The regulation of glutamate dehydrogenase,nitrite reductase,and nitrate reductase in excised pea roots by nitrite

Both nitrite reductase and nitrate reductase were induced by nitrite, but there were differences in the time course of induction and in the response to different NO₂ ^- concentrations between these enzymes. NH₄ ⁺ depressed the induction of nitrite reductase. NADH₂ dependent glutamate dehydrogenase activity was enhanced by those NO₂-concentrations in the medium at which unmetabolized NO₂ ^- occurred in the roots. NADPH₂ and NAD+ dependent GDh activities were not affected. In vivo modification and (or) in vivo activation were probably responsible for the increase in NADH₂ dependent GDH activity.     

Purification and properties of thiosulfate reductase from Desulfovibrio gigas

Thiosulfate reductase of the dissimilatory sulfate-reducing bacterium Desulfovibrio gigas has been purified 415-fold and its properties investigated. The enzyme was unstable during the different steps of purification as well as during storage at-15°C. The molecular weight of thiosulfate reductase estimated from the chromatographic behaviour of the enzyme on Sephadex G-200 was close to 220 000. The absorption spectrum of the purified enzyme exhibited a protein peak at 278 nm without characteristic features in the visible region. Thiosulfate reductase catalyzed the stoichiometric production of hydrogen sulfide and sulfite from thiosulfate, and exhibited tetrathionate reductase activity. It did not show sulfite reductase activity. The optimum pH of thiosulfate reduction occurred between pH 7.4 and 8.0 and its K _m value for thiosulfate was calculated to be 5·10^-4 M. The sensitivity of thiosulfate reductase to sulfhydryl reagent and the reversal of the inhibition by cysteine indicated that one or more sulfhydryl groups were involved in the catalytic activity. The study of electron transport between hydrogenase and thiosulfate reductase showed that the most efficient coupling was obtained with a system containing cytochromes c ₃ (M _r =13000) and c ₃ (M _r =26000).     

The Effect of Exogenous IAA and Kinetin on Nitrate Reductase,Nitrite Reductase and Glutamate Dehydrogenase Activities in Excised Pea Roots

Nitrate reductase (NO₃R) activity, nitrite reductase (NO₂R) activity and NADH₂ dependent glutamate dehydrogenase (GDH) activity were followed in extracts from excised pea roots incubated under aseptic conditions for 9 and 24 h in nitrate containing nutrient medium to which IAA was added in concentrations promoting lateral root formation (1 × 10^?5; 3 × 10^?5; 5 × 10^?5 M) and kinetin in concentrations which reduce lateral root formation (0.1; 1; 5 mg 1^?1, that is 4.65 × 10^?7;4.65 × 10^?6 and 2.3 × 10^?5 M). NO₃R activity was not influenced by IAA, NO₂R activity was slightly depressed by IAA after 24 h incubation and GDH activity was slightly increased after 24 h incubation in the presence of IAA. Kinetin decreased NO₃R activity significantly both after 9 h and 24 h incubation, slightly increased NO₂R activity after 9 h incubation but slightly decreased it after 24 h incubation, and did not affect GDH activity after 24 h incubation. However, when applied together with IAA, kinetin abolished the promoting effect of IAA on GDH activity. IAA neither reversed nor accentuated the effect of kinetin on NO₂R activity. Nevertheless the depressing effect of kinetin on NO₃R activity was emphasized by the presence of IAA after 9 h incubation. The results obtained indicate that reduced nitrate assimilation due to the depression of nitrate reductase activity caused by kinetin probably contributes to the negative growth effect of kinetin in pea root segments grown in nitrate medium.     

