The Role of Intracellular Glutathione in Inorganic Mercury-Induced Toxicity in Neuroblastoma Cells

It is well known that antioxidants containing sulfhydryl (−SH) groups are protective against the toxic effects of mercury. The current study was designed to elucidate the mechanism(s) of the cytoprotective effects of glutathione (GSH) and N-acetylcysteine (NAC) against the toxicity of inorganic mercury (HgCl₂) in neuroblastoma cells (N-2A). The obtained results demonstrated the protective effects of these compounds in a dose dependant manner up to 95 and 74% cell viability, respectively as compared to the control of HgCl₂ of 10%. The administration of buthionine sulfoximine (BSO), an inhibitor of GSH synthesis, increased the toxicity of HgCl₂ in a dose dependent manner. Moreover, BSO treatment attenuated the levels of the cellular free −SH concentrations at low concentrations (1–100 μM) of HgCl₂. The data also show that cellular thiol concentrations were augmented in the presence of GSH and NAC and these compounds were cytoprotective against HgCl₂ and this is due to up regulating of GSH synthesis. A reduction in intracellular levels of GSH was observed with treatment of HgCl₂. In addition, the ratio of GSH/GSSG increased from 16:1 to 50:1 from 1 to 10 μM concentration of HgCl_2. The ratio of GSH/GSSG then decreased from 4:1 to 0.5:1 with the increase of concentration of HgCl₂ between 100 μM and 1 mM due to the collapse of the N-2A cells. It was of interest to note that the synthesis of GSH was stimulated in cells exposed to low concentration of HgCl₂ when extra GSH is available. These data support the idea that the loss of GSH plays a contributing role to the toxic effects of HgCl₂ and that inorganic mercury adversely affects viability, through altering intracellular −SH concentrations. The data further indicate that the availability of GSH to the cells may not be sufficient to provide protection against mercury toxicity and the de novo synthesis of intracellular GSH is required to prevent the damaging effects of mercury.     

Water channel does not limit evaporation of water from plant cells

Evaporation of water from the cell surface of the internode ofChara corallina was not affected by HgCl₂ which is known to inhibit water channels. This makes a sharp contrast to the fact that most of osmotically driven water transport is inhibited by HgCl₂. Also in radish hypocotyls whose epidermis had been peeled off, evaporation of water was not inhibited by HgCl₂, while osmotic water transport was significantly inhibited. The cell wall tube was prepared by squeezing out the content of theChara internode. The rate of evaporation from the cell wall tube filled with 150 mM KCl was almost equal to that from the living cell. The apparent hydraulic conductivity of the cell calculated from evaporation rate was found to be 1–2×10⁻³ pm s⁻¹ Pa⁻¹ which is about 1/1000 times the hydraulic conductivity of the plasma membrane (L_p) and 1/40 times the L_p under maximal inhibition with HgCl₂. It is concluded that under the relative humidity of 53–70% the rate of evaporation of water from the cell surface is limited by the rate of evaporation from the cell wall which is so low that the loss of water can be supplemented without delay from the cell interior across the plasma membrane even when water channels are completely closed.     

Rhodobacter capsulatus magnesium chelatase subunit BchH contains an oxygen sensitive iron–sulfur cluster

Magnesium chelatase is the first unique enzyme of the bacteriochlorophyll biosynthetic pathway. It consists of three subunits (BchI, BchD, and BchH). Amino acid sequence analysis of the Rhodobacter capsulatus BchH revealed a novel cysteine motif (³⁹³CX₂CX₃CX₁₄C) that was found in only six other proteobacteria (CX₂CX₃CX_11–14C). The cysteine motif is likely to coordinate an unprecedented [Fe–S] cluster. Purified BchH demonstrated absorbance in the 460 nm region. This absorbance was abolished in BchH proteins with alanine substitutions at positions Cys396 and Cys414. These modified proteins were also EPR silent. In contrast, wild type BchH protein in the reduced state showed EPR signals resembling those of a [4Fe–4S] cluster with rhombic symmetry and g values at 1.90, 1.93, and 2.09, superimposed with a [3Fe–4S] cluster centered at g = 2.02. The [3Fe–4S] signal was observed independently of the [4Fe–4S] signal under oxidizing conditions. Mg-chelatase activity assays showed that the cluster is not catalytic. We suggest that the [4Fe–4S] and [3Fe–4S] signals originate from a single coordination site on the monomeric BchH protein and that the [4Fe–4S] cluster is sensitive to oxidation. It is speculated that the cluster participates in the switching between aerobic and anaerobic life of the proteobacteria.     

