Hemoprotein models based on a covalent helix-heme-helix sandwich. 3. Coordination properties, reactivity and catalytic application of Fe(III)- and Fe(II)-mimochrome I

 The coordination state of Fe(III)- and Fe(II)-mimochrome I, a covalent peptide-deuteroheme sandwich involving two nonapeptides bearing a histidine residue in a central position, was studied by UV-visible, EPR, and resonance Raman spectroscopy. The ferric and ferrous states of this new iron species mainly exist, at pH 7, in a low-spin hexacoordinate form with two axial histidine ligands coming from the peptide chains. A minor amount of high-spin form for the ferric state is also present at pH 7. However, it is mainly high-spin at pH 2 or in DMSO. Fe(II)-mimochrome I binds CO with an affinity comparable to that of myoglobin and hemoglobin. Fe(III)-mimochrome I reacts with alkylhydroxylamine and arylhydrazines, leading to the corresponding Fe(II)-nitrosoalkyl and Fe(III)-σ-aryl complexes, respectively. These reactions were greatly dependent on the solvent used and on the pH, and were much slower than the corresponding reactions performed by deuterohemin in the presence of excess imidazole. All these results indicate that the reactivity of iron-mimochrome I is controlled by the binding of the peptide chains to the iron. The reactivity shown by this complex at neutral pH is intermediate between that observed for iron porphyrins in the presence of excess imidazole and that of hemoproteins characterized by a strong bis-histidine axial coordination, such as cytochrome b ₅. Fe(III)-mimochrome I is able to catalyze styrene epoxidation by using a [Fe(III)-mimochrome I]/[H₂O₂]/[stryrene] ratio of 1 : 10 : 2000 in phosphate buffer solution (pH 7.2) containing 2% CTAB both under strictly anaerobic conditions and in the presence of oxygen, at 0  °C. Received: 26 May 1998 / Accepted: 20 August 1998     

Intramolecular electron transfer in the oxidation of amines by methylamine oxidase from Arthrobacter P1

 The intramolecular electron-transfer rate constant for the Cu(II)–topa_NH2⇌ Cu(I)–topa_SQ equilibrium in methylamine oxidase has been measured by temperature-jump relaxation techniques. At pH 7.0 the estimated k_obs = 150±30 s^–1 for both methylamine and benzylamine; assuming the equilibrium constant is ≈0.7–1 at pH 7.0 and 296 K, this would correspond to a forward electron-transfer rate constant k_ET≈ 60–75 s^–1. Although substantially slower than the previously determined k_ET≈ 20 000 s^–1 for pea seedling amine oxidase [5] steady-state kinetics measurements established that k_ET > k_cat≈ 4–10 s^–1. Thus the Cu(I)-semiquinone state is a viable intermediate in methylamine oxidase turnover. Received: 16 August 1995 / Accepted: 21 December 1995     

The amelioration of aluminium toxicity by silicon in wheat (Triticum aestivum L.): malate exudation as evidence for an in planta mechanism

Two wheat (Triticum aestivum L.) cultivars, one aluminium tolerant (Atlas 66) and one sensitive (Scout 66), were grown in a continuous-flow culture system (≤pH 5.0) containing aluminium (0–100 μM) and silicon (0–2000 μM) in factorial combination. Treatment with silicon resulted in a highly significant amelioration of aluminium toxicity as assessed by root growth in both cultivars. Amelioration was influenced by wheat cultivar and silicon concentration, as 2000 μM silicon significantly ameliorated the toxic effects of 100 μM aluminium in Atlas 66, and only 5 μM silicon alleviated the effect of 1.5 μM aluminium on Scout 66. Nutrient medium pH was critical, as an amelioration by silicon was apparent only at pH > 4.2 for Atlas 66, and at pH > 4.6 for Scout 66. Silicon neither reduced levels of toxic aluminium species in the growth solutions, nor the amount of aluminium taken up by roots. In experiments to assess exudation of malate by Atlas 66 roots treated with 100 μM aluminium, the presence of 2000 μM silicon (pH 4.6) was found to have a negligible effect on exudation. In contrast, citrate, a known aluminium chelator, reduced aluminium-induced exudation of malate at 5–40 μM and completely inhibited it at 100 μM citrate. The results indicate that silicon does not reduce aluminium phytotoxicity as a result of aluminium/silicon interactions in the external media, and that the mechanism of amelioration has an in planta component. Received: 22 April 1997 / Accepted: 16 August 1997     

