RESUMO
Twenty polymorphic microsatellite markers were developed for the Neotropical fish Leporinus obtusidens using a next generation sequencing approach and tested in two other characifomes species, Schizodon platae and Prochilodus lineatus. Microsatellite loci alleles in L. obtusidens ranged between 2 and 20 alleles per locus (mean = 5·7), with expected heterozygosity values ranging from 0·097 to 0·956 (mean = 0·578) and observed heterozygosity values ranging from 0·000 to 0·800 (mean = 0·400) in a sample of 20 specimens from the lower Paraná River (Argentina). Most of these markers will be a valuable tool for captive breeding and stocking programmes, as well as for analyses of population connectivity and genetic structure in this broadly distributed Neotropical migratory fish.
Assuntos
Caraciformes/genética , Repetições de Microssatélites , Alelos , Animais , Argentina , Heterozigoto , Sequenciamento de Nucleotídeos em Larga Escala , Polimorfismo Genético , RiosRESUMO
In ferredoxin-NADP(+) reductase (FNR), FAD is bound outside of an anti-parallel beta-barrel with the isoalloxazine lying in a two-tyrosine pocket. To elucidate the function of the flavin si-face tyrosine (Tyr-89 in pea FNR) on the enzyme structure and catalysis, we performed ab initio molecular orbital calculations and site-directed mutagenesis. Our results indicate that the position of Tyr-89 in pea FNR is mainly governed by the energetic minimum of the pairwise interaction between the phenol ring and the flavin. Moreover, most of FNR-like proteins displayed geometries for the si-face tyrosine phenol and the flavin, which correspond to the more negative free energy theoretical value. FNR mutants were obtained replacing Tyr-89 by Phe, Trp, Ser, or Gly. Structural and functional features of purified FNR mutants indicate that aromaticity on residue 89 is essential for FAD binding and proper folding of the protein. Moreover, hydrogen bonding through the Tyr-89 hydroxyl group may be responsible of the correct positioning of FAD and the substrate NADP(+)
Assuntos
Ferredoxina-NADP Redutase/química , Ferredoxina-NADP Redutase/metabolismo , Pisum sativum/enzimologia , Tirosina/química , Tirosina/metabolismo , Animais , Sítios de Ligação , DNA Complementar/metabolismo , Escherichia coli/metabolismo , Humanos , Ligação de Hidrogênio , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , NADP/química , Oxigênio/metabolismo , Fenol/química , Ligação Proteica , Estrutura Secundária de Proteína , Espectrofotometria , Especificidade por Substrato , Termodinâmica , Fatores de TempoRESUMO
Bicarbonate, an activating anion of ATP hydrolysis, inhibited ATP synthesis coupled to succinate oxidation in beef heart submitochondrial particles but diminished the lag time and increased the steady-state velocity of the (32)Pi-ATP exchange reaction. The latter effects exclude the possibility that bicarbonate is inducing an intrinsic uncoupling between ATP hydrolysis and proton translocation at the level of F(1)F(o) ATPase. The inhibition of ATP synthesis was competitive with respect to ADP at low fixed [Pi], mixed at high [Pi] and non-competitive towards Pi at any fixed [ADP]. From these results we can conclude that (i) bicarbonate does not bind to a Pi site in the mitochondrial F(1); (ii) it competes with the binding of ADP to a low-affinity site, likely the low-affinity non-catalytic nucleotide binding site. It is postulated that bicarbonate stimulates ATP hydrolysis and inhibits ATP synthesis by modulating the relative affinities of the catalytic site for ATP and ADP.
Assuntos
Trifosfato de Adenosina/metabolismo , Mitocôndrias Cardíacas/metabolismo , Bicarbonato de Sódio/farmacologia , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/biossíntese , Trifosfato de Adenosina/química , Animais , Sítios de Ligação , Ligação Competitiva , Bovinos , Hidrólise/efeitos dos fármacos , Fosfatos/química , ATPases Translocadoras de Prótons/metabolismoRESUMO
The contribution made by tyrosine 308 to the stability of pea ferredoxin-NADP+ reductase was investigated using site-directed mutagenesis. The phenol side chain of the invariant carboxyl terminal tyrosine is stacked coplanar to the isoalloxazine moiety of the FAD cofactor. Fluorescence measurements indicate that this interaction plays a significant role in FAD fluorescent quenching by the reductase apoprotein. Replacement of the tyrosine by tryptophan or phenylalanine caused only a minor increase in the quantum yields of bound FAD, whereas nonaromatic substitutions to serine and glycine resulted in a large fluorescent rise. Results from NADP+ titration experiments support a recent hypothesis [Karplus et al. (1991) Science 251, 60-66], suggesting that the phenol ring of Tyr 308 may fill the nicotinamide binding pocket in the absence of the nucleotide. The stability of the site-directed mutants, judged by thermal- and urea-induced denaturation studies, was lowered with respect to the wild-type enzyme. FNR variants harboring nonaromatic substitutions displayed more extensive destabilization. The decrease in thermodynamic stability correlated with the impairment of catalytic activities [Orellano et al. (1993) J. Biol. Chem 268, 19267-19273]. The results indicate that the presence of the electron-rich aromatic side chain adjacent to the isoalloxazine ring is essential for maximum stabilization of the FNR holoenzyme, resulting in a flavin conformation which optimizes electron flow between the prosthetic group and its redox partners.