Osmotic water permeability of isolated vacuoles

We measured the osmotic water permeability (P _os) of vacuoles isolated from onion (Allium cepa L.), rape (Brassica napus L.), petunia (Petunia hybrida Hook.) and red beet (Beta vulgaris L.). For all the vacuolar types investigated, P _os values were in the range 200–1000 μm s⁻¹. The change in membrane surface area induced by an osmotic gradient was smaller than 2–6%. The vacuolar P _os values for red beet and onion were reduced by 1 mM HgCl₂, to 14% and 30% of the control values, respectively, but were partially restored to 51% and 76% by 5 mM β-mercaptoethanol. These results suggest that aquaporins were present in all the vacuoles tested. In HgCl₂-treated onion vacuoles, the reduced P _os (56 μm s⁻¹) had a low activation energy (approx. 6 kJ mol⁻¹), indicating that water permeation was still occurring mainly via aquaporins, and that the water permeability of the lipid part of the vacuolar membrane is probably very low. Received: 18 February 1999 / Accepted: 21 June 1999     

The site of photoinhibition in leaves of Cucumis sativus L. at low temperatures is photosystem I,not photosystem II

Maximum quantum yields (QY) of photosynthetic electron flows through PSI and PSII were separately assessed in thylakoid membranes isolated from leaves of Cucumis sativus L. (cucumber) that had been chilled in various ways. The QY(PSI) in the thylakoids prepared from the leaves treated at 4° C in moderate light at 220 mol quanta·m^–2·s^–1 (400–700 nm) for 5 h, was about 20–30% of that in the thylakoids prepared from untreated leaves, while QY(PSII) decreased, at most, by 20% in response to the same treatment. The decrease in QY(PSI) was observed only when the leaves were chilled at temperatures below 10° C, while such a marked temperature dependency was not observed for the decrease in QY(PSII). In the chilling treatment at 4° C for 5 h, the quantum flux density that was required to induce 50% loss of QY (PSI) was ca. 50 umol quanta·m^–2·s^–1. When the chilling treatment at 4° C in the light was conducted in an atmosphere of N₂, photoinhibition of PSI was largely suppressed, while the damage to PSII was somewhat enhanced. The ferricyanide-oxidised minus ascorbate-reduced difference spectra and the light-induced absorbance changes at 700 nm obtained with the thylakoid suspension, indicated the loss of P700 to extents that corresponded to the decreases in QY(PSI). Accordingly, the decreases in QY(PSI) can largely be attributed to destruction of the PSI reaction centre itself. These results clearly show that, at least in cucumber, a typical chillingsensitive plant, PSI is much more susceptible to aerobic photoinhibition than PSII.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - P700 primary electron donor of PSI - PPFD photosynthetically active photon flux density - QY quantum yield We are grateful to invaluable comments by Prof. S. Katoh, K. Hikosaka and the members of our laboratory. We also thank A. Aoyama for technical assistance. This work was partly supported by the grants from the Ministry of Education, Science, and Culture, Japan, to I. Terashima (#03740342 and #04640621).     

Biodesulfurization of dibenzothiophene by growing cells of Gordonia sp. in batch cultures

A new isolate, identified as Gordonia sp. ZD-7 by 16S rDNA sequence analysis, grew in n-hexadecane containing dibenzothiophene (DBT) which was degraded from 2.8 mM to 0.2 mM within 48 h. Biodesulfurization could be repeatedly performed for more than 190 h, with average desulfurization rates of 5 mmol DBT kg cells (dry wt)⁻¹ h⁻¹.     