DNA molecular recognition and cellular selectivity of anticancer metal(II) complexes of ethylenediaminediacetate and phenanthroline: multiple targets

Abstract  

By inhibiting only two or three of 12 restriction enzymes, the series of [M(phen)(edda)] complexes [M(II) is Cu, Co, Zn; phen is 1,10-phenanthroline; edda is N,N′-ethylenediaminediacetate] exhibit DNA binding specificity. The Cu(II) and Zn(II) complexes could differentiate the palindromic sequences 5′-CATATG-3′ and 5′-GTATAC-3′, whereas the Co(II) analogue could not. This and other differences in their biological properties may arise from distinct differences in their octahedral structures. The complexes could inhibit topoisomerase I, stabilize or destabilize G-quadruplex, and lower the mitochondrial membrane potential of MCF7 breast cells. The pronounced stabilization of G-quadruplex by the Zn(II) complex may account for the additional ability of only the Zn(II) complex to induce cell cycle arrest in S phase. On the basis of the known action of anticancer compounds against the above-mentioned individual targets, we suggest the mode of action of the present complexes could involve multiple targets. Cytotoxicity studies with MCF10A and cisplatin-resistant MCF7 suggest that these complexes exhibit selectivity towards breast cancer cells over normal ones.     

Comparative estimation of the neurotropicity of trace elements in the organism of children living in industrial towns of Ukraine

In 10- to 16-year-old children, inhabitants of industrial towns of Ukraine, an X-ray fluorescence analysis of the content of some chemical elements in hair samples allowed us to find a deficiency of Zn and Cu against the background of excesses of Ca, Cr, Ni, Mo, and Cd. Comparison of the parameters of EEG frequency components and levels of Ca, Zn, Cu, Fe, Mn, Co, Cr, Mo, Ni, Sr, Pb, and Cd showed that there are significant correlations between the normalized values of spectral powers of many frequency components of the ongoing EEG recorded in different functional states (eyes closed/open, solving an arithmetic task, and phono-/photostimulation) and concentrations of ten of the above-mentioned trace elements. Comparative estimation of the neurotropicity of the elements showed the following sequence of numbers of significant correlations (shown in parentheses) from the total set of possible comparisons: Cd (35) > Ni (31) > Cr (19) > Sr (17) > Pb (16) > > Ca (10) > Cu (7) > Mo (3) > Zn (2) > Fe (1). The intensity of correlations (values of the correlation coefficients) varied from 0.26 to 0.42 at 0.05 < P < 0.001; more frequently, such correlations were observed under conditions of EEG recording with the eyes closed (39) and upon solving an arithmetic task (33).     

Helix-helix transitions in DNA: fibre X-ray study of the particular cases poly(dG-dC) · poly(dG-dC) and poly(dA) · 2poly(dT)

The helix-helix transitions which occur in poly(dG-dC) · poly(dG-dC) and in poly (dG-m⁵dC) · poly(dG-m⁵dC) are commonly assumed to be changes between the right-handed A- or B-DNA double helices and the left-handed Z-DNA structure. The mechanisms for such transconformations are highly improbable, especially when they are supposed to be active in long polynucleotide chains organised in semicrystalline fibres. The present alternative possibility assumes that rather than the Z-DNA it is a right-handed double helix (S-DNA) which actually takes part in these form transitions. Two molecular models of this S form, in good agreement with X-ray measurements, are proposed. They present alternating C(2′)-endo and C(3′)-endo sugar puckering like the “alternating B-DNA” put forward some years ago. Dihedral angles, sets of atomic coordinates and stereo views of the two S-DNA structures are given, together with curves of calculated diffracted intensities. Furthermore, we question the possibility of obtaining semicrystalline fibres with triple helices of poly(dA) · 2poly(dT) in a way which renders X-ray diffraction efficient. It is suggested that, up to now, only double helices of poly(dA) · poly(dT) can actually be observed by fibre X-ray diffraction measurements. Received: 30 March 1999 / Revised version: 30 June 1999 / Accepted: 30 June 1999     

