Spectrophotometric Determination of Glyoxylic Acid with o-Aminobenzaldehyde and Glycine,and Its Application to Enzyme Assay

Glyoxylic acid reacted with o-aminobenzaldehyde and glycine to yield a yellow product, whose absorption maximum was at 440 mµ. Glycine could be replaced by ω-amino acids, e.g., γ-aminobutyrate, and aliphatic amines, but not by α-amino acids. The effects of pH, kind and concentration of buffers, concentration of o-aminobenzaldehyde, and incubation time on the reaction were investigated to establish the optimum conditions. Microamounts of glyoxylate (0.05~1.5 µmoles) were determined rapidly by this simple method with satisfactory results, α-Keto acids such as α-ketoglutarate reacted far less effectively ; almost no appreciable interference with quantitative estimation of glyoxylate occurred with them. This procedure is applicable for the assay of enzymes which catalyze the formation of glyoxylate, e.g., isocitrate lyase and glycollate oxidase.     

Effect of Hill Reaction Inhibitors on the Photoreduction of Ferricyanide and Cyclic Photophosphorylation by Spinach Chloroplasts

Some derivatives of phenylurea, N-phenylcarbamate, s-triazine and acylanilide inhibited the cyclic photophosphorylation of spinach chloroplasts catalyzed by phenazine methosulfate and accelerated the photosystem I-dependent electron flow estimated as the disproportionation of diphenylcarbazone. Acceleration was slightly stimulated by the simultaneous addition of methylamine. Thus, these Hill reaction inhibitors act as uncouplers of cyclic photophosphorylation as does methylamine. The inhibiting activities of the chemicals on the photoreduction of ferricyanide and on photophosphorylation had a parabolic relation to the partition coefficient in the octanol-water system of the chemicals.     

A Phylogenomic Study of the OCTase Genes in Pseudomonas syringae Pathovars: The Horizontal Transfer of the argK–tox Cluster and the Evolutionary History of OCTase Genes on Their Genomes

Phytopathogenic Pseudomonas syringae is subdivided into about 50 pathovars due to their conspicuous differentiation with regard to pathogenicity. Based on the results of a phylogenetic analysis of four genes (gyrB, rpoD, hrpL, and hrpS), Sawada et al. (1999) showed that the ancestor of P. syringae had diverged into at least three monophyletic groups during its evolution. Physical maps of the genomes of representative strains of these three groups were constructed, which revealed that each strain had five rrn operons which existed on one circular genome. The fact that the structure and size of genomes vary greatly depending on the pathovar shows that P. syringae genomes are quite rich in plasticity and that they have undergone large-scale genomic rearrangements. Analyses of the codon usage and the GC content at the codon third position, in conjunction with phylogenomic analyses, showed that the gene cluster involved in phaseolotoxin synthesis (argK–tox cluster) expanded its distribution by conducting horizontal transfer onto the genomes of two P. syringae pathovars (pv. actinidiae and pv. phaseolicola) from bacterial species distantly related to P. syringae and that its acquisition was quite recent (i.e., after the ancestor of P. syringae diverged into the respective pathovars). Furthermore, the results of a detailed analysis of argK [an anabolic ornithine carbamoyltransferase (anabolic OCTase) gene], which is present within the argK–tox cluster, revealed the plausible process of generation of an unusual composition of the OCTase genes on the genomes of these two phaseolotoxin-producing pathovars: a catabolic OCTase gene (equivalent to the orthologue of arcB of P. aeruginosa) and an anabolic OCTase gene (argF), which must have been formed by gene duplication, have first been present on the genome of the ancestor of P. syringae; the catabolic OCTase gene has been deleted; the ancestor has diverged into the respective pathovars; the foreign-originated argK–tox cluster has horizontally transferred onto the genomes of pv. actinidiae and pv. phaseolicola; and hence two copies of only the anabolic OCTase genes (argK and argF) came to exist on the genomes of these two pathovars. Thus, the horizontal gene transfer and the genomic rearrangement were proven to have played an important role in the pathogenic differentiation and diversification of P. syringae. Received: 22 May 2001 / Accepted: 26 September 2001     

Protection against photooxidative injury of tobacco leaves by 2-alkenal reductase. Detoxication of lipid peroxide-derived reactive carbonyls