Assuntos
Ferredoxina-NADP Redutase/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Pisum sativum/enzimologia , Tirosina/metabolismo , Sítios de Ligação , Estabilidade Enzimática , Ferredoxina-NADP Redutase/química , Ferredoxina-NADP Redutase/genética , Temperatura Alta , Mutação , Desnaturação Proteica , Espectrometria de Fluorescência , Tirosina/químicaRESUMO
The flavoprotein ferredoxin-NADP+ reductase catalyzes the final step of the photosynthetic electron transport i.e., the reduction of NADP+ by ferredoxin. Expression and secretion of this enzyme was examined in Saccharomyces cerevisiae using a cDNA cloned from a pea library [Newman, B. J. & Gray, J. C. (1988) Plant Mol. Biol. 10, 511-520]. Two pea library cDNA sequences were employed, one corresponding to the mature enzyme and the other containing, in addition, the sequence of the transit peptide that directs ferredoxin-NADP+ reductase to the chloroplast. These sequences were introduced into a yeast shuttle vector in frame with the mating factor alpha 1 secretion-signal coding region under the control of its natural mating factor alpha 1 promoter. Saccharomyces cerevisiae cells transformed with the recombinant plasmids were able to synthesize and secrete fully active pea ferredoxin-NADP+ reductase. In both cases, a 35-kDa polypeptide was the major product. N-terminal sequencing of the secreted proteins indicates processing at position -1 with respect to the N-terminus of the pea mature enzyme. Yeast cells transformed with plasmid encoding the ferredoxin-NADP+ reductase precursor secrete four-times more ferredoxin-NADP+ reductase to the medium than cells transformed with the plasmid encoding the mature form of the enzyme. Ferredoxin-NADP+ reductases purified from culture medium showed structural and enzymatic properties that were identical, within the experimental error, to those of native plant ferredoxin-NADP+ reductase. The overall results indicate that pea ferredoxin-NADP+ reductase can be properly folded and its prosthetic group assembled in the yeast endoplasmic reticulum, and that its natural transit peptide favors its secretion.
Assuntos
Ferredoxina-NADP Redutase/genética , Ferredoxina-NADP Redutase/metabolismo , Regulação Enzimológica da Expressão Gênica , Plantas/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Northern Blotting , Eletroforese em Gel de Poliacrilamida , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Ferredoxina-NADP Redutase/biossíntese , Ferredoxina-NADP Redutase/química , Dados de Sequência Molecular , Peso Molecular , Plasmídeos , Dobramento de Proteína , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genéticaRESUMO
The carboxyl-terminal region of plant ferredoxin-NADP+ reductases is formed by an invariant alpha-helix/loop/beta-strand, culminating in a conserved tyrosine that displays extensive interaction with the prosthetic group FAD. We have investigated the potential role of the terminal region in reductase function, by introducing mutations and deletions on pea ferredoxin-NADP+ reductase overexpressed in Escherichia coli. Replacement of the terminal tyrosine by tryptophan, phenylalanine, serine, and glycine resulted in a 2.2-, 2.0-, 22-, and 302-fold reduction, respectively, in kcat for the diaphorase reaction, whereas elimination of the tyrosine caused a 846-fold decrease in kcat. Km values were largely unaffected by the substitutions. Similar results were obtained when the mutants were assayed for cytochrome c reduction, indicating that aromaticity is the most important factor to the function of the tyrosine in catalysis. The presence of the phenol ring at the carboxyl-terminal position of wild-type reductase is important, but not an absolute requirement for enzyme function or FAD assembly. Deletion of the alpha-helix/beta-strand region prevented reductase proper folding in the bacterial host, while shortening of the terminal region by splicing 3 amino acids at the beginning of the alpha-helix produced a moderately soluble reductase, devoid of FAD and enzymatic activity.
Assuntos
Fabaceae/enzimologia , Ferredoxina-NADP Redutase/metabolismo , Plantas Medicinais , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Sequência Conservada , Escherichia coli/genética , Ferredoxina-NADP Redutase/química , Ferredoxina-NADP Redutase/genética , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos , Plantas/enzimologia , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Difração de Raios XRESUMO
Southern blot hybridization techniques were used to estimate the extent of chloroplast DNA sequences present in the mitochondrial genome of cowpea (Vigna unguiculata L.) The entire mitochondrial chromosome was homogeneously labeled and used to probe blotted DNA fragments obtained by extensive restriction of the tobacco chloroplast genome. The strongest cross-homologies were obtained with fragments derived from the inverted repeat and the atpBE cluster regions, although most of the clones tested (spanning 85% of the tobacco plastid genome) hybridized to mitochondrial DNA. Homologous chloroplast DNA restriction fragments represent a total of 30 to 68 kilobase pairs, depending upon the presence or absence of tRNA-encoding fragments. Plastid genes showing homology with mitochondrial DNA include those encoding ribosomal proteins, RNA polymerase, subunits of photosynthetic complexes, and the two major rRNAs.
RESUMO
Suramin, a drug intensively used in the chemotherapy of African trypanosomiasis and onchocerciasis, is currently being tested in clinical trials for AIDS treatment. Its effects on mitochondrial energy metabolism in mammals were studied. At low concentrations it inhibited ATP synthesis and ATPase activity in submitochondrial particles, as well as ADP-stimulated oxygen consumption and the uncoupler-stimulated ATPase activity in intact rat liver mitochondria. At higher concentrations it also inhibited uncoupled electron transport in both submitochondrial particles and intact mitochondria. From comparison of the kinetic patterns of those inhibitions, evidence suggesting that the adenine nucleotide translocase may be another target for the action of suramin was obtained. The relevance of these findings to the understanding of the biochemical basis of suramin toxicity is discussed.