β-Glycosidase (amygdalase and linamarase) from Endomyces fibuliger (LU677): formation and crude enzyme properties

In our previous studies, the yeast Endomyces fibuliger LU677 was found to degrade amygdalin in bitter apricot seeds. The present investigation shows that E. fibuliger LU677 produces extracellular β-glycosidase activity when grown in malt extract broth (MEB). Growth was very good at 25 °C and 30 °C and slightly less at 35 °C. When grown in MEB of pH 5 and pH 6 with addition of 0, 10 or 100 ppm amygdalin, E. fibuliger produced only slightly more biomass at pH 5, and was only slightly inhibited in the presence of amygdalin. Approximately, 60% of the added amygdalin was degraded (fastest at 35 °C) during an incubation period of 5 days. Supernatants of cultures grown at 25 °C and pH 6 for 5 days were tested for the effects of pH and temperature on activity (using amygdalin, linamarin and prunasin as substrates). Prunase activity had two pH optima (pH 4 and pH 6), amygdalase and linamarase only one each at pH 6 and pH 4–5 respectively. The linamarase activity evolved earlier than amygdalase (2 days and 4 days respectively). The data thus indicate the presence of at least two different glycosidases having different pH optima and kinetics of excretion. In the presence of amygdalin, lower glycosidase activities were generally produced. However, the amygdalin was degraded from the start of the growth, strongly indicating an uptake of amygdalin by the cells. The temperature optimum for all activities was at 40 °C. Activities of amygdalase (assayed at pH 4) and linamarase (at pH 6) evolving during the growth of E. fibuliger were generally higher in cultures grown at 25 °C and 30 °C. TLC analysis of amygdalin degradation products show a two-stage sequential mechanism as follows: (1) amygdalin to prunasin and (2) prunasin to cyanohydrin. Received: 16 September 1997 / Received revision: 6 October 1997 / Accepted: 14 October 1997     

Effects of Wastewater Irrigation on Chemical and Physical Properties of <Emphasis Type="Italic">Petroselinum crispum</Emphasis>

The present study was carried out to assess the impact of wastewater on parsley (Petroselinum crispum). The parameters determined for soil were pH, electrical conductivity (EC), soil organic matter (SOM), nutrient elements (Ca, Mg, Na, K, Mn, Cu, Zn, and Fe), and heavy metals (Cd, Cr, Ni, and Pb), while the parameters determined for the plant included pigment content, dry matter, nutrient element, and heavy metals. SOM, EC, and clay contents were higher, and pH was slightly acidic in soil treated with wastewater compared to control soil. The enrichment factors (EF) of the nutrient elements in contaminated soil are in the sequence of Na (2) > Ca (1.32) > Mn = Mg (1.17) > Cu (1.11) > Zn (1.08) > Fe (1.07) > K (0.93), while EF in parsley are Na (6.63) > Ca (1.60) > Mg (1.34) > Zn (1.15) > Fe (0.95) > Cu = K (0.90) > Mn (0.85). Application of wastewater significantly decreased dry matter, while photosynthetic pigment content increased in parsley. The enrichment of the heavy metals is in the sequence: Cd (1.142) > Pb (1.131) > Ni (1.112) > Cr (1.095). P. crispum shows a high transfer factor (TF > 1) for Cd signifying a high mobility of Cd from soil to plant. Thus, although the wastewater irrigation in parsley production aims to produce socioeconomic benefits, study results indicated that municipal wastewater is not suitable for irrigation of parsley because it has negative effects on plant and causes heavy metal accumulation.     

