Effect of HgCl2 on acetylcholine,carbachol, and glutamate currents ofAplysia neurons

Summary 1. Using conventional two-microelectrode voltage-clamp techniques we studied the effects of inorganic mercury (HgCl₂) on acetylcholine-, carbachol-, and glutamate-activated currents onAplysia neurons. Hg²⁺ was applied with microperfusion.2. Acetylcholine and carbachol activated an inward, sodium-dependent current in the anterior neurons of the pleural ganglion. The medial neurons gave a biphasic current to acetylcholine and carbachol, which was outward at resting membrane potential. The faster component was Cl^– dependent and reversed at about –60 mV, while the slower component was K⁺ dependent and reversed at greater than –80 mV.3. Hg²⁺ (0.1–10 µM) caused a dramatic increase in the acetylcholine- and carbachol-induced inward current in anterior neurons and the fast Cl^– current in medial neurons. With only a 1-min preapplication of Hg²⁺, the acetylcholine- or carbachol-activated sodium or chloride currents were increased to 300% and the effect was only partly reversible. The threshold concentration was 0.1 µM Hg²⁺.4. Contrary to the effects on sodium and chloride currents, concentrations of 0.1–10 µM Hg²⁺ caused a complete and irreversible blockade of K⁺-dependent acetylcholine and carbachol currents. The block of the potassium current was relatively fast and increased with time. The concentration of HgCl₂ that gave a half-maximal blockade of the carbachol-activated potassium current was 0.89 µM. The chloride-dependent current elicited by glutamate on medial neurons was increased by HgCl₂ as well.5. These results suggest that actions at agonist-activated channels must be considered as contributing to mercury neurotoxicity. It is possible that the toxic actions of Hg²⁺ on synaptic transmission at both pre- and postsynaptic sites are important factors in the mechanism of Hg²⁺ toxicity.     

New Facts about CdCl2 Action on the Photosynthetic Apparatus of Spinach Chloroplasts and Its Comparison with HgCl2 Action

Using EPR spectroscopy it was found that CdCl₂ and HgCl₂ interact (1) with the intermediates , i.e. with the tyrosine radicals on the donor side of photosystem (PS) 2 situated in the 161^st position in D₁ and D₂ proteins; (2) with the primary donor of PS1 (P700) whereby the oxidation of chlorophyll (Chl) a dimer in the reaction centre of PS1 occurs yet in the dark; (3) with the manganese cluster which is situated in the oxygen evolving complex. Due to these interactions of investigated metal chlorides with the photosynthetic apparatus, the interruption of the photosynthetic electron transport through photosynthetic centres occurs. Monitoring of time dependence of EPR signal I of chloroplasts treated with CdCl₂ or HgCl₂ after switching off the light suggests that all mechanisms, i.e. direct, cyclic, and non-cyclic reductions of P700⁺ are damaged. The formation of complexes between mercury or cadmium ions and amino acid residues constituting photosynthetic peptides was suggested as possible mechanism of their inhibitory action. The higher HgCl₂ efficiency in comparison with that of CdCl₂ was explained by higher ability of mercury ions to form complexes with amino acids, what was demonstrated by their apparent binding constants: K = 10 200 M^–1 for Hg²⁺ ions, and K = 3 700 M^–1 for Cd²⁺ ions.     

Detoxification of mercury and organomercurials by nitrogen-fixing soil bacteria

Minimal inhibitory concentration values of HgCl₂ and 5 organomercurials were determined against 24 mercury-resistant N₂-fixing soil bacteria previously isolated from soil and identified in our laboratory. These bacterial strains also displayed multiple antibiotic resistant properties. Typical growth pattern of a highly mercury-resistantBeijerinckia sp (KDr₂) was studied in liquid broth supplemented with toxic levels of mercury compounds. Four bacterial strains were selected for determining their ability to volatilize mercury and their Hg-volatilizing capacity was different. Cell-free extracts prepared from overnight mercury-induced cells catalyzed Hg²⁺-induced NADPH oxidation. Specific activities of Hg²⁺-reductase which is capable of catalyzing conversion of Hg²⁺ →Hg(o) of 10 Hg-resistant bacterial strains are also reported.     

