by small angle X-ray scattering (SAXS) measurements. The molecular parameters derived for the core (Rg=2.09 nm, D_max=6.5 nm) and for the intact enzyme (Rg=4.27 nm, D_max=18 nm) indicate very different shapes. The resulting models show a tadpole-like structure for the intact enzyme where the isotropic part coincides with the core protein and the flexible tail part should be identified with the C-terminal glycopeptide. Thus in this enzyme, functional differentiation is reflected in structural peculiarities.Abbreviations SAXS small angle X-ray scattering - SDS-PAGE SDS-polyacrylamide gel electrophoresis - IEF-PAG polyacrylamide gel isoelectric focusing; cellobiohydrolase (CBH, 1,4--glucan cellobio hydrolase (E.C.3.2.1.91)) - D_max maximum diameter - Rg radius of gyration