Rhythms in magnesium ion inhibition and hysteretic properties of nitrate reductase in the CAM plant Bryophyllum fedtschenkoi

Nitrate reductase (NR, EC 1.6.6.1) was tested in crude extracts of leaves from Bryophyllum fedtschenkoi plants growing under alternating light/darkness as well as in excised leaves kept in continuous light or darkness. In most extracts NR activity was inhibited 20–80% by 5 m M Mg²⁺ A light or darkness shift (30 min darkness) during the first part of the photoperiod gave an increase in the Mg²⁺ inhibition and a decrease in NR activity. Magnesium ion inhibition of NR also showed diurnal variations. Strongest inhibition was found in extracts made during the latter part of the photoperiod and start of the dark period. Pre-incubation of crude extracts with ATP increased Mg²⁺ inhibition, indicating that phosphorylation of NR is involved in regulation of NR in Crassulacean acid metabolism (CAM) plants. In continuous light an increase in Mg²⁺ inhibition occurred after 20 h and 40 h, indicating a rhythm in the phosphorylation of NR. A delay in the production of nitrite in the assay (hysteresis) was generally seen in extracts susceptible to Mg²⁺ inhibition. The rhythms related to NR activity showed the same period length (20 h) as the rhythm in CO₂ exchange. However, in contrast to the rhythm in CO₂ exchange, NR rhythms were strongly damped in continuous light. In constant darkness the rhythms were even more damped. The results show that post-translational modification of CAM NR is influenced by light/darkness and by an endogenous rhythm.     

Changes in malondialdehyde content and in superoxide dismutase, catalase and glutathione reductase activities in sunflower seeds as related to deterioration during accelerated aging

Sunflower ( Helianthus annuus L.) seeds progressively lost their ability to germinate at 25°C, the optimal temperature for germination, after accelerated aging was carried out at 45°C (a temperature too high to permit germination) in water or at 76 or 100% relative humidity (RH). The deleterious effects of the high-temperature treatment increased with increasing seed moisture content. Incubation of seeds at 45°C in water resulted in electrolyte leakage, which indicated a loss of membrane integrity. A relationship between leakage and loss of seed viability could not be assumed, since no increase in electrolyte efflux occurred after aging al 100% RH. Accelerated aging induced accumulation of malondialdehyde, suggesting that seed deterioration was associated with lipid peroxidation. However, there was no direct relationship between lipid peroxidation and deterioration in membrane integrity. Loss of seed viability was also associated with a decrease in superoxide dismutase, catalase and glutathione reductase activities. Finally, the results obtained suggest that sunflower seed deterioration during accelerated aging is closely related to a decrease in the activities of detoxifying enzymes and to lipid peroxidation.     

Regulation of nitrate reductase and phosphoenolpyruvate carboxylase activities in barley leaf protoplasts

Barley leaf protoplasts were incubated in light or darkness in the presence of various inhibitors, metabolites or weak acids/bases. Nitrate reductase (NR) and phosphoenolpyruvate carboxylase (PEPCase) were rapidly extracted from the protoplasts and assayed under sub-optimal conditions, i.e. in the presence of Mg²⁺ and malate, respectively. Under these conditions changes in activities are thought to reflect changes in the phosphorylation states of the enzymes. The NR was activated by illumination to 90% of its maximal activity within 10 min. Photosynthetic electron transport appeared necessary for light activation of NR since activation was inhibited by the photosynthetic electron-transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), and, additionally, an electron acceptor (HCO ₃ ^- ) was required. The PEPCase was also activated by light. However, this activation was not prevented by DCMU or lack of HCO ₃ ^- . Loading of protoplasts in the dark with a weak acid resulted in activation of both NR and PEPCase. For NR, full activation was completed within 5 min, whereas for PEPCase a slower, modest activation continued for at least 40 min. Incubation of protoplasts with a weak base also gave activation of PEPCase, but not of NR. On the contrary, base loading counteracted light activation of NR. Since several treatments tested resulted in the modulation of either NR or PEPCase activity, but not both, signal transduction cascades leading to changes in activities appear to be very different for the two enzymes.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea (diuron) - DMO 5,5-dimethyl-2,4 oxazolidinedione - NR nitrate reductase - PEPCase Phosphoenolpyruvate carboxylase This work was supported by the Norwegian Research Council by a Grant to C.L: L.H.S. was supported by the Biotechnology and Biological Sciences Research Council.     