Characterization of a HoxEFUYH type of [NiFe] hydrogenase from <Emphasis Type="Italic">Allochromatium vinosum</Emphasis> and some EPR and IR properties of the hydrogenase module

A soluble hydrogenase from Allochromatium vinosum was purified. It consisted of a large (M _r = 52 kDa) and a small (M _r = 23 kDa) subunit. The genes encoding for both subunits were identified. They belong to an open reading frame where they are preceded by three more genes. A DNA fragment containing all five genes was cloned and sequenced. The deduced amino acid sequences of the products characterized the complex as a member of the HoxEFUYH type of [NiFe] hydrogenases. Detailed sequence analyses revealed binding sites for eight Fe–S clusters, three [2Fe–2S] clusters and five [4Fe–4S] clusters, six of which are also present in homologous subunits of [FeFe] hydrogenases and NADH:ubiquione oxidoreductases (complex I). This makes the HoxEFUYH type of hydrogenases the one that is evolutionary closest to complex I. The relative positions of six of the potential Fe–S clusters are predicted on the basis of the X-ray structures of the Clostridium pasteurianum [FeFe] hydrogenase I and the hydrophilic domain of complex I from Thermus thermophilus. Although the HoxF subunit contains binding sites for flavin mononucleotide and NAD(H), cell-free extracts of A. vinosum did not catalyse a H₂-dependent reduction of NAD⁺. Only the hydrogenase module (HoxYH) could be purified. Its electron paramagnetic resonance (EPR) and IR spectral properties showed the presence of a Ni–Fe active site and a [4Fe–4S] cluster. Its activity was sensitive to carbon monoxide. No EPR signals from a light-sensitive Ni_a–C* state could be observed. This study presents the first IR spectroscopic data on the HoxYH module of a HoxEFUYH type of [NiFe] hydrogenase.     

The peroxidase activity of cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>6</Subscript><Emphasis Type="Italic">f</Emphasis> complex from spinach chloroplasts

The cytochrome b ₆ f (Cyt b ₆ f) complex, which functions as a plastoquinol-plastocyanin oxidoreductase and mediates the linear electron flow between photosystem II (PSII) and photosystem I (PSI) and the cyclic electron flow around PSI, was isolated from spinach (Spinacia oleracea L.) chloroplasts using n-octyl-β-D-glucopyranoside (β-OG). The preparation was also able to catalyze the peroxidase-like reaction in the presence of hydrogen peroxide (H₂O₂) and guaiacol. The optimal conditions for peroxidase activity of the preparation included: pH 3.6, ionic strength 0.1, and temperature 35°C. The apparent Michaelis constant (K _m) values for H₂O₂ and guaiacol were 50 mM and 2 mM, respectively. The bimolecular rate constant (k _obs) was about 26 M⁻¹ s⁻¹ and the turnover number (K _cat) was about 60 min⁻¹ (20 mM guaiacol, 100 mM sodium phosphate, pH 3.6, 25°C, [H₂O₂]<100mM). These parameters were similar to those of several other heme-containing proteins, such as myoglobin and Cyt c.     

Effects of selective destruction of iron-sulfur center B on electron transfer and charge recombination in Photosystem I

Incubation of spinach thylakoids with HgCl₂ selectively destroys Fe–S center B (F_B). The function of electron acceptors in F_B-less PS I particles was studied by following the decay kinetics of P700⁺ at room temperature after multiple flash excitation in the absence of a terminal electron acceptor. In untreated particles, the decay kinetics of the signal after the first and the second flashes were very similar (t _1/22.5 ms), and were principally determined by the concentration of the artificial electron donor added. The decay after the third flash was fast (t _1/20.25 ms). In F_B-less particles, although the decay after the first flash was slow, fast decay was observed already after the second flash. We conclude that in F_B-less particles, electron transfer can proceed normally at room temperature from F_X to F_A and that the charge recombination between P700⁺ and F_X ^-/A₁ ^- predominated after the second excitation. The rate of this recombination process is not significantly affected by the destruction of F_B. Even in the presence of 60% glycerol, F_B-less particles can transfer electrons to F_A at room temperature as efficiently as untreated particles.Abbreviations DCIP 2, 6-dichlorophenol indophenol - F_A, F_B, F_X iron-sulfur center A, B and X, respectively - PMS phenazine methosulfate     

Absence of PsaC subunit allows assembly of photosystem I core but prevents the binding of PsaD and PsaE in Synechocystis sp. PCC6803

