Isolation of photosynthetic catalysts from cyanobacteria.

Methods are described for the isolation of ferredoxins I and II, cytochrome c-553, cytochrome f, cytochrome c-550 and plastocyanin from large quantities of various cyanobacteria. The amino acid composition of cytochrome c-550 is reported. There is a variation in the relative amounts of these proteins in different batches of cells which may relate to the nutritional status of the organisms.

Inhibitory effect of the antioxidant ethoxyquin on electron transport in the mitochondrial respiratory chain.

Ethoxyquin (6-ethoxy-2,2,4-trimethyl-1,2-dihydroquinoline, EQ) is an antioxidant used as a preservative in animal and human foods. In a previous work, we demonstrated that EQ induces an inhibition of renal secretory mechanisms that are dependent on metabolic energy; EQ inhibits renal ATPases. In the present study, we analyzed the effects of EQ on the metabolic pathways of renal and hepatic rat cells, as well as on mitochondrial and submitochondrial particles isolated from bovine heart and kidney. EQ induced a mild inhibition of oxygen uptake when it was added to whole homogenates of rat renal cortex in the presence of glucose. In contrast, a strong concentration-dependent inhibition was produced when EQ was added to preparations of intact liver mitochondria or to submitochondrial particles isolated from renal cortex. In the presence of NADH, 90% inhibition was attained at a final concentration of 1 mM EQ. The direct inhibitory effect of EQ on NADH dehydrogenase was a most relevant finding, since no inhibitor for the partial reaction of NADH-ferricyanide on this complex has been reported previously.

Inhibition of the renal uptake of p-aminohippurate and tetraethylammonium by the antioxidant ethoxyquin in the rat.

Ethoxyquin (6-ethoxy-2,2,4-trimethyl-1,2-dihydroquinolein, EQ) is an antioxidant used in animal foodstuffs and to prevent superficial scalding in some fruits. In renal cortical slices prepared from male rats that had consumed a diet containing EQ, EQ inhibited the specific uptake of 14C-labelled p-aminohippurate ([14C]PAH) and tetraethylammonium ([14C]TEA), markers of organic anion and cation tubular secretion, respectively. The specific uptake of [14C]TEA was five-fold more sensitive to EQ than [14C]PAH uptake (IC50 0.33 and 1.51 mM, respectively). EQ (1 mM) decreased Na+/K(+)-ATPase activity from 1.58 to 1.0 mumol inorganic phosphate/mg protein/min in renal microsomes. The activity of this enzyme provides the energy for the function of both secretory systems. These results suggest that the mechanisms by which EQ inhibits both anion and cation tubular secretion involves a decrease in the Na+/K(+)-ATPase activity. This effect leads to interference with the energy supply required for these tubular secretory mechanisms. Our results indicate that the exposure of animals or humans to high concentrations of ethoxyquin should be avoided.

Preparation of a highly active ATPase of the mesophilic cyanobacterium Spirulina maxima.

In this work, we report new studies on the ATPase attached to the photosynthetic membranes of the mesophilic cyanobacterium Spirulina maxima. This enzyme does not display persistent latency as had been previously reported for the ATPase of Spirulina platensis. The enzyme is readily activated by the careful application of methods currently used to activate chloroplast CF1. Photosynthetic membranes of Spirulina maxima show a Mg(2+)-dependent ATPase activity of 195±25 µmol Pi (mg chl)(-1) h(-1) after a light plus dithiothreitol (DDT) treatment. Methanol treatment of these membranes elicits Mg(2+)-dependent ATPase activity of 222±18 µmol Pi (mg chl)(-1) h(-1).Here, we also describe the purification of the soluble coupling factor AF1 of Spirulina maxima. This enzyme is unique among mesophilic cyanobacterial F1 preparations in regard to its high specific Ca(2+)-dependent ATPase activity after heat treatment (14.75±1.91 µmol Pi (mg prot)(-1) min(-1)) and its room temperature stability.