Miscibility of phosphatidylethanolamine-phosphatidylglycerol mixtures as a function of pH and acyl chain length

We have examined the mixing properties of phosphatidylethanolamine (PE) and phosphatidylglycerol (PG), the major components of many bacterial membranes. The phase transition behavior of dilute aqueous suspensions of PE:PG mixtures with different chain lengths (n = 14, 16) in 0.1 M NaCl at pH 7 and pH 2 was investigated by differential scanning calorimetry (DSC). The DSC curves were simulated using an approach which takes into account the broadening of the phase transition in addition to symmetric, non-ideal mixing in the gel and the liquid-crystalline phase. Based on the temperatures for onset and end of “melting” obtained by the simulations, the phase diagrams were constructed and then refined using a regular solution model with non-symmetric mixing in both phases. The mixing properties of PE:PG mixtures were analyzed as a function of pH and acyl chain length. In almost all cases, non-symmetric mixing behavior was observed, i.e. the non-ideality parameters are different for bilayers with low PG content compared to bilayers with high PG content. For equimolar mixtures at pH 7, when PG is negatively charged, the non-ideality parameters are negative for both phases, indicating preferential formation of mixed pairs. This mixed pair formation is more pronounced for the gel phase. At pH 2, when PG is partly protonated, the non-ideality parameter is less negative and the formation of mixed pairs is reduced compared to pH 7. The formation of PE:PG mixed pairs at pH 7 might be of benefit to a bacterial membrane, because it prevents demixing of lipid components with a concomitant destabilization of the membrane. Received: 3 August 1998 / Revised version: 4 October 1999 / Accepted: 12 October 1999     

Bacterial siderophores: the solution stoichiometry and coordination of the Fe(III) complexes of pyochelin and related compounds

Pyochelin, its analog 3′′-nor-NH-pyochelin, and the related methyl hydroxamate, 2-(2′-hydroxyphenyl)-4,5-dihydrothiazol-4-carboxylic acid methoxymethyl amide, have been prepared together with their Fe(III) complexes. The solution stoichiometry and the coordination of the three Fe(III) complexes in methanol or buffered (pH∼2) 50:50 (v/v) methanol–water mixtures were determined using various spectroscopic methods: UV–vis absorption, X-ray absorption, extended X-ray absorption fine structure and electron paramagnetic resonance. All three systems showed both a 1:1 and 2:1 ligand–Fe(III) stoichiometry, but presented different coordination properties. Conditional formation constants (pH∼2) were determined for both the 1:1 and 2:1 complexes in all three systems. Computation of the coordination-conformational energies by semiempirical methods indicated that the coordination in the case of the 2:1 complexes of pyochelin–Fe(III) and 3′′-nor-NH-pyochelin–Fe(III) was asymmetrical, with one molecule of pyochelin (or 3′′-nor-NH-pyochelin) tetradentately coordinated (O1, N1, N2 and O3) to the Fe(III), and the second molecule bound bidentately (O1, N1 or N2, O3), to complete the octahedral geometry. In contrast, two molecules of the methyl hydroxamate each provided a set of tridentate ligand atoms in the formation of the 2:1 ligand–Fe(III) complex. These results are consistent with the role of pyochelin in the uptake of iron by the FptA receptor in the outer membrane of Pseudomonas aeruginosa and in several gram-negative bacteria.     

Identification of a Novel Vanadium-binding Protein by EST Analysis on the Most Vanadium-rich Ascidian, Ascidia gemmata

Ascidians are known to accumulate extremely high levels of vanadium in their blood cells (up to 350 mM). The branchial sac and the intestine are thought to be the first tissues to contact the outer environment and absorb vanadium ions. The concentration of vanadium in the branchial sac and the intestine of the most vanadium-rich ascidian Ascidia gemmata were determined to be 32.4 and 11.9 mM, respectively. Using an expressed sequence tag (EST) analysis of a cDNA library from the intestine of A. gemmata, we determined 960 ESTs and found 55 clones of metal-related gene orthologs, 6 redox-related orthologs, and 18 membrane transporter orthologs. Among them, two genes, which exhibited significant similarity to the vanadium-binding proteins of other vanadium-rich ascidian species, were designated AgVanabin1 and AgVanabin2. Immobilized metal ion affinity chromatography revealed that recombinant AgVanabin1 bound to metal ions with an increasing affinity for Cu(II) > Zn(II) > Co(II) and AgVanabin2 bound to metal ions with an increasing affinity for Cu(II) > Fe(III) > V(IV). To examine the use of AgVanabins for a metal absorption system, we constructed Escherichia coli strains that expressed AgVanabin1 or AgVanabin2 fused to maltose-binding protein and secreted into the periplasmic space. We found that the strain expressing AgVanabin2 accumulated about 13.5 times more Cu(II) ions than the control TB1 strain. Significant accumulation of vanadium was also observed in the AgVanabin2-expressing strain as seen by a 1.5-fold increase.     