Degradation of lipid peroxides leads to the formation of cytotoxic 2-alkenals and oxenes (collectively designated reactive carbonyls). The novel NADPH-dependent oxidoreductase 2-alkenal reductase (AER; EC 1.3.1.74) from Arabidopsis (Arabidopsis thaliana), which is encoded by the gene At5g16970, catalyzes the reduction of the alpha,beta-unsaturated bond of reactive carbonyls, and hence is presumed to function in antioxidative defense in plants. Here we show that Arabidopsis AER (At-AER) has a broad substrate spectrum to biologically relevant reactive carbonyls. Besides 2-alkenals, the enzyme recognized as substrates the lipid peroxide-derived oxenes 9-oxo-octadeca-(10E),(12Z)-dienoic acid and 13-oxo-octadeca-(9E),(11Z)-dienoic acid, as well as the potent genotoxin 4-oxo-(2E)-nonenal, altogether suggesting AER has a key role in the detoxification of reactive carbonyls. To validate this conclusion by in vivo studies, transgenic tobacco (Nicotiana tabacum) plants that had 100- to 250-fold higher AER activity levels than control plants were generated. The engineered plants exhibited significantly less damage from either (1) the exogenously administered 4-hydroxy-(2E)-nonenal, (2) treatment with methyl viologen plus light, or (3) intense light. We further show that the At-AER protein fused with the Aequorea victoria green fluorescent protein localizes in cytosol and the nucleus in Bright-Yellow 2 cells. These results indicate that reactive carbonyls mediate photooxidative injury in leaf cells, and At-AER in the cytosol protects the cells by reducing the alpha,beta-unsaturated bond of the photoproduced reactive carbonyls.     

The NADPH:quinone oxidoreductase P1-zeta-crystallin in Arabidopsis catalyzes the alpha,beta-hydrogenation of 2-alkenals: detoxication of the lipid peroxide-derived reactive aldehydes

P1-zeta-crystallin (P1-ZCr) is an oxidative stress-induced NADPH:quinone oxidoreductase in Arabidopsis thaliana, but its physiological electron acceptors have not been identified. We found that recombinant P1-ZCr catalyzed the reduction of 2-alkenals of carbon chain C(3)-C(9) with NADPH. Among these 2-alkenals, the highest specificity was observed for 4-hydroxy-(2E)-nonenal (HNE), one of the major toxic products generated from lipid peroxides. (3Z)-Hexenal and aldehydes without alpha,beta-unsaturated bonds did not serve as electron acceptors. In the 2-alkenal molecules, P1-ZCr catalyzed the hydrogenation of alpha,beta-unsaturated bonds, but not the reduction of the aldehyde moiety, to produce saturated aldehydes, as determined by gas chromatography/mass spectrometry. We propose the enzyme name NADPH:2-alkenal alpha,beta-hydrogenase (ALH). A major portion of the NADPH-dependent HNE-reducing activity in A. thaliana leaves was inhibited by the specific antiserum against P1-ZCr, indicating that the endogenous P1-ZCr protein has ALH activity. Because expression of the P1-ZCr gene in A. thaliana is induced by oxidative stress treatments, we conclude that P1-ZCr functions as a defense against oxidative stress by scavenging the highly toxic, lipid peroxide-derived alpha,beta-unsaturated aldehydes.     

Phenotypic characteristics and pathogenicity of Aeromonas genomospecies isolated from common carp (Cyprinus carpio L.)

AIMS: To evaluate the relationship between the genomospecies, phenotypic profile and pathogenicity for carp of 37 motile Aeromonas strains. METHODS AND RESULTS: Aeromonas strains were identified to genomospecies level by the 16S rDNA restriction fragment length polymorphism (RFLP) method and characterized phenotypically by the API 20E and API Zym systems and by conventional tube or plate methods. 16S rDNA RFLP analysis showed that the strains belonged to five species, Aeromonas bestiarum (5), Aerom. salmonicida (13), Aerom. veronii (11), Aerom. sobria (6) and Aerom. encheleia (2). Most strains of Aerom. bestiarum (80%) and Aerom. salmonicida (85%) could be separated by growth at 4 and 42 degrees C, autoagglutination after boiling, reaction for lipase (C14) and naphthol-AS-BI-phosphohydrolase. All strains of Aerom. veronii corresponded to Aerom. veronii biotype sobria and could be separated from Aerom. sobria by citrate utilization, growth at 37 and 42 degrees C, amygdalin and cellobiose fermentation. All strains of Aerom. bestiarum and most strains of Aerom. salmonicida (76.9%) and Aerom. veronii (63.6%) were pathogenic for carp. CONCLUSIONS: The biochemical identification of carp Aeromonas strains is not entirely clear. Some association between Aeromonas species, phenotypic profile and specific disease signs was observed. SIGNIFICANCE AND IMPACT OF THE STUDY: The results will be useful for ichthyopathology laboratories in the diagnosis of motile aeromonad septicaemia in carp.     

Donation of Electrons to Plastoquinone by NAD(P)H Dehydrogenase and by Ferredoxin-Quinone Reductase in Spinach Chloroplasts