Evidence for multiple phosphorylation sites in rat liver 3-hydroxy-3-methylglutaryl Coenzyme A reductase

Microsomal 3-hydroxy-3-methylglutaryl Coenzyme A reductase (EC 1.1.1.34) was inactivated by [γ-³²P]ATP in the presence of endogenous reductase kinases, solubilized, and purified 575-fold with retention of³²P to a state where phosphoreductase was the only³²P-labeled protein present.³²P comigrated with reductase activity under nondenaturing conditions (polyacrylamide gets, isoelectric focusing gels) and with reductase monomer under denaturing conditions (sodium dodecyl sulfate-polyacrylamide gels). Polyfunctional antibody to homogeneous reductase precipitated all of the³²P present. The phosphate-reductase bond was acid-stable and base-labile. Following acid hydrolysis and high-voltage electrophoresis,³²P label migrated solely with phosphoserine and inorganic orthophosphate. Exhaustive (>100 h) tryptic digestion of phosphoreductase denatured in 2 M urea yielded two major phosphorylated components as judged by high-performance liquid chromatography or Sephadex G-25 chromatography. 3-Hydroxy-3-methylglutaryl Coenzyme A reductase inactivated in the microsomal state by [γ-³²P]ATP is thus phosphorylated exclusively at seryl residues and contains two structurally distinct phosphorylation sites.     

Nitrate reductase of Dunaliella parva

Nitrate reductase of the salt tolerant alga Dunaliella parva, in contrast to that of most green algae, can use NADPH as well as NADH as electron donor. Extracts of cells contained various amounts of latent nitrate reductase. The latent enzyme could be activated at 45°C but only in the presence of flavine adenine dinucleotide. The heat activated enzyme did not require flavine adenine dinucleotide for activity and was fully active with NADH, NADPH or reduced flavine mononucleotide as electron donors.     

Enzyme(s) responsible for tellurite reducing activity in a moderately halophilic bacterium,Salinicoccus iranensis strain QW6

Oxyanions of tellurium, like tellurate (TeO₄ ^2?) and tellurite (TeO₃ ^2?), are highly toxic for most microorganisms. There are a few reports on the bacterial tellurite resistance mechanism(s). Salinicoccus iranensis, a Gram-positive halophilic bacterium, shows high tellurite resistance and NADH-dependent tellurite reduction activity in vitro. Since little is known regarding TeO₃ ^2? resistance mechanisms in halophilic microorganisms, here one of the enzymatic reduction activities presented in this microorganism is investigated. To enhance the enzymatic activity during purification, the effect of different parameters including time, inoculation, different pHs, different tellurite concentrations and different salts were optimized. We also examined the tellurite removal rates by diethyldithiocarbamate (DDTC) during optimization. In the culture medium the optimum conditions obtained showed that at 30 h, 2 % inoculum, pH 7.5, without tellurite and with 5 % NaCl (w/v) the highest enzyme activity and tellurite removal were observed. Results of the purification procedure done by hydroxyapatite batch-mode, ammonium sulfate precipitation, followed by phenyl-Sepharose and Sephadex G-100 column chromatography, showed that the enzyme consisted of three subunits with molecular masses of 135, 63 and 57 kDa. In addition to tellurite reduction activity, the enzyme was able to reduce nitrate too. Our study extends the knowledge regarding this process in halophilic microorganisms. Besides, this approach may suggest an application for the organism or the enzyme itself to be used for bioremediation of polluted areas with different contaminants due to its nitrate reductase activity.     

Leucine aminopeptidase and ferricyanide reductase activities in radish microsomes

A NADH-ferricyanide reductase activity found in radish microsomes isolated from germinated seeds has been shown to be stimulated by pCMB and pCMBS which are both strong nactivators of many plant proteolytic enzymes. In the same preparation a leucine aminopeptidase was found while endoprotease and carboxypeptidase activities were not detected using exogenous substrates. The aminopeptidase, highly active at the same optimal pH-condition of FeCN reductase, was stimulated by CoCl₂ and non-polar detergents (Triton X-100 and Brij 35). It was inhibited by sulphydryl reagents. By gel filtration of microsomal detergent extract two peaks of activity were separated: red I coeluted with LeuAPase and red II, free of aminopeptidase. Red I, a protein, was inhibited by sulphydral reagents and stimulated by duroquinone. Red II, stimulated by pCMB, is not a protein because of the small size and the noninfluence of heating treatment on catalytic activity.     

Nitrate content and nitrate reductase activity in Rumex obtusifolius L.