Microcalorimetric Study of <Emphasis Type="Italic">Tetrahymena</Emphasis> Growth Affected by Copper(II) Complexes

By means of microcalorimetry, the effect of four copper(II) complexes on Tetrahymena growth was investigated. The extent and duration of the inhibitory effect on the metabolism, judged by the rate constant, k, and the half inhibition concentration, IC₅₀, varied with the different complexes. The results showed that the half inhibition concentrations IC₅₀ of CuCl₂, (C₉H₆NO)₂Cu and [Cu(phen)₂]Cl₂⋅6H₂O were 9.9 × 10⁻⁴, 2.0 × 10⁻⁴, and 2.6 × 10⁻⁴ mol/L, respectively. The sequence of antibiotic activity of these three complexes was: (C₉H₆NO)₂Cu > [Cu(phen)₂]Cl₂⋅6H₂O > CuCl₂. The growth rate constants of [Cu(phen)₃]Cl₂⋅6H₂O did not change obviously with the increase of concentrations, but [Cu(phen)₃]Cl₂⋅6H₂O also can prolong the time of Tetrahymena growth.     

Identification of a four copper folding intermediate in mammalian copper metallothionein by electrospray ionization mass spectrometry

 Mammalian metallothioneins (MT) are known to maximally bind 12 copper ions in two six-Cu(I) ion clusters. Using electrospray ionization mass spectrometry of MT at pH 4.5, a four-Cu(I) ion cluster was observed intermediate to a fully formed six Cu(I) in a single domain or a fully formed Cu₁₂MT species. The four-Cu(I) cluster was observed in both MT1 and MT3 isoforms. Addition of increasing amounts of Cu(I) to MT at pH 4.5 resulted in prominent ions whoses masses were consistent with apo-MT, Cu₄MT, Cu₆MT, and Cu₁₂MT. The cooperativity of cluster formation was reduced at pH 2.5. Addition of Cu(I) to apo-MT at a reduced pH resulted in a series of ions consistent with Cu₄ to Cu₁₂MT species. However, formation of the tetracopper MT species remained cooperative at low pH, suggesting that this species is very stable. To determine whether the tetracopper cluster was formed in either the α or β domain, domain peptides of MT3 were used. Addition of Cu(I) to the apo β domain resulted in a peak consistent with the formation of a four-Cu(I) cluster. This is consistent with reports that Cu(I) ions bind preferentially to the β domain of MTs. Received: 2 June 1998 / Accepted: 21 August 1998     

Isolation and characterization of highly (R)-specific N-acetyl-1-phenylethylamine amidohydrolase, a new enzyme from Arthrobacter aurescens AcR5b

A new amidohydrolase deacetylating several N-acetyl-1-phenylethylamine derivatives (R)-specifically was found in Arthrobacter aurescens AcR5b. The strain was isolated from a wet haystack by enrichment culture with (R)-N-acetyl-1-phenylethylamine as the sole carbon source. (R) and (S )-N-acetyl-1-phenylethylamine do not serve as inducers for acylase formation. By improving the growth conditions the enzyme production was increased 47-fold. The amidohydrolase was purified to homogeneity leading to a 5.2-fold increase of the specific activity with a recovery of 67%. A molecular mass of 220 kDa was estimated by gel filtration. Sodium dodecyl sulfate/polyacrylamide gel electrophorosis shows two subunits with molecular masses of 16 kDa and 89 kDa. The optimum pH and temperature were pH 8 and 50 °C, respectively. The enzyme was stable in the range of pH 7–9 and at temperatures up to 30 °C. The enzyme activity was inhibited by Cu²⁺, Co²⁺, Ni²⁺, and Zn²⁺, and this inhibition was reversed by EDTA.M Received: 20 September 1996 / Received version: 23 December 1996 / Accepted: 30 December 1996     