The reduction of plastoquinone by NADPH was detected as an increasein the dark level of Chi fluorescence in osmotically rupturedchloroplasts of spinach. This activity was observed only whenthe chloroplasts were ruptured in a medium containing a highconcentration of MgCl₂. The activity was suppressed by inhibitorsof the respiratory NADH dehydrogenase (NDH) complex in mitochondria,capsaicin and amobarbital, suggesting that the activity wasmediated by chloroplastic NDH complex. Antimycin A, an inhibitorof ferredoxin-quinone reductase (FQR), and the protonophorenigericin also inhibited the increase in Chi fluorescence byNADPH. By contrast, JV-ethylmaleimide (NEM), an inhibitor offerredoxin-NADP⁺ reductase (FNR), did not suppress the fluorescenceincrease, showing that FNR is not involved in this reaction.When the osmotically ruptured chloroplasts were washed by centrifugation,a further addition of ferredoxin as well as NADPH was requiredfor an increase in fluorescence. This ferredoxin-de-pendentactivity also was suppressed by antimycin A, but only partlyinhibited by capsaicin or amobarbital, suggesting that thisis mediated mainly by FQR. These findings suggest that the NADPH-bindingsubunit of NDH complex is easily dissociated from the thylakoidmembranes during the process of the washing the thylakoids bycentrifugation. ³Present address: Shanghai Institute of Plant Physiology, AcademiaSinica, 300 Fenglin Road, Shanghai 200032, China ⁵Present address: Department of Biotechnology, Faculty of Engineering,Fukuyama University, 1 Gakuen-cho, Fukuyama, Hiroshima, 729-02Japan     

Molecular Structure of a High-Potential Form of Thylakoid Cytochrome b-559 as Determined by Radiation Inactivation

Cytochrome b-559 in photosystem II can be characteristicallyconverted from a high- to a low-potential form. Taking thisresponse of Cyt b-559 as evidence for the denaturation of proteinmolecules, the sizes of the structures that stabilize the high-potentialform of Cyt b-559 in PS II membranes and thylakoids from spinachwere determined by radiation inactivation. When a target of26 kDa was inactivated in PS II membranes, Cyt b-559 was convertedto the low-potential form. The size was consistent with a molecularweight of Cyt b-559 in a proposed tetrameric structure thatconsists of two sets of 9.2-kDa and 4.3-kDa subunits [Widgeret al. (1985) FEBS Lett. 191: 186–190]. In contrast tothe functional size of 26 kDa in the PS II membranes, the functionalsize was 116 kDa in thylakoid membranes. The results suggestthe presence of an extra 90-kDa electron carrier between a redoxtitrator outside the membranes and the Cyt b-559, which maynot expose its active site to the surface of the thylakoids. (Received March 9, 1989; Accepted June 23, 1989)     

Selective Iodo-Labeling of an Intrinsic Electron Donor of Photosystem II in Illuminated Tris-Treated Thylakoids

Peptides that participate in the oxidizing side of PS II wereselectively tagged with iodine, activated by illuminated Tris-treatedthylakoids. A peptide of 29 kDa was iodinated, with less labelingof 33- and 58-kDa peptides. The iodination was inhibited byDCMU, and suppressed when the Tris-treated thylakoids were supportedby diphenylcarbazide. No peptide in the untreated thylakoidswas iodinated. These findings indicate that the oxidation ofI^– at the oxidizing side of photosystem II results iniodination. The iodinated peptide of 29 kDa was identified to be one ofthe major components of the isolated PS II reaction center complexfrom the following characteristics of the peptide: 1) polymerizationon heat-treatment (95C, 5 min) of the iodinatcd thylakoidsin the presence of SDS and 2-mercaptoethanol, 2) thylakoid-bindingafter NaSCN-washing, and 3) migration distinct from that ofthe herbicide-binding protein in SDS-polyacrylamide gel electrophoresis.Thus, the iodinated 29-kDa peptide is a secondary electron donorof PS II or the nearest one to a site where I^– is oxidized. (Received March 13, 1985; Accepted June 5, 1985)     

Intact chloroplasts display pH 5 optimum of O2-reduction in the absence of methyl viologen: Indirect evidence for a regulatory role of superoxide protonation

The pH-dependence of light-driven O₂-reduction in intact spinach chloroplasts is studied by means of chlorophyll fluorescence quenching analysis and polarographic O₂-uptake measurements. Most experiments are carried out in presence of KCN, which blocks activities of Calvin cycle, ascorbate peroxidase and superoxide dismutase. pH is varied by equilibration with external buffers in presence of nigericin. Vastly different pH-optima for O₂-dependent electron flow are observed in the presence and absence of the redox catalyst methyl viologen. Both fluorescence quenching analysis and O₂-uptake reveal a distinct pH 5 optimum of O₂-reduction in the absence of methyl viologen. In the presence of this catalyst, O₂-reduction is favoured in the alkaline region, with an optimum around pH 8, similar to other types of Hill reaction. It is suggested that in the absence of methyl viologen the extent of irreversibility of O₂-reduction is determined by the rate of superoxide protonation. This implies that O₂-reduction takes place within the aprotic phase of the thylakoid membrane and that superoxide-reoxidation via oxidized PS I donors competes with protonation. Superoxide protonation is proposed to occur at the internal surface of the thylakoid membrane. There is no competition between superoxide reoxidation and protonation when in the presence of methyl viologen the site of O₂-reduction is shifted into the protic stroma phase. In confirmation of this interpretation, fluorescence measurements in the absence of KCN reveal, that non-catalysed O₂-dependent electron flow is unique in beingstimulated by the transthylakoidal pH-gradient. On the basis of these findings a major regulatory role of O₂-dependent electron flow under excess light conditions is postulated.