The purification and preliminary X-ray diffraction studies of recombinant Synechococcus ribulose-1,5-bisphosphate carboxylase/oxygenase from Escherichia coli

X-ray crystallographic diffraction data has been collected for recombinant hexadecameric ribulose-P2 carboxylase from the cyanobacterium Synechococcus PCC6301 expressed in Escherichia coli. The enzyme has been purified and then crystallized in a number of crystal forms from polyethylene glycol solutions. The best crystals were obtained with enzyme that was first activated with the cofactors CO2 and Mg2+ in the presence of the tight-binding intermediate analogue, 2'-carboxyarabinitol 1,5-bisphosphate. One crystal form with plate-like morphology diffracts beyond 2.5 A but has one axis greater than 350 A. A second crystal form that diffracts to similar resolution grows with space group P212121 and unit cell dimensions of a = 223.9 A, b = 111.9 A, and c = 199.7 A. The crystal forms used to collect the diffraction data have been redissolved to determine that the recombinant ribulose-P2 carboxylase L8S8 molecule is indeed composed of equal numbers of large and small subunits and also that a quaternary complex between activated ribulose-P2 carboxylase E.CO2.Mg2+, and the analogue was present in the crystals. Denaturation of the redissolved enzyme in the absence of thiol-reducing agents established that the L-subunits of the L8 core are substantially dimeric, cross-linked by a disulfide bridge. Crystals of spinach ribulose-P2 carboxylase were likewise analyzed to show that dimers of the L-subunit were also predominant. This report identifies a single cysteine residue in the L-subunit that forms a bridge between those L-monomers that compose the four putative functional dimers of the L8 core.     

Detection of salmonellas in confectionery products by conductance

A modified lysine decarboxylase broth has been developed which could be used with a Bactometer M123 to differentiate salmonellas from other bacteria by the characteristics of the conductance detection curve. The medium was used in combination with a selenite cystine trimethylamine oxide dulcitol medium to screen 50 strains of salmonellas and 42 strains of other organisms to establish detection curve magnitude and rate values which could be used to identify curves specific to salmonellas. The combination of media detected all salmonellas tested except Salmonella pullorum . The two media were used to screen 100 inoculated product samples with the Bactometer instrument, in parallel with traditional plating procedures, and using various combinations of pre-enrichment and selective enrichment incubation periods. After 24 h pre-enrichment, the Bactometer system detected more positive samples than the conventional plating procedures after pre-enrichment and selective enrichment. It is considered that these media used in parallel in the Bactometer after conventional pre-enrichment could provide a 48 h screening procedure for salmonellas with a sensitivity comparable to present plating procedures.     

Superoxide dismutase and Fenton chemistry. Reaction of ferric-EDTA complex and ferric-bipyridyl complex with hydrogen peroxide without the apparent formation of iron(II).

A ferric-EDTA complex, prepared directly from FeCl3 or from an oxidized ferrous salt, reacts with H2O2 to form hydroxyl radicals (.OH), which degrade deoxyribose and benzoate with the release of thiobarbituric acid-reactive material, hydroxylate benzoate to form fluorescent dihydroxy products and react with 5,5-dimethylpyrrolidine N-oxide (DMPO) to form a DMPO-OH adduct. Degradation of deoxyribose and benzoate and the hydroxylation of benzoate are substantially inhibited by superoxide dismutase and .OH-radical scavengers such as formate, thiourea and mannitol. Inhibition by the enzyme superoxide dismutase implies that the reduction of the ferric-EDTA complex for participation in the Fenton reaction is superoxide-(O2.-)-dependent, and not H2O2-dependent as frequently implied. When ferric-bipyridyl complex at a molar ratio of 1:4 is substituted for ferric-EDTA complex (molar ratio 1:1) and the same experiments are conducted, oxidant damage is low and deoxyribose and benzoate degradation were poorly if at all inhibited by superoxide dismutase and .OH-radical scavengers. Benzoate hydroxylation, although weak, was, however, more effectively inhibited by superoxide dismutase and .OH-radical scavengers, implicating some role for .OH. The iron-bipyridyl complex had available iron-binding capacity and therefore would not allow iron to remain bound to buffer or detector molecules. Most .OH radicals produced by the iron-bipyridyl complex and H2O2 are likely to damage the bipyridyl molecules first, with few reacting in free solution with the detector molecules. Deoxyribose and benzoate degradation appeared to be mediated by an oxidant species not typical of .OH, and species such as the ferryl ion-bipyridyl complex may have contributed to the damage observed.     