Prolonged incubation with low concentrations of mercury alters energy transfer and chlorophyll (Chl) a protein complexes in Synechococcus 6301: changes in Chl a absorption and emission characteristics and loss of the F695 emission band

Synechococcus PCC 6301 cells grown in the presence of low sublethal levels of (about 2 m) mercury induced alterations in chlorophyll (Chl) a absorption without significant alterations in phycocyanin. Chl a fluorescence emission in Hg²⁺ -raised cells showed a large (about 18 nm) blue shift in the peak emission. No major spectral changes in phycobilisome (PBsome) emission characteristic were noticed, indicating major structural alterations in Chl-protein complexes by incubation with Hg²⁺ ions. Low temperature (77 K) emission spectra of cells grown in the presence of Hg²⁺ showed a loss of the characteristic Chl a emission band at 695 nm (F695), which is known to be linked to photosystem II photochemistry and to originate from the Chl a of core antenna polypeptide CP 47 of photosystem II. The SDS-PAGE polypeptide profile of thylakoids indicates a loss of a polypeptide(s) with a molecular mass between 40 and 60 k Da by Hg²⁺ incubation of cells. Our results suggest that prolonged incubation of Synechococcus 6301 cells with low concentrations of Hg²⁺ affects the Chl a spectral properties and the structure of Chl-protein complexes.     

Action of selected heavy metal ions on the photosystem 2 activity of the cyanobacteriumSpirulina platensis

Addition of different concentrations of heavy metal ions (Hg²⁺, Cu²⁺, Ni²⁺ and Pb²⁺) inhibited the photosystem 2 catalyzed electron transport activity (H₂O→p-benzo-quinone) of the cyanobacteriumSpirulina platensis. Hg²⁺ caused the inhibition in electron transport activity in very low concentrations compared to the other metal ions. Hg²⁺ at this low concentration specifically altered the spectral properties of phycocyanin of the phycobilisomes in the intact cells ofSpirulina, whereas other heavy metal ions were ineffective in this sense.     

Biotransformation of mercury in pH-stat cultures of eukaryotic freshwater algae

Eukaryotic algae were studied to determine their ability to biotransform Hg^II under aerated and pH controlled conditions. All algae converted Hg^II into β-HgS and Hg⁰ to varying degrees. When Hg^II was administered as HgCl₂ to the algae, biotransformation by species of Chlorophyceae (Selenastrum minutum and Chlorella fusca var. fusca) was initiated with β-HgS synthesis (K _1/2 of hours) and concomitant Hg° evolution occurred in the first hour. Hg° synthesis was impeded by the formation of β-HgS and this inhibition was released in C. fusca var. fusca when cellular thiols were oxidized by the addition of dimethylfumarate (DMF). The diatom, Navicula pelliculosa (Bacillariophyceae), converted a substantially greater proportion of the applied Hg^II into Hg⁰, whereas the thermophilic alga, Galdieria sulphuraria (Cyanidiophyceae), rapidly biotransformed as much as 90% of applied Hg^II into β-HgS (K _1/2 ≈ 20 min). This thermophile was also able to generate Hg⁰ even after all exogenously applied HgCl₂ had been biotransformed. The results suggest that β-HgS may be the major dietary mercurial for grazers of contaminated eukaryotic algae.     

Mercury Tolerance of Thermophilic <Emphasis Type="Italic">Bacillus</Emphasis> sp. and <Emphasis Type="Italic">Ureibacillus</Emphasis> sp

Although resistance of microorganisms to Hg(II) salts has been widely investigated and resistant strains have been reported from many eubacterial genera, there are few reports of mercuric ion resistance in extremophilic microorganisms. Moderately thermophilic mercury resistant bacteria were selected by growth at 62 °C on Luria agar containing HgCl₂. Sequence analysis of 16S rRNA genes of two isolates showed the closest matches to be with Bacillus pallidus and Ureibacillus thermosphaericus. Minimum inhibitory concentration (MIC) values for HgCl₂ were 80 μg/ml and 30 μg/ml for these isolates, respectively, compared to 10 μg/ml for B. pallidus H12 DSM3670, a mercury-sensitive control. The best-characterised mercury-resistant Bacillus strain, B. cereus RC607, had an MIC of 60 μg/ml. The new isolates had negligible mercuric reductase activity but removed Hg from the medium by the formation of a black precipitate, identified as HgS by X-ray powder diffraction analysis. No volatile H₂S was detected in the headspace of cultures in the absence or presence of Hg²⁺, and it is suggested that a new mechanism of Hg tolerance, based on the production of non-volatile thiol species, may have potential for decontamination of solutions containing Hg²⁺ without production of toxic volatile H₂S.     