Summary With Rumex obtusifolius L., the influence of some environmental conditions on nitrate uptake and reduction were investigated. Nitrate concentrations of plant material were determined by HPLC, the activity of nitrate reductase by an in vivo test. As optimal incubation medium, a buffer containing 0.04 M KNO₃; 0.25 M KH²PO₄; 1.5% propanol (v/v); pH 8.0 was found. Vacuum infiltration caused an increase of enzyme activity of up to 40%.High nitrate concentrations were found in roots and leaf petioles. Nitrate reductase activity of these organs, however, was low. On the other hand, the highest nitrate reductase activity was observed in leaf laminae, which contained lowest nitrate concentrations.In leaves, nitrate content and nitrate reductase activity exhibited inverse diurnal fluctuations. During darkness, decreasing activities of the enzyme were followed by increasing nitrate concentrations, while during light the contrary was true. In petioles diurnal fluctuations in nitrate content were observed, too. No significant correlations with illumination, however, could be found.Our results prove that Rumex obtusifolius is characterized by an intensive nitrate turnover. Theoretically, internal nitrate content of the plant would be exhausted within a few hours, if a supply via the roots would be excluded.     

Cloning,purification and characterization of novel Cu-containing nitrite reductase from the <Emphasis Type="Italic">Bacillus firmus</Emphasis> GY-49

Nitrite is generated from the nitrogen cycle and its accumulation is harmful to environment and it can be reduced to nitric oxid by nitrite reductase. A novel gene from Bacillus firmus GY-49 is identified as a nirK gene encoding Cu-containing nitrite reductase by genome sequence. The full-length protein included a putative signal peptide of 26 amino acids and shown 72.73% similarity with other Cu-containing nitrite reductase whose function was verified. The 993-bp fragment encoding the mature peptide of NirK was cloned into pET-28a (+) vector and overexpressed as an active protein of 36.41 kDa in the E.coli system. The purified enzyme was green in the oxidized state and displayed double gentle peaks at 456 and 608 nm. The specific activity of purified enzyme was 98.4 U/mg toward sodium nitrite around pH 6.5 and 35 °C. The K _m and K _cat of NirK on sodium nitrite were 0.27 mM and 0.36?×?10³ s^?1, respectively. Finally, homology model analysis of NirK indicated that the enzyme was a homotrimer structure and well conserved in Cu-binding sites for enzymatic functions. This is a first report for nitrite reductase from Bacillus firmus, which augment the acquaintance of nitrite reductase.     

The activities of hydrogenase and enoate reductase in two Clostridium species,their interrelationship and dependence on growth conditions

The enzyme activities of Clostridium La 1 and Clostridium kluyveri involved in the stereospecific hydrogenation of ,-unsaturated carbonyl compounds with hydrogen gas were measured. In C. La 1 the specific activities of hydrogenase and enoate reductase depended heavily on the growth phase and the composition of the medium. During growth in batch cultures on 70 mM crotonate the specific activity of hydrogenase increased and then dropped to about 10% of its maximum value, whereas the activity of enoate reductase reached its maximum in cells of the stationary phase. Under certain conditions during growth the activity ratio hydrogenase: enoate reductase changed from 120 to 1. Thus, the rate limiting enzyme for the hydrogenation can be either the hydrogenase or the enoate reductase, depending on the growth conditions of the cells.The specific activities of ferredoxin-NAD reductase and butyryl-CoA dehydrogenase increased 3-4-fold during growth on crotonate. By turbidostatic experiments it was shown that at constant input of high crotonate concentrations (200 mM) the enoate reductase activity was almost completely suppressed; it increased steadily with decreasing crotonate down to an input concentration of 35 mM.Glucose as carbon source led to high hydrogenase and negligible enoate reductase activities. The latter could be induced by changing the carbon source of the medium from glucose to crotonate. Tetracycline inhibited the formation of enoate reductase.A series of other carbon sources was tested. They can be divided into ones which result in high hydrogenase and rather low enoate reductase activities and others which cause the reverse effect.When the Fe²⁺ concentration in crotonate medium was growth limiting, cells with relatively high hydrogenase activity and very low enoate reductase activity in the stationary phase were obtained. At Fe²⁺ concentrations above 3·10^-7 M enoate reductase increased and hydrogenase activity reached its minimum. The ratio of activities changes by a factor of about 200. In a similar way the dependence of enzyme activities on the concentration of sulfate was studied.In batch cultures of Clostridium kluyveri a similar opposite time course of enoate reductase and hydrogenase was found.The possible physiological significance of this behavior is discussed.Non Standard Abbreviations O.D.₅₇₈ Optical density at 578 nm Dedicated to Professor Dr. O. Kandler on the occasion of his 60th birthday     