In photosystem I (PSI) of oxygenic photosynthetic organisms the psaC polypeptide, encoded by the psaC gene, provides the ligands for two [4Fe-4S] clusters, F_A and F_B. Unlike other cyanobacteria, two different psaC genes have been reported in the cyanobacterium Synechocystis 6803, one (copy 1) with a deduced amino acid sequence identical to that of tobacco and another (copy 2) with a deduced amino acid sequence similar to those reported for other cyanobacteria. Insertion of a gene encoding kanamycin resistance into copy 2 resulted in a photosynthesis-deficient strain, CDK25, lacking the PsaC, PsaD and PsaE polypeptides in isolated thylakoid membranes, while the PsaA/PsaB and PsaF subunits were found. Growth of the mutant cells was indistinguishable from that of wild-type cells under light-activated heterotrophic growth (LAHG). A reversible P700⁺ signal was detected by EPR spectroscopy in the isolated thylakoids during illumination at low temperature. Under these conditions, the EPR signals attributed to F_A and F_B were absent in the mutant strain, but a reversible F_x signal was present with broad resonances at g=2.079, 1.903, and 1.784. Addition of PsaC and PsaD proteins to the thylakoids gave rise to resonances at g=2.046, 1.936, 1.922, and 1.880; these values are characteristic of an interaction-type spectrum of F_A ^- and F_B ^-. In room-temperature optical spectroscopic analysis, addition of PsaC and PsaD to the thylakoids also restored a 30 ms kinetic transient which is characteristic of the P700⁺ [F_A/F_B]^- backreaction. Expression of copy 1 was not detected in cells grown under LAHG and under mixotrophic conditions. These results demonstrate that copy 2 encodes the PsaC polypeptide in PSI in Synechocystis 6803, while copy 1 is not involved in PSI; that the PsaC polypeptide is necessary for stable assembly of PsaD and PsaE into PSI complex in vivo; and that PsaC, PsaD and PsaE are not needed for assembly of PsaA-PsaB dimer and electron transport from P700 to F_x.     

Characteristics of Photosystem I Complexes in the End Membranes of Pea Granal Thylakoids

Two fractions of the light fragments enriched in the photosystem I (PSI) complexes were obtained from pea (Pisum sativum L.) thylakoids by digitonin treatment and subsequent differential centrifugation. The ratio of chlorophyll a to chlorophyll b, chlorophyll/P700 spectra of low-temperature fluorescence, and excitation spectra of long-wave fluorescence were measured. These characteristics were shown to be different due to variation in the size and composition of the light-harvesting antenna of PSI complexes present in the particles obtained. The larger antenna size of one of the fractions was related to the incorporation of the pool of light-harvesting complex II (LHCII). A comparison with the data available allowed us to identify these particles as fragments of intergranal thylakoids and end membranes of granal thylakoids. The suggestion that an increase in the PSI light-harvesting antenna in intergranal thylakoids is related to the attachment of phosphorylated LHCII is discussed.     

The different effects of chilling stress under moderate light intensity on photosystem II compared with photosystem I and subsequent recovery in tropical tree species

Tropical plants are sensitive to chilling temperatures above zero but it is still unclear whether photosystem I (PSI) or photosystem II (PSII) of tropical plants is mainly affected by chilling temperatures. In this study, the effect of 4°C associated with various light densities on PSII and PSI was studied in the potted seedlings of four tropical evergreen tree species grown in an open field, Khaya ivorensis, Pometia tomentosa, Dalbergia odorifera, and Erythrophleum guineense. After 8 h chilling exposure at the different photosynthetic flux densities of 20, 50, 100, 150 μmol m⁻² s⁻¹, the maximum quantum yield of PSII (F _v /F _m) in all of the four species decreased little, while the quantity of efficient PSI complex (P _m) remained stable in all species except E. guineense. However, after chilling exposure under 250 μmol m⁻² s⁻¹ for 24 h, F _v /F _m was severely photoinhibited in all species whereas P _m was relative stable in all plants except E. guineense. At the chilling temperature of 4°C, electron transport from PSII to PSI was blocked because of excessive reduction of primary electron acceptor of PSII. F _v /F _m in these species except E. guineense recovered to ~90% after 8 h recovery in low light, suggesting the dependence of the recovery of PSII on moderate PSI and/or PSII activity. These results suggest that PSII is more sensitive to chilling temperature under the moderate light than PSI in tropical trees, and the photoinhibition of PSII and closure of PSII reaction centers can serve to protect PSI.     