Copper salt-dependent hydroxyl radical formation. Damage to proteins acting as antioxidants

Cupric ions (Cu2+) and ferric ions (Fe3+) added to hydrogen peroxide generate hydroxyl radicals (OH) capable of degrading deoxyribose with the formation of thiobarbituric acid-reactive products. This damage can be inhibited by catalase, OH radical scavengers and specific metal ion chelators. All proteins tested nonspecifically inhibited copper-dependent damage but have little effect on the iron-dependent reaction. Copper ions appear to bind to the proteins which prevents formation of OH radicals in free solution. However, OH radicals are still generated at a site-specific location on the protein molecule. Protein damage is detected as fluorescent changes in amino acid residues.     

Electron-paramagnetic-resonance parameters of molybdenum(V) in sulphite oxidase from chicken liver.

A study has been made of e.p.r. signals due to Mo(V) in reduced sulphite oxidase (EC 1.8.3.1) from chicken liver. Reduction by SO3(2-), or photochemically in the presence of a deazaflavin derivative, produces spectra indistinguishable from one another. Three types of spectra from the enzyme were distingusihed and shown to correspond to single chemical species, since they could be simulated at both 9 and 35 GHz by using the same parameters. These were the low-pH form of the enzyme, with gav. 1.9805, the high-pH form, with gav. 1.9681 and a phosphate complex, with gav. 1.9741. The low-H form shows interaction with a single exchangeable proton, with A(1H)av. (hyperfine coupling constant) = 0.98 mT, probably in the form of an MoOH group. Parameters of the signals are compared with those for signals from xanthine oxidase and nitrate reductase. The signal from the phosphate complex of sulphite oxidase in unique among anion complexes of Mo-containing enzymes in showing no hyperfine coupling to protons. There is no evidence for additional weakly coupled protons or nitrogen nuclei in the sulphite oxidase signals. The possibility is considered that the enzymic mechanism involves abstraction of a proton and two electrons from HSO3- by a Mo = O group in the enzyme.     

566C80, an antimalarial hydroxynaphthoquinone with broad spectrum: experimental activity against opportunistic parasitic infections of AIDS patients.

Hydroxynaphthoquinone 566C80 was synthesised and initially developed as an antimalarial with potent activity against drug-resistant strains of the human malaria parasite, Plasmodium falciparum. Subsequent studies have revealed that in addition, this compound has experimental activity, both in vitro and in vivo, against Pneumocystis carinii and Toxoplasma gondii; the data obtained thus far for Cryptosporidium parvum are equivocal. Currently 566C80 is being assessed clinically not only against malaria, but also against P. carinii pneumonia, toxoplasmosis and cryptosporidiosis.     

Action of lead(II) and aluminium (III) ions on iron-stimulated lipid peroxidation in liposomes, erythrocytes and rat liver microsomal fractions

Lead (Pb2+) ions accelerate the lipid peroxidation observed when Fe2+ ions are added to phospholipid liposomes at pH 5.5 or pH 7.4, although Pb2+ ions alone do not induce any peroxidation. Similarly, aluminium (Al3+) ions increase Fe2+-dependent liposomal peroxidation at pH 5.5. Both Pb2+ and Al3+ accelerate the peroxidation of erythrocytes induced by high concentrations of H2O2 in the presence of azide, and they also increase the peroxidation that occurs when Fe2+ or Fe2+-ADP is added to rat liver microsomes at pH 7.4. It is proposed that increased lipid peroxidation may contribute to the toxic actions of Pb2+ in humans.