Effect of cadmium on photosystem II activity in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>: alteration of O–J–I–P fluorescence transients indicating the change of apparent activation energies within photosystem II

In this study, we evaluated how cadmium inhibitory effect on photosystem II and I electron transport may affect light energy conversion into electron transport by photosystem II. To induce cadmium effect on the photosynthetic apparatus, we exposed Chlamydomonas reinhardtii 24 h to 0–4.62 μM Cd²⁺. By evaluating the half time of fluorescence transients O–J–I–P at different temperatures (20–30°C), we were able to determine the photosystem II apparent activation energies for different reduction steps of photosystem II, indicated by the O–J–I–P fluorescence transients. The decrease of the apparent activation energies for PSII electron transport was found to be strongly related to the cadmium-induced inhibition of photosynthetic electron transport. We found a strong correlation between the photosystem II apparent activation energies and photosystem II oxygen evolution rate and photosystem I activity. Different levels of cadmium inhibition at photosystem II water-splitting system and photosystem I activity showed that photosystem II apparent activation energies are strongly dependent to photosystem II donor and acceptor sides. Therefore, the oxido-reduction state of whole photosystem II and I electron transport chain affects the conversion of light energy from antenna complex to photosystem II electron transport.     

Site-specific Inhibition of Photophosphorylation in Isolated Spinach Chloroplasts by Mercuric Chloride

Photophosphorylation associated with noncyclic electron transport in isolated spinach (Spinacia oleracea) chloroplasts is inhibited to approximately 50% by low concentrations of HgCl₂ (less than 1 μmole Hg²⁺/mg chlorophyll) when the electron transport pathway includes both sites of energy coupling. Reactions involving only a part of the electron transport system can give a functional isolation of at least two sites coupled to phosphorylation. Only one of these sites, located between the oxidation of plastoquinone and the reduction of cytochrome f, is sensitive to mercuric chloride. The energy conservation site located before plastoquinone and close to photosystem II is unaffected by HgCl₂ concentrations up to 10-fold those required to inhibit phosphorylation by the coupling site after plastoquinone. This site-specific inhibition may reflect a mechanistic difference in the mode of energy coupling at the two coupling sites or a variable accessibility of HgCl₂ to these sites.     

Calcium and magnesium effect on divalent cation deficientHydrilla verticillata thylakoid electron transport activity

The role of divalent cations like magnesium (Mg²⁺) and calcium (Ca²⁺) was irrvestigated on energy distribution process ofHydrilla verticillata thylakoids. Effect of these cations was tested on relative quantum yield of photosystem (PS) II catalyzed electron transport activity, room and liquid nitrogen temperature fluorescence emission properties and thylakoid light scattering characteristics. The electron transport activity was found to be stimulated in the presence of these cations in a light intensity independent manner. The concentration of cation required for maximum stimulation was nearly 10–12 mM. Comparatively, Ca²⁺ was more effective than Mg²⁺. Cation induced stimulation in electron transport activity was not accompanied by increase in chlorophylla fluorescence intensity either at room (25°C) or liquid nitrogen (77°K) temperatures. Furthermore, 540 nm absorption and 90° light scattering properties of thylakoids remained insensitive towards divalent cations. These facts together suggest that divalent cations inHydrilla thylakoids are not effective in supporting the excitation distribution between the interacting photosystem complexes.     