Expression,purification and characterization of pectate lyase A from <Emphasis Type="Italic">Aspergillus nidulans</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Pectate lyase A (PelA) of Aspergillus nidulans was successfully expressed in Escherichia coli and effectively purified using a Ni²⁺-nitrilotriacetate-agarose column. Enzyme activity of the recombinant PelA could reach 360 U ml⁻¹ medium. The expressed PelA exhibited its optimum level of activity over the range of pH 7.5–10 at 50°C. Mn²⁺, Ca²⁺, Fe²⁺, Mg²⁺ and Fe³⁺ ions stimulated the pectate lyase activity, but Cu²⁺ and Zn²⁺ inhibited it. The recombinant PelA had a V _max of 77 μmol min⁻¹ mg⁻¹ and an apparent K _m of 0.50 mg ml⁻¹ for polygalacturonic acid. Low-esterified pectin was the optimum substrate for the PelA, whereas higher-esterified pectin was hardly cleaved by it. PelA efficiently macerated mung bean hypocotyls and potato tuber tissues into single cells.     

Structure and coordination of CuB in the Acidianus ambivalens aa 3 quinol oxidase heme–copper center

The coordination environment of the Cu_B center of the quinol oxidase from Acidianus ambivalens, a type B heme–copper oxygen reductase, was investigated by Fourier transform (FT) IR and extended X-ray absorption fine structure (EXAFS) spectroscopy. The comparative structural chemistry of dinuclear Fe–Cu sites of the different types of oxygen reductases is of great interest. Fully reduced A. ambivalens quinol oxidase binds CO at the heme a ₃ center, with ν(CO)=1,973 cm⁻¹. On photolysis, the CO migrated to the Cu_B center, forming a Cu_B^I–CO complex with ν(CO)=2,047 cm⁻¹. Raising the temperature of the samples to 25°C did not result in a total loss of signal in the FTIR difference spectrum although the intensity of these signals was reduced sevenfold. This observation is consistent with a large energy barrier against the geminate rebinding of CO to the heme iron from Cu_B, a restricted limited access at the active-site pocket for a second binding, and a kinetically stable Cu_B–CO complex in A. ambivalens aa ₃. The Cu_B center was probed in a number of different states using EXAFS spectroscopy. The oxidized state was best simulated by three histidines and a solvent O scatterer. On reduction, the site became three-coordinate, but in contrast to the bo ₃ enzyme, there was no evidence for heterogeneity of binding of the coordinated histidines. The Cu_B centers in both the oxidized and the reduced enzymes also appeared to contain substoichiometric amounts (0.2 mol equiv) of nonlabile chloride ion. EXAFS data of the reduced carbonylated enzyme showed no difference between dark and photolyzed forms. The spectra could be well fit by 2.5 imidazoles, 0.5 Cl⁻ and 0.5 CO ligands. This arrangement of scatterers would be consistent with about half the sites remaining as unligated Cu(his)₃ and half being converted to Cu(his)₂Cl⁻CO, a 50/50 ratio of Cu(his)₂Cl⁻ and Cu(his)₃CO, or some combination of these formulations. Electronic Supplementary Material Supplementary material is available for this article at .     

Enhancement of susceptivity to photoinhibition and photooxidation in rice chlorophyll <Emphasis Type="Italic">b</Emphasis>-less mutants