Thioredoxin/thioredoxin reductase system involvement in cerebellar granule cell apoptosis

The involvement of thioredoxin/thioredoxin reductase system has been investigated in cerebellar granule cells (CGCs), a cellular system in which neurons are induced in apoptosis by the physiological stimulus of lowering extracellular potassium. Clarifying the sequence of events that occur during apoptosis is a critical issue as it can lead to the identification of those key events that, if blocked, can slow down or reverse the death process. The results reported in this work show that TrxR is involved in the early phase of CGC apoptosis with an increase in activity that coincides with the increased expression of the TrxR1 isoform and guarantees the maintenance of adequate level of Trx in its reduced, active form. However, in late apoptosis, when about 50 % of cells are dead, partial proteolysis of TrxR1 by calpain occurs and the reduction of TrxR1 mRNA, together with the overall decrease in TrxR activity, contribute to increase the levels of the oxidized form of Trx. When the reduced form of Trx is externally added to apoptotic cultures, a significant reduction in cell death is achieved confirming that a well-functioning thioredoxin/thioredoxin reductase system is required for survival of CGCs.     

Cadmium,mercury, and lead effects on gill tissue of freshwater crayfishProcambarus clarkii (girard)

Intermolt adult crayfishP. clarkii were used for this work. After acclimatation to laboratory conditions crayfish were exposed to sublethal concentrations of cadmium, mercury, and lead for 96 h. Gills of control and exposed crayfish were removed and ATPase activity and oxygen uptake rate were determined. Structural damage of gill filaments was also observed. Gill tissue respiration rates were measured for individual crayfish using a Gilson differential respirometer. Lead causes a decrease of gill oxygen uptake, but neither cadmium nor mercury seems to affect it at the concentrations employed. Although all metals studied alter gill filament structure, lead damage is the most apparent. In the same way, significant differences in gill ATPase activity owing to metal exposure were only observed in lead treated crayfish.     

Effect of sublethal doses of copper sulphate and bayluscide on survival and oxygen consumption of the snail biomphalaria alexandrina

The survival rate and oxygen consumption of Biomphalaria alexandrina snails exposed to sublethal concentrations of copper sulphate and Bayluscide were investigated. Prolonged exposure periods to the sublethal concentrations increased the percentage mortality among the exposed snails. Although the snails exposed to lower concentrations of the sublethal doses, were apparently normal, their oxygen consumption was markedly lower than the control ones. The rate of reduction of oxygen consumption by the experimental snails increased significantly at higher concentrations. The reduction of oxygen consumption was also presistent at prolonged exposure periods.     

Aflatoxin in human and camel milk in Abu Dhabi,United Arab Emirates

During an initial survey, using thin layer chromatography, 10 of 64 samples of mothers’ breast milk, collected from donors at the Corniche Maternity Hospital and the Al-Nehyan Clinic for Maternity and Childhood, were found to contain Aflatoxin M₁ at concentrations ranging from 0.3 to 1.3 ng mL^?1. A second survey using HPLC showed Aflatoxin M₁ at concentrations ranging from 7 to 23 pg mL^?1 in all of the 15 samples collected. 6 of 20 samples of camel milk collected from several sources in Abu Dhabi were also found to contain Aflatoxin M₁ at levels ranging from 0.25 to 0.8 ng mL^?1.     