Assessment of Minerals in Obesity-related Diseases in the Chandigarh (India) Population

Excessive Zn but normal Cu and Mg in the staple food consumed by the people of Chandigarh (Union territory and capital of Punjab and Haryana States of India) has been considered to be the major risk factor for the prevalence of obesity (33.15%) and obesity-related diseases in this region. Therefore, in the present investigations, in obesity-related diseases, the status of these minerals was estimated in their tissues, including hair, nails, and blood serum and urine, and compared with those of normal subjects. They were grouped as: normal subjects in control Group A, middle-aged diabetics in Group D_M, older diabetics in Group D_O, and diabetics with osteoarthritis in Group D+ OA, osteoarthritis in Group OA and rheumatoid arthritis in Group RA, respectively. The results evaluated in the order as: hair Zn, group D+OA>D_M>OA>A (control)>RA>D_O (p < 0.001); hair Cu, group A (control)>D_M>OA>D+OA>D_O>RA (p < 0.001); hair Mg, group A (control)>D_M>OA>D+OA>RA>D_O (p < 0.001, 0.01); hair Mn, group A (control)>RA>OA>D-OA>D_M>D_O (p < 0.001); nail Zn, group D_M>D+OA>OA>A (control)>RA>D_O (p < 0.001, 0.05); nail Cu, group A (control)>OA>D_M>D+OA>RA>D_O (p < 0.001); nail Mg, group A (control)>OA>D_M>D_O>D+OA >RA (p < 0.001); nail Mn, group A (control) >RA>OA>D+OA>D_M>D_O (p < 0.01); urine Zn, group D_O>D_M>D+OA>A (control)>RA>OA (p < 0.01); urine Cu, group RA>D+OA>D_O>OA> D_M>A (control) (p<0.001); urine Mg, group RA>OA>D+OA>D_O>D_M>A (control; p < 0.001); urine Mn, group D_O>D_M>OA>D+OA>RA>A (control; p < 0.001), respectively. The analysis of the mineral status in serum of diabetics further showed their highly significant rise from lower mean age subgroup to higher mean age subgroup than their control counter parts (p < 0.001, 0.01, and 0.05) with coincident deficiencies of Cu, Mg, and Mn in their tissues. This study would be helpful considering the status of minerals in these obesity-related diseases depending on the choice of the food consumed to improve the quality of life and prognosis for the diseases.     

Effects of Ni(II) on respirometric oxygen uptake

The effects of Ni(II), substrate and initial biomass concentrations on biochemical oxygen demand (BOD) were studied by using an electrolytic respirometer. The effects of Ni(II) (2.5, 5.0, 10.0, 25.0 mg/l) and substrate (325, 650, 1300 mg/l as chemical oxygen demand) in a synthetic wastewater with differing initial biomass concentrations (1, 10, 100 mg/l) were investigated. The biomass-to-metal ratio was found to be the most important parameter affecting the measured BOD values. The maximum specific growth rates were calculated and the results of batch respirometric experiments were analysed both by graphical and statistical methods. In statistical analyses, a factorial experimental design approach was followed and results were treated by multiple regression techniques. A mathematical model was developed to express the maximum oxygen uptake in terms of nickel, substrate and initial biomass concentrations and their magnitudes of their effects were compared. The biomass-to-metal ratio was found to be very significant so that another model that expresses oxygen uptake in relation to the biomass-to-metal ratio and also to substrate concentration was developed. Finally, the effect of Ni(II) was demonstrated to depend on both substrate and initial biomass concentrations. This effect was stimulatory at low concentrations of Ni(II), and complete inhibition was never observed even at the highest concentration of Ni(II) studied, which was 25.0 mg/l. Received: 4 January 1997 / Received revision: 10 June 1997 / Accepted: 29 June 1997     

Study on the Trace Elements in <Emphasis Type="Italic">Swertia chirayita</Emphasis> (Roxb.) H. Karsten

An attempt has been made to analyze some trace elements and electrolytes like Zn, Cu, Mn, Fe, Co, Na, K, Ca, and Li present in the Swertia chirayita roots and leaves. The concentration of Ca in all the samples was more than 1,346.0 mg/kg and the concentration of other elements was found in the order K > Ca > Fe > Na > Mn > Zn > Co > Cu > Li in different samples of S. chirayita.     