Two rice chlorophyll (Chl) b-less mutants (VG28-1, VG30-5) and the respective wild type (WT) plant (cv. Zhonghua No. 11) were analyzed for the changes in Chl fluorescence parameters, xanthophyll cycle pool, and its de-epoxidation state under exposure to strong irradiance, SI (1 700 μmol m⁻² s⁻¹). We also examined alterations in the chloroplast ultrastructure of the mutants induced by methyl viologen (MV) photooxidation. During HI (0–3.5 h), the photoinactivation of photosystem 2 (PS2) appeared earlier and more severely in Chl b-less mutants than in the WT. The decreases in maximal photochemical efficiency of PS2 in the dark (F_v/F_m), quantum efficiency of PS2 electron transport (Φ_PS2), photochemical quenching (q_P), as well as rate of photochemistry (P_rate), and the increases in de-epoxidation state (DES) and rate of thermal dissipation of excitation energy (D_rate) were significantly greater in Chl b-mutants compared with the WT plant. A relatively larger xanthophyll pool and 78–83 % conversion of violaxanthin into antheraxanthin and zeaxanthin in the mutants after 3.5 h of HI was accompanied with a high ratio of inactive/total PS2 (0.55–0.73) and high 1–q_P (0.57–0.68) which showed that the activities of the xanthophyll cycle were probably insufficient to protect the photosynthetic apparatus against photoinhibition. No apparent difference of chloroplast ultrastructure was found between Chl b-less mutants and WT plants grown under low, LI (180 μmol m⁻² s⁻¹) and high, HI (700 μmol m⁻² s⁻¹) irradiance. However, swollen chloroplasts and slight dilation of thylakoids occurred in both mutants and the WT grown under LI followed by MV treatment. These typical symptoms of photooxidative damage were aggravated as plants were exposed to HI. Distorted and loose scattered thylakoids were observed in particular in the Chl b-less mutants. A greater extent of photoinhibition and photooxidation in these mutants indicated that the susceptibility to HI and oxidative stresses was enhanced in the photosynthetic apparatus without Chl b most likely as a consequence of a smaller antenna size.     

Characterization of PSI recovery after chilling-induced photoinhibition in cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) leaves

By simultaneously analyzing the chlorophyll a fluorescence transient and light absorbance at 820 nm as well as chlorophyll fluorescence quenching, we investigated the effects of different photon flux densities (0, 15, 200 μmol m⁻² s⁻¹) with or without 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) on the repair process of cucumber (Cucumis sativus L.) leaves after treatment with low temperature (6°C) combined with moderate photon flux density (200 μmol m⁻² s⁻¹) for 6 h. Both the maximal photochemical efficiency of Photosystem II (PSII) (F _v/F _m) and the content of active P700 (ΔI/I _o) significantly decreased after chilling treatment under 200 μmol m⁻² s⁻¹ light. After the leaves were transferred to 25°C, F _v/F _m recovered quickly under both 200 and 15 μmol m⁻² s⁻¹ light. ΔI/I _o recovered quickly under 15 μmol m⁻² s⁻¹ light, but the recovery rate of ΔI/I _o was slower than that of F _v/F _m. The cyclic electron transport was inhibited by chilling-light treatment obviously. The recovery of ΔI/I _o was severely suppressed by 200 μmol m⁻² s⁻¹ light, whereas a pretreatment with DCMU effectively relieved this suppression. The cyclic electron transport around PSI recovered in a similar way as the active P700 content did, and the recovery of them was both accelerated by pretreatment with DCMU. The results indicate that limiting electron transport from PSII to PSI protected PSI from further photoinhibition, accelerating the recovery of PSI. Under a given photon flux density, faster recovery of PSII compared to PSI was detrimental to the recovery of PSI or even to the whole photosystem.     

A steady-state and pre-steady-state kinetics study of the tungstoenzyme formaldehyde ferredoxin oxidoreductase from <Emphasis Type="Italic">Pyrococcus furiosus</Emphasis>

Formaldehyde ferredoxin oxidoreductase from Pyrococcus furiosus is a homotetrameric protein with one tungstodipterin and one [4Fe–4S] cubane per 69-kDa subunit. The enzyme kinetics have been studied under steady-state conditions at 80 °C and pre-steady state conditions at 50 °C, in the latter case via monitoring of the relatively weak (ε ≈ 2 mM⁻¹ cm⁻¹) optical spectrum of the tungsten cofactor. The steady-state data are consistent with a substrate substituted-enzyme mechanism for three substrates (formaldehyde plus two ferredoxin molecules). The K _M value for free formaldehyde (21 μM) with ferredoxin as an electron acceptor is approximately 3 times lower than the value measured when benzyl viologen is used as an acceptor. The K _M of ferredoxin (14 μM) is an order of magnitude less than previously reported values. An explanation for this discrepancy may be the fact that high concentrations of substrate are inhibitory and denaturing to the enzyme. Pre-steady-state difference spectra reveal peak shifts and a lack of isosbestic points, an indication that several processes happen in the first seconds of the reaction. Two fast processes (k _obs1 = 4.7 s⁻¹, k _obs2 = 1.9 s⁻¹) are interpreted as oxidation of the substrate followed by rearrangement of the active site. Alternatively, these processes could be the entry/binding of the substrate followed by its oxidation. The release of the product and the electron shuffling over the tungsten and iron–sulfur center in the absence of an external electron acceptor are slower (k _obs3 = 6.10 × 10⁻² s⁻¹, k _obs4 = 2.18 × 10⁻² s⁻¹). On the basis of these results in combination with results from previous electron paramagnetic resonance studies an activation route plus catalytic redox cycle is proposed.     

