Francisco Córdoba

Zonal Changes in Ascorbate and Hydrogen Peroxide Contents, Peroxidase, and Ascorbate-Related Enzyme Activities in Onion Roots

Onion (Allium cepa) roots growing hydroponically show differential zonal values for intra- (symplastic) and extra- (apoplastic) cellular ascorbate (ASC) and dehydroascorbate (DHA) contents and for related enzyme activities. In whole roots, ASC and DHA concentrations were higher in root apex and meristem and gradually decreased toward the root base. Guaiacol peroxidase, ASC peroxidase, monodehydroascorbate oxidoreductase, DHA reductase, catalase, and glutathione reductase activities showed differential activity patterns depending on the zone of the root and their apoplastic or symplastic origin. An in vivo staining of peroxidase activity also revealed a specific distribution pattern along the root axis. Using electron microscopy, hydrogen peroxide was found at different locations depending on the root zone but was mainly located in cell walls from epidermal and meristematic cells and in cells undergoing lignification. A balanced control of all of these molecules seems to exist along the root axis and may be directly related to the mechanisms in which the ASC system is involved, as cell division and elongation. The role of ASC on growth and development in relation to its presence at the different zones of the root is discussed.

Purification and Characterization of Two Distinct NAD(P)H Dehydrogenases from Onion (Allium cepa L.) Root Plasma Membrane.

Highly purified plasma membrane fractions were obtained from onion (Allium cepa L.) roots and used as a source for purification of redox proteins. Plasma membranes solubilized with Triton X-100 contained two distinct polypeptides showing NAD(P)H-dependent dehydrogenase activities. Dehydrogenase I was purified by gel filtration in Sephacryl S-300 HR, ion-exchange chromatography in DEAE-Sepharose CL-6B, and dye-ligand affinity chromatography in Blue-Sepharose CL-6B after biospecific elution with NADH. Dehydrogenase I consisted of a single polypeptide of about 27 kD and an isoelectric point of about 6. Dehydrogenase II was purified from the DEAE-unbound fraction by chromatography in Blue-Sepharose CL-6B and affinity elution with NADH. Dehydrogenase II consisted of a single polypeptide of about 31 kD and an isoelectric point of about 8. Purified dehydrogenase I oxidized both NADPH and NADH, although higher rates of electron transfer were obtained with NADPH. Maximal activity was achieved with NADPH as donor and juglone or coenzyme Q as acceptor. Dehydrogenase II was specific for NADH and exhibited maximal activity with ferricyanide. Optimal pH for both dehydrogenases was about 6. Dehydrogenase I was moderately inhibited by dicumarol, thenoyltrifluoroacetone, and the thiol reagent N-ethyl-maleimide. A strong inhibition of dehydrogenase II was obtained with dicumarol, thenoyltrifluoroacetone, and the thiol reagent p-hydroxymercuribenzoate.

Genetic Evidence for Coenzyme Q Requirement in Plasma Membrane Electron Transport