Protection of cytochrome c oxidase against cyanide inhibition by pyruvate and alpha-ketoglutarate: effect of aeration in vitro.

Toxicity of cyanide is related to its inhibitory action on cytochrome c oxidase (COx). The alpha-keto acids pyruvate and alpha-ketoglutarate are known to exert in vivo certain protective effect against CN- toxicity if present when the poison is administered. We characterized in vitro their protection of oxidative phosphorylation and of the activity of COx in rat testis, heart, and liver mitochondria, as well as that of the beef heart enzyme in the pure state. In all cases the keto acids proved to have a protective action, even when KCN was previously added to the incubation mixtures and inhibition had already been established. However, the extent of the protection seemed to depend upon the degree of aeration. In the presence of the alpha-keto acids, O2 succeeded with 77% efficiency in reversing CN- inhibition of [gamma-32P]ATP synthesis under high aeration, whereas only 15% was achieved if aeration was poor. In poisoned heart mitochondria (1 mM KCN) simultaneous estimations of ATP synthesis and COx activity in a closed oxygraph chamber showed a recovery of only 6% of both activities upon the addition of 12 mM pyruvate. Our results suggest that O2 displaced cyanide from the enzyme and the poison was then trapped by the keto acids to form the respective nontoxic cyanohydrin. The combination of both high oxygen concentration and the presence of either pyruvate or alpha-ketoglutarate was necessary to effectively protect COx against cyanide poisoning.

Participation of plastoquinone, cytochrome c 553 and ferrodoxin-NADP (+) oxido-reductase in both photosynthesis and respiration in Spirulina maxima.

Dark and light oxidation of NADPH was measured in Spirulina maxima thylakoid membranes. The dark reaction was more cyanide sensitive than the light reaction. In light, 83% of the electrons from NADPH produced H2O2 on reducing oxygen, whereas in the dark this number was only 36%. These results are explained by assuming the presence of an electron transport segment common to the photosynthetic and the respiratory chains, so that electrons flowing through the cyanide sensitive oxidase in the dark are diverted to the photosytem (PS) I reaction center (P700). In addition, cytochrome (cyt) c 553 was found to be an electron donor for both cyt oxidase and P700. Half maximum reduction rates were obtained with 7 µM cyt c 553. The intrathylakoidal concentration of cyt c 553 was determined to be 83 µM. About 60% of the respiratory NADPH oxidation activity was lost by extracting the membranes with pentane and was restored by adding plastoquinone (the main photosythetic quinone). NADPH oxidation activity was also inhibited upon washing the membranes with a low salt buffer. This activity was restored by adding partially purified ferredoxin-NADP(+) oxido-reductase (FNR). A model for the electron transport in thylakoids, in which cyt c 553, plastoquinone and FNR participate in both photosynthesis and respiration is proposed.

Growth of Phormidium sp. in aerobic secondary piggery waste-water.

The potential of a cyanobacterium Phormidium sp., for the tertiary treatment of piggery waste-water chemical oxygen demand > or = 3000 mg.l-1, using an aerobically stabilized secondary effluent, was studied. Batch cultures were carried out in 2-l glass reactors and in a 30-l glass-fibre carousel reactor. The nutrient removal efficiency as well as biomass production were compared in synthetic mineral medium and in different concentrations of aerobically stabilized piggery effluent. The best performance of Phormidium sp. occurred in diluted stabilized secondary effluent (1:1). Removal efficiencies were 100% for P-PO4(-3), 50% for N-NH4+ and 35% for N-NO3- in small-volume cultures; and 31% for P-PO4(-3), 100% for N-NH4+ and 70% for N-NO3- in an open carousel reactor. Biomass production on the aerobically stabilized effluent was six times higher in the 2-l reactors and 1.7 times higher in the carousel reactor when compared to the synthetic medium.

