Characterization of a carbon monoxide complex of reduced dopamine beta-hydroxylase. Evidence for inequivalence of the Cu(I) centers

Ascorbate-reduced dopamine beta-hydroxylase (DBH) is inhibited by CO in a competitive manner with respect to molecular O2. Measurement of the stoichiometry of CO binding indicates 0.50 CO bound per Cu(I), which provides the first evidence that the Cu(I) centers in the reduced enzyme are structurally inequivalent. FTIR spectroscopy has been used to detect an infrared absorption band characteristic of coordinated CO, with v(CO) = 2089 cm-1. Comparison of this frequency with those of other Cu(I)-carbonyls in both inorganic and protein systems suggests a coordination site with fewer or less basic ligands than the 3-histidine site of carbon-monoxy hemocyanin.     

Carbonmonoxy dopamine beta-hydroxylase. Structural characterization by Fourier transform infrared, fluorescence, and x-ray absorption spectroscopy

The carbon monoxide complex of ascorbate-reduced dopamine beta-hydroxylase has been prepared and characterized by Fourier transform infrared, fluorescence, and x-ray absorption spectroscopies. CO has previously been shown to be a competitive inhibitor with respect to O2, and binds to only one of the two copper atoms/active site (Blackburn, N. J., Pettingill, T. M., Seagraves, K. S., and Shigeta, R. T. (1990) J. Biol. Chem. 265, 15383-15386). Thus, it acts as an excellent probe of the O2-binding site. A single C-O infrared absorption band is observed at 2089 cm-1, shifting by 46 cm-1 to lower energy on substitution with either 13C16O or 12C18O. The 13C isotope shift is reversed to the position expected for 12CO upon vacuum flushing with 12CO gas, indicating that formation of the CO adduct is a fully reversible process. Binding of the substrate tyramine does not eliminate the infrared peak but causes a 3-cm-1 shift to lower energy. On the other hand, binding of a bifunctional inhibitor which cross-links the substrate and O2-binding site does eliminate the CO peak. These data, in conjunction with the competitive nature of CO binding with respect to O2, identify the CO-binding site as the O2-binding site, and place it in close proximity to the substrate-binding site. CO-dopamine beta-hydroxylase exhibits no luminescence in the visible region, suggesting a structure different from carbonmonoxy hemocyanin, and in all probability mononuclear. Analysis of extended x-ray absorption spectroscopy data is most consistent with an average coordination per Cu of 2-3 histidines, 0.5 CO, and 0.5 S atoms as ligands, and absorption edge comparisons indicates pseudo-4 coordination as the most likely geometry at each Cu(I) center. The results can be interpreted by a model involving inequivalent 4-coordination at each Cu(I) center in the CO adduct with CuAHis3S...CuBHis2CO-X as the coordination most consistent with all of the data.     

Preparation of soybean seed samples for FT-IR microspectroscopy

Typical preparation of seed samples for infrared (IR) microspectroscopy involves imbibition of the seed for varying time periods followed by cryosectioning. Imbibition, however, may initiate germination even at 4° C with associated changes in the chemistry of the sample. We have found that it is possible to section seeds that are sufficiently hard, such as soybeans, on a standard laboratory microtome without imbibition. The use of dry sectioning of unimbibed seeds is reported here, as well as a comparison of different mounting media and modes of analysis. Glycerol, Tissue-Tek, and ethanol were used as mounting media, and the quality of the resulting spectra was assessed. Ethanol was the preferred mountant, because it dried quickly with no residue and thus did not interfere with the spectrum of interest. Analysis in transmission mode using barium fluoride windows to hold the samples was compared with transmission-reflection analysis with sections mounted on special infrared-reflecting slides. The two modes of analysis performed well in different regions of the spectrum. The mode of analysis (transmission vs. transmission-reflection) should be based on the components of greatest interest in the sample.     

Copper K-extended x-ray absorption fine structure studies of oxidized and reduced dopamine beta-hydroxylase. Confirmation of a sulfur ligand to copper(I) in the reduced enzyme.