Effect of Light Quality on the Composition, Function, and Structure of Photosynthetic Thylakoid Membranes of Asplenium australasicum (Sm.) Hook

The effect of light quality on the composition, function and structure of the thylakoid membranes, as well as on the photosynthetic rates of intact fronds from Asplenium australasicum, a shade plant, grown in blue, white, or red light of equal intensity (50 microeinsteins per square meter per second) was investigated. When compared with those isolated from plants grown in white and blue light, thylakoids from plants grown in red light have higher chlorophyll a/chlorophyll b ratios and lower amounts of light-harvesting chlorophyll a/b-protein complexes than those grown in blue light. On a chlorophyll basis, there were higher levels of PSII reaction centers, cytochrome f and coupling factor activity in thylakoids from red light-grown ferns, but lower levels of PSI reaction centers and plastoquinone. The red light-grown ferns had a higher PSII/PSI reaction center ratio of 4.1 compared to 2.1 in blue light-grown ferns, and a larger apparent PSI unit size and a lower PSII unit size. The CO₂ assimilation rates in fronds from red light-grown ferns were lower on a unit area or fresh weight basis, but higher on a chlorophyll basis, reflecting the higher levels of electron carriers and electron transport in the thylakoids.

The structure of thylakoids isolated from plants grown under the three light treatments was similar, with no significant differences in the number of thylakoids per granal stack or the ratio of appressed membrane length/nonappressed membrane length. The large freeze-fracture particles had the same size in the red-, blue-, and white-grown ferns, but there were some differences in their density. Light quality is an important factor in the regulation of the composition and function of thylakoid membranes, but the effects depend upon the plant species.

     

Characterization of an ecto-ATPase activity in Fonsecaea pedrosoi

In this work, we characterized an ecto-ATPase activity in intact mycelial forms of Fonsecaea pedrosoi, the primary causative agent of chromoblastomycosis. In the presence of 1 mM EDTA, fungal cells hydrolyzed adenosine-5′-triphosphate (ATP) at a rate of 84.6 ± 11.3 nmol Pi h⁻¹ mg⁻¹ mycelial dry weight. The ecto-ATPase activity was increased at about five times (498.3 ± 27.6 nmol Pi h⁻¹ mg⁻¹) in the presence of 5 mM MgCl₂, with values of V _max and apparent K _m for Mg-ATP²⁻corresponding to 541.9 ± 48.6 nmol Pi h⁻¹ mg⁻¹ cellular dry weight and 1.9 ± 0.2 mM, respectively. The Mg²⁺-stimulated ecto-ATPase activity was insensitive to inhibitors of intracellular ATPases such as vanadate (P-ATPases), bafilomycin A₁ (V-ATPases)_, and oligomycin (F-ATPases). Inhibitors of acid phosphatases (molybdate, vanadate, and fluoride) or alkaline phosphatases (levamizole) had no effect on the ecto-ATPase activity. The surface of the Mg²⁺-stimulated ATPase in F. pedrosoi was confirmed by assays in which 4,4′-diisothiocyanostylbene-2,2′-disulfonic acid (DIDS), a membrane impermeant inhibitor, and suramin, an inhibitor of ecto-ATPase and antagonist of P₂ purinoreceptors. Based on the differential expression of ecto-ATPases in the different morphological stages of F. pedrosoi, the putative role of this enzyme in fungal biology is discussed.