Plasma membranes isolated from wild-type Saccharomyces cerevisiae crude membrane fractions catalyzed NADH oxidation using a variety of electron acceptors, such as ferricyanide, cytochrome c, and ascorbate free radical. Plasma membranes from the deletion mutant strain coq3Δ, defective in coenzyme Q (ubiquinone) biosynthesis, were completely devoid of coenzyme Q6 and contained greatly diminished levels of NADH–ascorbate free radical reductase activity (about 10% of wild-type yeasts). In contrast, the lack of coenzyme Q6 in these membranes resulted in only a partial inhibition of either the ferricyanide or cytochrome-c reductase. Coenzyme Q dependence of ferricyanide and cytochrome-c reductases was based mainly on superoxide generation by one-electron reduction of quinones to semiquinones. Ascorbate free radical reductase was unique because it was highly dependent on coenzyme Q and did not involve superoxide since it was not affected by superoxide dismutase (SOD). Both coenzyme Q6 and NADH–ascorbate free radical reductase were rescued in plasma membranes derived from a strain obtained by transformation of the coq3Δ strain with a single-copy plasmid bearing the wild type COQ3 gene and in plasma membranes isolated form the coq3Δ strain grown in the presence of coenzyme Q6. The enzyme activity was inhibited by the quinone antagonists chloroquine and dicumarol, and after membrane solubilization with the nondenaturing detergent Zwittergent 3–14. The various inhibitors used did not affect residual ascorbate free radical reductase of the coq3Δ strain. Ascorbate free radical reductase was not altered significantly in mutants atp2Δ and cor1Δ which are also respiration-deficient but not defective in ubiquinone biosynthesis, demonstrating that the lack of ascorbate free radical reductase in coq3Δ mutants is related solely to the inability to synthesize ubiquinone and not to the respiratory-defective phenotype. For the first time, our results provide genetic evidence for the participation of ubiquinone in NADH–ascorbate free radical reductase, as a source of electrons for transmembrane ascorbate stabilization.

Regulation of nitrite uptake and nitrite reductase expression in Chlamydomonas reinhardtii

Expression of nitrite uptake and nitrite reductase activities has been studied in Chlamydomonas reinhardtii under different nutritional conditions. Both activities were expressed at a low level in derepressed cells (with no nitrogen source) and at a high level in induced cells (with nitrate or nitrite). Nitrate was required for both activities to be maximally expressed. Ammonium-grown cells did not show nitrite uptake capability and had a basal nitrite reductase activity. Nitrite uptake but not nitrite reductase levels decreased very significantly in nitrate-induced cells subject to cycloheximide treatment, which suggests that protein(s) involved in the uptake are under a rapid turnover. Nitrite uptake expression was strongly inhibited by the presence of the glutamine synthetase inhibitor L-methionine-D,L-sulfoximine under either derepression or induction conditions, whereas that of nitrite reductase was not affected under the same conditions. Our results indicate that nitrite uptake expression is regulated primarily by ammonium, and that of nitrite reductase by both ammonium and ammonium derivative(s).

Changes in neurotransmitter amino acids and protein in CNS areas of mice subjected to differential housing conditions

The amino acid and protein content of mice exposed to enriched, restricted and impoverished environments have been studied in six discrete CNS areas. Differences between enriched and either restricted or impoverished groups were found whereas no difference was observed between restricted and impoverished ones. In the first case, a significant increase for aspartate was found in spinal cord, whereas glutamate significantly decreased in colliculi and cerebral cortex. Similarly, glycine increased in cerebral cortex and decreased in colliculi and pons-medulla, and gamma-aminobutyrate (GABA) increased in spinal cord, pons-medulla and cerebellum and decreased in thalamus-hypothalamus. No changes in concentrations of five non-transmitter amino acids (serine, threonine, alanine, isoleucine, leucine) were observed. Significant increases of the protein concentration in cerebellum and spinal cord were found. The changes were due to enrichment, not to aggregation conditions. The results corroborate the proposed plasticity of the aminoacidergic system.

Ascorbate and the plasma membrane. A new view of cell growth control.

Ascorbate is needed for different physiological functions of living organisms and is thus an essential nutrient for animals lacking its synthesis pathway. From the variety of cellular functions affected by ascorbate (Padh, 1990; Gershoff, 1993), its role in the synthesis of collagen has been clearly established. The maintenance of metal ions in their reduced form is another function in cells. Ascorbic acid can act as a scavenger of reactive oxygen species by its reducing capacity; it also acts a prooxidant under certain conditions. Thus, another important function of ascorbate is to protect tissues from harmful oxidative products.