Directional cloning of native PCR products with preformed sticky ends (Autosticky PCR)

A novel method for the directional cloning of native PCR products was developed. Abasic sites in DNA templates make DNA polymerases stall at the site during synthesis of the complementary strand. Since the 5′ ends of PCR product strands contain built-in amplification primers, abasic sites within the primers result in the formation of 5′ single-stranded overhangs at the ends of the PCR product, enabling its direct ligation to a suitably cleaved cloning vector without any further modification. This “autosticky PCR” (AS-PCR) overcomes the problems caused by end sensitivity of restriction enzymes, or internal restriction sites within the amplified sequences, and enables the generation of essentially any desired 5′ overhang. Received: 11 August 1998 / Accepted: 2 October 1998     

Nitrate reductase activation state in barley roots in relation to the energy and carbohydrate status

The NADH-dependent nitrate reductase (NR, EC 1.6.6.1) in roots of hydroponically grown barley seedlings was extracted, desalted and the activity measured in buffer containing either Mg²⁺ (10 mM) or EDTA (5 mM). The former gives the actual NR activity (NR_act) equivalent to dephospho-NR, whereas the latter gives the maximum NR capacity of the dephospho-form (NR_max). Both values together permit an estimation of the NR-phosphorylation state. Changes in NR_act and NR_max were followed in response to root aeration or to shoot illumination or shoot removal, and were correlated with sugar contents and adenylate levels. Ethanol formation was also measured in roots differing in NR activity in order to obtain information on the relation between anaerobic alcoholic fermentation and nitrate reduction. In aerated roots, NR was highly phosphorylated (about 80%) and largely inactive. It was partly dephosphorylated (activated) by anoxia or by cellular acidification (pH 4.8 plus propionic acid). Anaerobic activation (dephosphorylation) of NR was stronger at acidic external pH (5) than at slightly alkaline pH (8), although ATP levels decreased and AMP levels increased at pH 5 and at pH 8 to the same extent. Thus, rapid changes in the NR-phosphorylation state in response to anaerobiosis were not directly triggered by the adenylate pool, but rather by cytosolic pH. Under prolonged darkness (24 h) or after shoot removal, NR_max decreased slowly without a large change in the phosphorylation state. This decrease of NR_max was correlated with a large decrease in the sugar content, and was prevented by glucose feeding, which had only minor effects on the phosphorylation state. Cycloheximide also prevented the decrease in NR_max without affecting the phosphorylation state. In contrast, anaerobiosis or cellular acidification prevented the decrease of NR_max and at the same time decreased the NR-phosphorylation state. It is suggested that NR turnover in roots is controlled by several factors: NR synthesis appears to depend on sugar availability, which has little effect on the phosphorylation state; in addition, NR degradation appears to be strongly affected by the phosphorylation state in such a way that the inactive phospho-NR is a better substrate for NR degradation than the dephospho-form. The rate of anaerobic ethanol formation was not affected by NR activity, indicating that the purpose of NR activation under hypoxia or anoxia is not to decrease or prevent alcoholic fermentation. Received: 29 August 1996 / Accepted: 8 November 1996     

Effects of Ca(II) ions on Cu(II) ion-phytic acid interactions

In vitro interactions among phytic acid (PA), Cu(II) ions, and Ca(II) ions were examined as functions of PA:Cu(II):Ca(II) molar ratios and pH. Ca(II) ions competed with Cu(II) ions for binding by the soluble phytate species for PA:Cu(II) molar ratios ranging from 10:1 to 1:6 and pH values in the 2.4-5.9 range. At pH values where precipitation occurred, Ca(II) ions potentiated Cu(II) ion binding by the precipitated phytate species for PA:Cu(II) molar ratios of 10:1 to 1:3. At lower PA:Cu(II) molar ratios, Ca(II) ions competed with Cu(II) ions for binding by the precipitated phytate species. Compositions of the precipitated copper-calcium phytates are reported.