Carlos Gómez-Lojero

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.

The Use of Ionophores and Channel Formers in the Study of the Function of Biological Membranes

Publisher Summary This chapter discusses the effects and mechanism of action of molecules that upon incorporation into model and biological lipid bilayers increase the permeability of the membrane to ions. As the molecules act similarly in biological and synthetic lipid membranes, it is assumed that these compounds operate in the lipid milieu of the membrane. The biological membranes are a fluid mosaic composed of lipids arranged in a bilayer in which proteins either go across the lipid phase or are partially submerged in the membrane. The membranes formed exclusively with lipids are highly impermeable to ions and the impermeability is determined by the existence of the lipid bilayer. Compounds that lower ion permeability have been used to explore the properties and structure of biological membranes. The ATP synthesis can be driven by a proton-motive force of sufficient magnitude

Charge transfer mediated by nigericin in black lipid membranes

Nigericin, in the concentration range (10−6M 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.

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.

Variability of the K+−Na+ discrimination of beauvericin in mitochondrial membranes

In intact mitochondria supplemented with succinate or β-hydroxybutyrate, the rates of oxygen consumption induced by beauvericin followed the ionic selectivity pattern: Na+>Rb+, Cs+, K+, Li+.When the respiratory substrate is glutamate plus malate in the absence of phosphate, the selectivity pattern is: K+>Rb+>Cs+>Li+>Na+.When the media are supplemented with phosphate, the Na+/K+ discrimination of beauvericin is considerably modified with all the respiratory substrates, being K+>Na+ with succinate and Na+>K+ with glutamate plus malate, whereas no significant ionic selectivity differences were obtained with β-hydroxybutyrate.The respiratory control induced by oligomycin in submitochondrial particles is released by beauvericin only in the presence of a nigericin-like carboxylic antibiotic and an alkali metal cation, being far more effective in K+ than in Na+.This selectivity is maintained regardless of whether NADH or succinate is used as a respiratory substrate.Release of respiratory control can also be obtained with a combination of beauvericin and NH4Cl.This information indicates that the ionic selectivity pattern obtained with beauvericin in mitochondrial membranes is an intrinsic property of the antibiotic which, however, can be significantly modified by factors such as the nature of the translocatable substrate anion or other anionic species, as well as the possible operation of a Na+/H+ antiporter existent in the membrane.

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) c553 was found to be an electron donor for both cyt oxidase and P700. Half maximum reduction rates were obtained with 7 μM cyt c553. The intrathylakoidal concentration of cyt c553 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 c553, plastoquinone and FNR participate in both photosynthesis and respiration is proposed.

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.

Photosynthetic phosphorylation by a membrane preparation of the cyanobacterium Spirulina Maxima

Abstract A new method for the isolation of photosynthetic membranes from the cyanobacterium Spirulina maxima has been developed. When illuminated, these membranes evolve oxygen in the presence of ferricyanide (Hill reaction) and consume oxygen in the presence of methyl viologen (Mehler reaction). When the membranes are left to stand at 4°C for 30 min, they develop the ability to consume oxygen in the light without an added, artificial electron acceptor. The Hill and Mehler reactions are not affected by the presence of ADP or uncouplers, but are inhibited by triphenyltin chloride. We have detected a cryptic ATPase activity stimulated by trypsin in the 2000×g supernatant fraction of the membrane preparation. In addition, the membrane vesicles contain an ATPase activity which is enhanced by treatment with dithiothreitol in the presence of light. These observations of ATPase led us to try a careful titration of the membrane vesicles with both triphenyltin chloride and N,N′-dicyclohexylcarbodiimide. When the vesicles were sealed with these reagents, we could observe both cyclic and stoichiometric photosynthetic phosphorylation.

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 Mg2+-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 Mg2+-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 Ca2+-dependent ATPase activity after heat treatment (14.75±1.91 μmol Pi (mg prot)−1 min−1) and its room temperature stability.

Distribution of isoforms of ferredoxin-NADP+ reductase (FNR) in cyanobacteria in two growth conditions

Ferredoxin-NADP+ reductase (FNR) transfers reducing equivalents between ferredoxin and NADP(H) in the photosynthetic electron transport chains of chloroplasts and cyanobacteria. In most cyanobacteria, FNR is coded by a single petH gene. The structure of FNR in photosynthetic organisms can be constituted by FAD-binding and NADPH-binding domains (FNR-2D), or by these and an additional N-terminal domain (FNR-3D). In this article, biochemical evidence is provided supporting the induction of FNR-2D by iron or combined nitrogen deficiency in the cyanobacteria Synechocystis PCC 6803 and Anabaena variabilis ATCC 29413. In cell extracts of these cyanobacteria, most of FNR was associated to phycobilisomes (PBS) or phycocyanin (PC), and the rest was found as free enzyme. Free FNR activity increased in both cyanobacteria under iron stress and during diazotrophic conditions in A. variabilis. Characterization of FNR from both cyanobacteria showed that the PBS-associated enzyme was FNR-3D and the free enzyme was mostly a FNR-2D isoform. Predominant isoforms in heterocysts of A. variabilis were FNR-2D; where its N-terminal sequence lacked an initial (formyl)methionine. This means that FNR-3D is targeted to thylakoid membrane, and anchored to PBS, and FNR-2D is found as a soluble protein in the cytoplasm, when iron or fixed nitrogen deficiencies prevail in the environment. Moreover, given that Synechocystis and Anabaena variabilis are dissimilar in genotype, phenotype and ecology, the presence of these two-domain proteins in these species suggests that the mechanism of FNR induction is common among cyanobacteria regardless of their habitat and morphotype.