NADH-specific dehydrogenase from onion root plasma membrane: purification and characterization

SummaryPlasma membranes isolated from onion roots by twophase partition contain at least two different NAD(P)H-dehydrogenases. A 27 kDa electron transport protein oxidises both NADH and NADPH and exhibits maximal activity with quinones as electron acceptors. A distinct 31 kDa dehydrogenase is specific for NADH as donor and shows maximal activity with ferricyanide. This novel enzyme is responsible for most NADH-ferricyanide oxidoreductase activity of solubilized onion root plasma membranes and exhibits properties different to other purified NAD(P)H-dehydrogenases.

Cooperative regulation by ammonium and ammonium derivatives of nitrite uptake in Chlamydomonas reinhardtii

Abstract The role of ammonium in the regulation of nitrite uptake in Chlamydomonas reinhardtii has been investigated under conditions that prevented ammonium assimilation. Prolonged carbon-starvation or inhibition of glutamine synthesis with l -methionine- dl -sulfoximine partially relieved ammonium inhibition of nitrite uptake. However, nitrite uptake was inhibited in both methionine sulfoximine-treated and carbon-starved cells preincubated with ammonium, the inhibition extent in the two cases being directly dependent on the ammonium concentration in the preincubation media. Methionine sulfoximine treatment caused an increase of intracellular ammonium levels. When methionine sulfoximine-treated cells were transferred to ammonium media there existed a linear correlation between intracellular and extracellular ammonium concentration. Addition of methionine sulfoximine to cells with their nitrite uptake system inhibited by ammonium counteracted the effect of ammonium and restored nitrite uptake rate. These results strongly suggest that ammonium itself and a (some) product(s) of its metabolism must act together to block completely nitrite uptake by C. reinhardtii cells. Partial inhibition of nitrite uptake by methylammonium, a structural analogue of ammonium incapable of being used for cell nutrition, supports the above conclusion.

Differential distribution of neurotransmitter amino acids from the limbic system of aggressive and non-aggressive bull strains

The amino acid content of crude synaptosomal fractions from the limbic system and related CNS regions showed significant differences between the aggressive Spanish fighting-bull and the non-aggressive Friesan bull breeds. Neurotransmitter amino acids (glutamate, aspartate, GABA and glycine) were the most unequally distributed. A higher ratio of excitatory to inhibitory neurotransmitter amino acids was always found in all the CNS regions studied in the aggressive breed. The concentrations of five non-transmitter amino acids (threonine, alanine, serine, leucine and isoleucine) showed minor variations between both studied bull strains and cannot be ascribed to differences in central energy metabolism. The results are explained in terms of a possible relationship between the amino acid neurotransmitter levels and the innate aggressiveness of the Spanish fighting-bull.

Role of the diaphorase moiety on the reversible inactivation of the Chlamydomonas reinhardii nitrate reductase complex

Abstract Nitrate reductase (NAD(P)H: nitrate oxidoreductase, EC 1.6.6.2) of Chlamydomonas reinhardii mutant 305. lacking NAD(P)H-cytochrome c reductase activity, became rapidly and reversibly inactivated in vitro upon incubation with dithionite and reactivated by oxidation with ferricyanide. Nitrate protected against the inactivation. Unlike the native nitrate reductase complex of its parental wild strain, the enzyme of 305 was not inactivated by reduced pyridine nucleotides. In contrast to that of wild-type cells, the in vivo reversible inactivation of mutant 305 nitrate reductase was observed only after complete elimination of nitrate from the media and subsequent transfer to ammonium medium or darkness conditions. The inactive enzyme was reactivated by addition of nitrate to the media without previous removal of ammonium, which indicates that, unlike in the wild-type cells, ammonium does not prevent nitrate uptake by 305 cells. This different regulation pattern is due to the structural modification of 305 nitrate reductase. We conclude that in vitro an active diaphorase moiety is required for the inactivation by reduced pyridine nucleotides of the nitrate reductase of C. reinhardii , and that in vivo the absence of nitrate rather than the presence of ammonium is the triggering event for nitrate reductase inactivation, which can be also achieved by reductants other than NAD(P)H.