The structure of the copper sites in oxidized and reduced dopamine beta-hydroxylase has been studied by extended x-ray absorption fine structure spectroscopy using a restrained refinement approach to data analysis. An histidine-rich active site has been found to be present with an average histidine coordination of between two and three histidine ligands per copper. In the oxidized protein, the data support four-coordination, involving two to three imidazole groups at 1.99 A with additional ligands derived from water or exogenous O-donor groups at an average distance of 1.94 A. Studies on the reduced enzyme have focused on resolving the controversy in the literature (Scott, R. A., Sullivan, R. J., De Wolfe, W. E., Dolle, R. E., and Kruse, L. I. (1988) Biochemistry 27, 5411-5417; Blumberg, W. E., Desai, P. R., Powers, L., Freedman, J. H., and Villafranca, J. J. (1989) J. Biol. Chem. 264, 6029-6032) as to whether a S/Cl scatterer is a ligand to Cu(I). Five independent samples of reduced enzyme prepared under conditions designed to probe the Cu(I) ligand environment have been measured and analyzed. All five samples gave identical spectra and could be simulated by two to three imidazoles (1.93 A) and 0.5 S/Cl (2.25 A) per Cu(I). The spectra were insensitive to the presence of added bromide or to exclusion of chloride during preparation. The results establish that the heavy atom scatterer is derived from a sulfur donor. Some evidence was found for an additional O/N scatterer at 2.6 A in the reduced enzyme. A hypothesis for the structure of the copper sites has been proposed involving inequivalent CuA(His)3(H2O)...CuB-(His)2X(H2O) coordination in the oxidized enzyme, which upon reduction loses coordinated water and coordinates a sulfur probably from a methionine.     

Anti-tumor therapy with macroencapsulated endostatin producer cells

Background  

Theracyte is a polytetrafluoroethylene membrane macroencapsulation system designed to induce neovascularization at the tissue interface, protecting the cells from host's immune rejection, thereby circumventing the problem of limited half-life and variation in circulating levels. Endostatin is a potent inhibitor of angiogenesis and tumor growth. Continuous delivery of endostatin improves the efficacy and potency of the antitumoral therapy. The purpose of this study was to determine whether recombinant fibroblasts expressing endostatin encapsulated in Theracyte immunoisolation devices can be used for delivery of this therapeutic protein for treatment of mice bearing B16F10 melanoma and Ehrlich tumors.     

Possible Role of Coryneform Bacteria in Age Gelation of Ultrahigh-Temperature-Processed Milk

Two strains of psychrotrophic, gram-positive, proteolytic bacteria isolated from raw milk were identified as Arthrobacter or closely related coryneform species. We inoculated raw-milk samples with the strains (one strain per sample) and compared rates of gelation after ultrahigh-temperature processing with that of ultrahigh-temperature-processed controls. The trial indicated that either organism could play a role in age gelation of ultrahigh-temperature-processed milk.     

Adsorption of Bacteriophage eb7 on Streptococcus cremoris EB7

When M17 broth was used as growth medium before preparation of cell walls, adsorption of phage eb7 on Streptococcus cremoris EB7 at pH 4.0 was stimulated. When preparation included treatment with trypsin, absorption of phage was 65%. Without trypsin treatment, absorption was 81%. Only 10 to 20% of the adsorption was irreversible. Treatment with pepsin or commercial rennet but not pure chymosin prevented adsorption on non-trypsin-treated cell walls. d-Galactosamine treatment of phage eb7 had an inactivating effect which was enhanced by l-rhamnose.     

Enhanced auditory neuron survival following cell-based BDNF treatment in the deaf guinea pig

Exogenous neurotrophin delivery to the deaf cochlea can prevent deafness-induced auditory neuron degeneration, however, we have previously reported that these survival effects are rapidly lost if the treatment stops. In addition, there are concerns that current experimental techniques are not safe enough to be used clinically. Therefore, for such treatments to be clinically transferable, methods of neurotrophin treatment that are safe, biocompatible and can support long-term auditory neuron survival are necessary. Cell transplantation and gene transfer, combined with encapsulation technologies, have the potential to address these issues. This study investigated the survival-promoting effects of encapsulated BDNF over-expressing Schwann cells on auditory neurons in the deaf guinea pig. In comparison to control (empty) capsules, there was significantly greater auditory neuron survival following the cell-based BDNF treatment. Concurrent use of a cochlear implant is expected to result in even greater auditory neuron survival, and provide a clinically relevant method to support auditory neuron survival that may lead to improved speech perception and language outcomes for cochlear implant patients.