The kinetics of reactions around the cytochrome bf complex studied in an isolated system

The kinetics of oxidation and reduction of P700, plastocyanin, cytochrome f and cytochrome b-563 were studied in a reconstituted system consisting of Photosystem I particles, cytochrome bf complex and plastocyanin, all derived from pea leaf chloroplasts. Decyl plastoquinol was the reductant of the bf complex. Turnovers of the system were initiated by laser flashes. The reaction between oxidised P700 and plastocyanin was non-homogeneous in that a second-order rate coefficient of c. 5×10^–7 M^–1 s^–1 applied to 80% of the P700⁺ and c. 0.7×10⁷ M^–1 s^–1 to the remainder. In the presence of bf complex, but without quinol, the electron transfer between cytochrome f and oxidised plastocyanin could be described by a second-order rate coefficient of c. 4×10⁷ M^–1 s^–1 (forward), and c. 1.6×10⁷ M^–1 s^–1 (reverse). The equilibrium coefficient was thus 2.5. Unexpectedly, there was little reduction of cytochrome f ⁺ or plastocyanin⁺ by electrons from the Rieske centre. With added quinol, reduction of cytochrome b-563 occurred. Concomitantly, electrons appeared in the oxidised species. It was inferred that either the Rieske centre was not involved in the high-potential chain of electron transfer events, or that, only in the presence of quinol, electrons were quickly passed from the Rieske centre to cytochrome f ⁺. Additionally, the presence of quinol altered the equilibrium coefficient for the cyt f/PC interaction from 2.5 to c. 5. The reaction between quinol and the bf complex was describable by a second-order rate coefficient of about 3×10⁶ M^–1 s^–1. The pattern of the redox reactions around the bf complex could be simulated in detail with a Q-cycle model as previously found for chloroplasts.Abbreviations AQS anthraquinone sulphonate - cyt cytochrome - cyt b-563(H) high-potential cyt b-563 - cyt b-563(L) low potential cyt b-563 - FeS(R) the Rieske protein of the cyt bf complex, containing an Fe₂S₂ centre - PC plastocyanin - PS photosystem - P700 reaction centre in PS I     

Inorganic mercury (Hg2+) transport through lipid bilayer membranes

Summary Diffusion of inorganic mercury (Hg²⁺) through planar lipid bilayer membranes was studied as a function of chloride concentration and pH. Membranes were made from egg lecithin plus cholesterol in tetradecane. Tracer (²⁰³Hg) flux and conductance measurements were used to estimate the permeabilities to ionic and nonionic forms of Hg. At pH 7.0 and [Cl^–] ranging from 10–1000mm, only the dichloride complex of mercury (HgCl₂) crosses the membrane at a significant rate. However, several other Hg complexes (HgOHCl, HgCl ₃ ^– and HgCl ₄ ^2– ) contribute to diffusion through the aqueous unstirred layer adjacent to the membrane. The relation between the total mercury flux (J _Hg), Hg concentrations, and permeabilities is: 1/J _Hg=1/P ^ul[Hg ^t ]+1/P ^m [HgCl₂], where [Hg ^t ] is the total concentration of all forms of Hg,P ^ul is the unstirred layer permeability, andP ^m is the membrane permeability to HgCl₂. By fitting this equation to the data we find thatP ^m =1.3×10^–2 cm sec^–1. At Cl^– concentrations ranging from 1–100mm, diffusion of Hg ^t through the unstirred layer is rate limiting. At Cl^– concentrations ranging from 500–1000mm, the membrane permeability to HgCl₂ becomes rate limiting because HgCl₂ comprises only about 1% of the total Hg. Under all conditions, chemical reactions among Hg²⁺, Cl^– and/or OH^– near the membrane surface play an important role in the transport process. Other important metals, e.g., Zn²⁺, Cd²⁺, Ag⁺ and CH₃Hg⁺, form neutral chloride complexes under physiological conditions. Thus, it is likely that chloride can facilitate the diffusion of a variety of metals through lipid bilayer and biological membranes.     

Contribution of plastocyanin isoforms to photosynthesis and copper homeostasis in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> grown at different copper regimes

In land plants plastocyanin is indispensable and therefore copper (Cu) availability is a prerequisite for growth. When Cu supply is limited, higher plants prioritize the Cu delivery to plastocyanin by down-regulation of other Cu proteins. Arabidopsis has two plastocyanin genes (PETE1 and PETE2). PETE2 is the predominant isoform in soil-grown plants and in hydroponic cultures it is accumulated in response to Cu addition. It functions as a Cu sink when more Cu is available, in addition to its role as an electron carrier. PETE1 is not affected by Cu feeding and it is the isoform that drives electron transport under Cu-deficiency. Cu feeding rescued the defect in photosystem II electron flux (Φ_PSII) in the pete1 mutant whereas Φ_PSII was not changed in the pete2 mutant as Cu was added. Plants with mutations in the plastocyanin genes had altered Cu homeostasis. The pete2 mutant accumulated more Cu/Zn superoxide dismutase (CSD2 and CSD1) and Cu chaperone (CCS) whereas the pete1 mutant accumulated less. On the other hand, less iron superoxide dismutase (FeSOD) and microRNA398b were observed in the pete2 mutant, whereas more were accumulated in the pete1 mutant. Our data suggest that plastocyanin isoforms are different in their response to Cu and the absence of either one changes the Cu homeostasis. Also a small amount of plastocyanin is enough to support efficient electron transport and more PETE2 is accumulated as more Cu is added, presumably, to buffer the excess Cu. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