Anaerobic metabolism of 2-hydroxybenzoic acid (salicylic acid) by a denitrifying bacterium

The anaerobic metabolism of 2-hydroxybenzoic acid (salicylic acid) was studied in a denitrifying bacterium. Cells grown with 2-hydroxybenzoate were simultaneously adapted to degrade benzoate. Extract of these cells formed benzoate or benzoyl-CoA when incubated under reducing conditions with salicylate, MgATP, and coenzyme A, suggesting a degradation of 2-hydroxybenzoate via benzoate or benzoyl-CoA. This suggestion was supported by enzyme activity measurements. In extracts of 2-hydroxybenzoate-grown cells, the following enzyme activities were detected: two CoA ligases, one specific for 2-hydroxybenzoate, the other for benzoate, and two different enzyme activities catalyzing the reductive transformation of 2-hydroxybenzoyl-CoA. These findings suggest a degradation of salicylic acid by two new enzymes, 2-hydroxybenzoate-CoA ligase (AMP-forming) and 2-hydroxybenzoyl-CoA reductase (dehydroxylating), catalyzing (1) 2-hydroxybenzoate + MgATP + CoASH → 2-hydroxybenzoyl-CoA + MgAMP + PP_i (2) 2-hydroxybenzoyl-CoA + 2[H] → benzoyl-CoA + H₂O Benzoyl-CoA was dearomatized by reduction of the ring. This represents another case in which benzoyl-CoA is a central intermediate in anaerobic aromatic metabolism. Received: 1 February 1996 / Accepted: 24 February 1996     

Reduction of methylglyoxal in Escherichia coli K12 by an aldehyde reductase and alcohol dehydrogenase

Two enzymes, one NADPH-dependent and another NADH-dependent which catalyze the reduction of methylglyoxal to acetol have been isolated and substantially purified from crude extracts of Escherichia coli K₁₂ cells. Substrate specificity and formation of acetol as the reaction product by both the enzymes, reversibility of NADH-dependent enzyme with alcohols as substrates and inhibitor study with NADPH-dependent enzyme indicate that NADPH-dependent and NADH-dependent enzymes are identical with an aldehyde reductase (EC 1.1.1.2) and alcohol dehydrogenase (EC 1.1.1.1) respectively. The K_m for methylglyoxal have been determined to be 0.77 mM for NADPH-dependent and 3.8 mM for NADH-dependent enzyme. Stoichiometrically equimolar amount of acetol is formed from methylglyoxal by both NADPH- and NADH-dependent enzymes. In phosphate buffer, both the enzymes are active in the pH range of 5.8–6.6 with no sharp pH optimum. Molecular weight of both the enzymes were found to be 100,000 ± 3,000 by gel filtration on a Sephacryl S-200 column. Both NADPH- and NADH-dependent enzymes are sensitive to sulfhydryl group reagents.     

The role of sulphur in detoxication of cadmium in young sugar beet plants

In young sugar beet plants cadmium suppressed the activity of nitrate reductase, glutamine synthetase and glutamate dehydrogenase, whereas sulphur exhibited a protective role towards activity of these enzymes, except of glutamine synthetase. Protein synthesis was suppressed in the absence of S in nutrient medium; the lowest level was at 10^-3 M Cd²⁺. Chloroplast pigment contents were increased by S while Cd²⁺, even in the lowest concentration, (10⁻⁵ M) showed a repressive effect. The highest concentrations of Cd²⁺ (10−3 M) caused a decrease in dry mass, whereas S induced its increase. Nitrate content was increased in the presence of Cd²⁺ and decreased by increased concentration of S. Acknowledgement: The authors acknowledge financial support of the Ministry for Science and Technology of Serbia. The paper was presented at 9th Congress of the Federation of European Societies of Plant Physiology, Brno, Czech Republic, 3–8 July 1994.     

Purification and characterization of ferredoxin-sulfite reductases from leek (Allium tuberosum) leaves

Ferredoxin-sulfite reductases (Fd-SiRs) [hydrogen-sulfide: ferredoxin oxidoreductase, EC 1.8.7.1] from leek leaves have been purified to homogeneity. The enzymes (SiR 1, SiR 2 and SiR 3) were separated by Mono Q chromatography. The collective molecular mass of the enzymes was estimated to be 65 kDa by gel filtration. In all three cases, subunit analysis by SDS-PAGE yielded a single protein band corresponding to a molecular mass of 64 kDa, indicating that the enzymes each exist as a monomer. In the oxidized forms, SiR 1, SiR 2 and SiR 3 all exhibited nearly identical absorption maxima at 279∼280, 389∼390, 588 and 714 nm, indicating that siroheme is involved in the catalysis of sulfite reduction. On enzymatic properties, SiR 1, SiR 2 and SiR 3 could only react with the physiological electron donor, feriedoxin. The enzymes exhibited different heat stabilities. The pH active curve obtained from SiR 2 was different from the others. Moreover, SiR 1 exhibited a lower Km value for ferredoxin than SiR 2. Although the N-terminal sequence was the same, the results of some enzymatic properties, amino acid analysis, and peptide mapping suggested the presence of the Fd-SiR isozymes in leek leaves.     