DBHBM (3,5-dibromo-4-hydroxy-benzylidenemalonitrile) is a novel inhibitor of electron transfer through the QN center of the mitochondrial bc1 complex.

DBHBM (3,5-dibromo-4-hydroxy-benzylidenemalonitrile) inhibited the NADH- or succinate-supported rate of O2 consumption in beef heart submitochondrial particles (Ki = 7 x 10(-7) M). Oxygen comsumption was restored with the addition of ascorbate/TMPD, indicating that the inhibitory effect was on the ubiquinol-cytochrome c reductase activity of the respiratory chain. Difference spectra with submitochondrial particles indicated that DBHBM blocked electron transport through the cytochrome bc1 complex, in a mode closely similar to that of antimycin A. The reduction rates of cytochrome b by succinate were strongly inhibited in the presence of DBHBM plus myxothiazol, but not by DBHBM plus antimycin A. These data suggest that DBHBM may bind primarily to the QN center. In the purified bc1 complex, DBHBM and antimycin A induced a red shift from 562 to 566 nm of the alpha peak of cytochrome b, supporting the idea that DBHBM influences predominantly the ligand field of the b562 (bh) heme. Difference spectra in the presence or absence of myxothiazol showed that DBHBM induced the same red shift with a maximum at 565 nm and a minimum at 559 nm. We conclude that DBHBM blocks electron transfer at the QN center and thus may be considered a novel group III inhibitor of the bc1 complex.

Charge transfer mediated by nigericin in black lipid membranes.

Nigericin, in the concentration range (10(-6) M or higher) at which it uncouples intact mitochondria, was found to increase the conductance of black lipid membranes (BLM) by several orders of magnitude. The dependence of the membrane conductance on pH and K+ concentration suggests a mechanism for the transfer of charge mediated by this ionophore based on a mobile dimer with both nigericin molecules protonated and complexed with one K+. This charged complex accounts for the uncoupling effect observed in intact mitochondria.

An atypical cytochrome b in the colorless alga Polytomella spp.: the high potential bH heme exhibits a double transition in the alpha-peak of its absorption spectrum.

Polytomella spp. is a colorless alga of the family Chlamydomonadaceae that lacks chloroplasts and cell wall. A highly active ubiquinol-cytochrome c oxidoreductase (bc1 complex), sensitive to antimycin and myxothiazol, has been purified and characterized from this alga (Gutiérrez-Cirlos et al., 1994, J. Biol. Chem. 269, 9147-9154). Both in mitochondrial membranes and in the isolated complex, the visible spectrum of cytochrome b from Polytomella spp. exhibits an atypical alpha-band with a maximum at 567 nm. This maximum is shifted 3-4 nm to the red when compared with b-type cytochromes from other organisms. Analysis of the b hemes of the bc1 complex by high performance liquid chromatography revealed no differences in the retention time and in the absorption spectra of the b-type hemes from Polytomella spp. and hemin, indicating that the prosthetic group in this alga is protoheme and thus ruling out the possibility that the red-shift could be due to different chemical substitutions in the porphyrin rings of the bL or bH hemes. The two b hemes were characterized by electrochemical redox titration; at pH 7.8-8.0, the midpoint potential for bL was-143 mV and for bH +25 mV. The spectra of the two b-type hemes were recorded in the presence of different reductants, at selected electrochemical potentials, and in the presence of antimycin A, to distinguish between the contribution of bL and bH to the visible spectrum. Both hemes bL and bH of the algal cytochrome b contribute to the observed bathochromic absorption maximum in the alpha-band of the spectrum. The data also show that the low potential bL heme from Polytomella spp. is spectroscopically similar to that of other organisms, with two transitions in the alpha-peak at 558.7 and 568.4 nm. The high-potential heme bH also exhibits a spectrum with two transitions at 557.2 and 568.9 nm, which surprisingly differs from the spectra of cytochrome bH of mammals, plants, yeasts, and bacteria, which all exhibit a single transition centered around 560 nm.