Formyl-met-tRNA inff supMet,a questionable initiator tRNA in eukaryotic systems

Met-tRNA _inff ^supMet is a much more efficient initiator in cell-free eukaryotic protein synthesis than the formylated species. Chemically and enzymatically formylated Met-tRNA _inff ^supMet exhibit the same activity. In contrast acetyl Met-tRNA _inff ^supMet is unable to initiate polypeptides in vitro.     

The occurrence of nitrate reductase in apple leaves

Nitrate reductase utilizing NADH or reduced flavin mononucleotide (FMNH₂) as electron donor was extracted from the leaves, stems and petioles, and roots of apple seedlings. Successful extraction was made possible by the use of insoluble polyvinylpyrrolidone (Polyclar AT) which forms insoluble complexes with polyphenols and tannins. The level of nitrate reductase per gram fresh weight was highest in the leaf tissue although the nitrate content of the roots was much higher than that of the leaves. Nitrite reductase activity was detected only in leaf extracts and was 4 times higher than nitrate reductase activity. Nitrate was found in all parts of young apple trees and trace amounts were also detected in mature leaves from mature trees. Nitrate reductase was induced in young leaves of apple seedlings and in mature leaves from 3 fruit-bearing varieties. An inhibitor of polyphenoloxidase, 2-mercaptobenzothiazole was used in both the inducing medium and the extracting medium in concentrations from 10⁻³ to 10⁻⁵m with no effect upon nitrate reductase activity.     

Purification and properties of N 5 ,N 10 -methylenetetrahydromethanopterin reductase (coenzyme F420-dependent) from the extreme thermophile Methanopyrus kandleri

Methanopyrus kandleri belongs to a novel group of abyssal methanogenic archaebacteria that can grow at 110°C on H₂ and CO₂ and that shows no close phylogenetic relationship to any methanogens known so far. N ⁵ N ¹⁰ -Methylenetetrahydromethanopterin reductase, an enzyme involved in methanogenesis from CO₂, was purified from this hyperthermophile. The apparent molecular mass of the native enzyme was found to be 300 kDa. Sodium dodecylsulfate/polyacrylamide gel electrophoresis revealed the presence of only one polypeptide of apparent molecular mass 38 kDa. The ultraviolet/visible spectrum of the enzyme was almost identical to that of albumin indicating the absence of a chromophoric prosthetic group. The reductase was specific for reduced coenzyme F₄₂₀ as electron donor; NADH, NADPH or reduced dyes could not substitute for the 5-deazaflavin. The catalytic mechanism was found to be of the ternary complex type as deduced from initial velocity plots. V _max at 65°C and pH 6.8 was 435 U/mg (k_cat=275 s^-1) and the K _m for methylenetetrahydro-methanopterin and for reduced F₄₂₀ were 6 M and 4 M, respectively. From Arrhenius plots an activation energy of 34 kJ/mol was determined. The Q ₁₀ between 40°C and 90°C was 1.5.The reductase activity was found to be stimulated over 100-fold by sulfate and by phosphate. Maximal stimulation (100-fold) was observed at a sulfate concentration of 2.2 M and at a phosphate concentration of 2.5 M. Sodium-, potassium-, and ammonium salts of these anions were equally effective. Chloride, however, could not substitute for sulfate or phosphate in stimulating the enzyme activity.The thermostability of the reductase was found to be very low in the absence of salts. In their presence, however, the reductase was highly thermostable. Salt concentrations between 0.1 M and 1.5 M were required for maximal stability. Potassium salts proved more effective than ammonium salts, and the latter more effective than sodium salts in stabilizing the enzyme activity. The anion was of less importance.The N-terminal amino acid sequence of the reductase from M. kandleri was determined and compared with that of the enzyme from Methanobacterium thermoautotrophicum and Methanosarcina barkeri. Significant similarity was found.Abbreviations H₄MPT tetrahydromethanopterin - CH₂=H₄MPT N ⁵ ,N ¹⁰ -methylene-H₄MPT - CH₃-H₄MPT N ⁵-methyl-H₄MPT - CHH₄MPT⁺ N ⁵ ,N ¹⁰ -methenyl-H₄MPT - F₄₂₀ coenzyme F₄₂₀; 1 U=1 mol/min