编码人疱疹病毒－6型核糖核苷酸还原酶和P41蛋白基因的克隆和序列测定

本文报道人疱疹病豢－６型（ＨＨＶ－６）ｐＳＴＹ２８ＤＮＡ片段的序列测定。应用分子克隆、缺损突变体（Ｄｃｌｅｔｉｏｎｍｕｔａｎｔ）制备和序列测定等技术,完成了３．９ｋｂＨＨＶ－６ｐＳＴＹ２８ＤＮＡ片段的全序列测定。经ＤＮＡＳＩＳ核酸蛋白软件分析,该片段含有两个开读框架（ＯＲＦ）核糖核苷酸还原酶（ＲＩＲ）ＯＲＦ有２４１４个核苷酸,可编码８０５个氨基酸;Ｐ４１蛋白由１１００个核苷酸组成。与其他疱疹病毒作氨基酸同源性比较,ＨＨＶ－６ＲｉＲ与人巨细胞病毒（ＨＣＭＶ）有高度同源性,最适记分（Ｏｐｔｉｍｉｚｅｄｓｃｏｒｅ）达４５９。实验结果支持Ｅｓｆｔａｔｈｉｏｕ提出的论点,ＨＨＶ－６属于β－疱疹病毒。     

Reversible ADP-ribosylation as a mechanism of enzyme regulation in procaryotes

Several cases of ADP-ribosylation of endogenous proteins in procaryotes have been discovered and investigated. The most thoroughly studied example is the reversible ADP-ribosylation of the dinitrogenase reductase from the photosynthetic bacteriumRhodospirillum rubrum and related bacteria. A dinitrogenase reductase ADP-ribosyltransferase (DRAT) and a dinitrogenase reductase ADP-ribose glycohydrolase (DRAG) fromR. rubrum have been isolated and characterized. The genes for these proteins have been isolated and sequences and show little similarity to the ADP-ribosylating toxins. Other targets for endogenous ADP-ribosylation by procaryotes include glutamine synthetase inR. rubrum andRhizobium meliloti and undefined proteins inStreptomyces griseus andPseudomonas maltophila.     

Mechanism of dissimilatory sulfite reduction by Desulfovibrio desulfuricans: purification of a membrane-bound sulfite reductase and coupling iwth cytochrome c 3 and hydrogenase

The localization of the dissimilatory sulfite reductase in Desulfovibrio desulfuricans strain Essex 6 was investigated. After treatment of the cells with lysozyme, 90% of the sulfite reductase activity was found in the membrane fraction, compared to 30% after cell rupture with the French press. Sulfite reductase was purified from the membrane (mSiR) and the soluble (sSiR) fractiion. On SDS-PAGE, both mSiR and sSiR exhibited three bands at 50, 45 and 11 kDa, respectively. From their UV/VIS properties (distinct absorption maxima at 391, 410, 583, 630 nm, enzymes as isolated) and the characteristic red fluorescence in alkaline solution, mSiR and sSiR were identified as desulfoviridin. Sulfite reductase (HSO₃ ^-H₂S) activity was reconstituted by coupling of mSiR to hydrogenase and cytochrome c ₃ from D. desulfuricans. The specific activity of mSiR was 103 nmol H₂ min^-1 mg^-1, and sulfide was the major product (72% of theoretical yield). No coupling was found with sSiR under these conditions. Furthermore, carbon monoxide was used to diferentiate between the membrane-bound and the soluble sulfite reductase. In a colorimetric assay, with photochemically reduced methyl viologen as redox mediator, CO stimulated the activity of sSiR significantly. CO had no effect in the case of mSiR. These studies documented that, as isolated, both forms of sulfite reductase behaved differently in vitro. Clearly, in D. desulfuricans, the six electron conversion HSO₃ ^-H₂S was achieved by a membranebound desulfoviridin without the assistance of artificial redox mediators, such as methyl viologen.Abbreviations SiR sulfite reductase - mSiR sulfite reductase purified from membranes - sSiR sulfite reductase purified from the soluble fraction Enzymes Sulfite reductase, EC 1.8.99.1 Cytochrome c ₃ hydrogenase, EC 1.12.2.1