On the functional organization of electron transport from plastoquinone to Photosystem I

Signal I, the EPR signal of P-700, induced by long flashes as well as the rate of linear electron transport are investigated at partial inhibition of electron transport in chloroplasts. Inhibition of plastoquinol oxidation by dibromothymoquinone and bathophenanthroline, inhibition of plastocyanin by KCN and HgCl₂, and inhibition by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide are used to study a possible electron exchange between electron-transport chains after plastoquinone. (1) At partial inhibition of plastocyanin the reduction kinetics of P-700⁺ show a fast component comparable to that in control chloroplasts and a new slow component. The slow component indicates P-700⁺ which is not accessible to residual active plastocyanin under these conditions. We conclude that P-700 is reduced via complexed plastocyanin. (2) The rate of linear electron transport at continuous illumination decreases immediately when increasing amounts of plastocyanin are inhibited by KCN incubation. This is not consistent with an oxidation of cytochrome f by a mobile pool of plastocyanin with respect to the reaction rates of plastocyanin being more than an order of magnitude faster than the rate-limiting step of linear electron transport. It is evidence for a complex between the cytochrome b₆ - f complex and plastocyanin. The number of these complexes with active plastocyanin is concluded to control the rate-limiting plastoquinol oxidation. (3) Partial inhibition of the electron transfer between plastoquinone and cytochrome f by dibromothymoquinone and bathophenanthroline causes decelerated monophasic reduction of total P-700⁺. The P-700 kinetics indicate an electron transfer from the cytochrome b₆ - f complex to more than ten Photosystem I reaction center complexes. This cooperation is concluded to occur by lateral diffusion of both complexes in the membrane. (4) The proposed functional organization of electron transport from plastoquinone to P-700 in situ is supported by further kinetic details and is discussed in terms of the spatial distribution of the electron carriers in the thylakoid membrane.     

Mercury detoxifying enzymes within endospores of a broad-spectrum mercury resistantBacillus pasteurii strain DR2

Bacillus pasteurii DR₂, a broad-spectrum Hg-resistant bacterial strain, exhibited delayed sporulation and less mercury volatilization in the presence of mercury compounds. However, Hg-sensitiveBacillus subtilis sporulated quickly in the presence of HgCl₂ and volatilized no mercury. Levels of Hg²⁺-reductase and organomercurial lyase in the endospores ofBacillus pasteurii DR₂ were lower than those in vegetative cells     

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.     

The origin of photosystem-I-mediated electron transport stimulation in heat-stressed chloroplasts

Exposure of isolated chloroplasts of pea (Pisum sativum L.) to temperatures above 35° C leads to a stimulation of photosystem-I-mediated electron transport from dichlorophenolindophenol to methyl viologen. The threshold temperature for this stimulation coincides closely with that for heat-induced inhibition of photosystem-II activity in such chloroplasts. This coincidence is explained in terms of a rearrangement of the thylakoid membrane resulting in the exposure of a new set of donor sites for dichlorophenolindophenol within the cytochrome f/b ₆ complex of the electron-transport chain linking the two photosystems.Abbreviations cyt cytochrome - DBMIB 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCPIP (H₂) 2,6-dichlorophenolindophenol - EDAC ethyldimethylaminopropyl-carbodiimide - MV methyl viologen - PSI, II photosystem I, II - PCy plastocyanin - PQ(H₂) plastoquinone     

Immediate effects of pyridazinone herbicides on photosynthetic electron transport in algal systems

Pyridazinone herbicides, SANDOZ 9785 (4-chloro-5-dimethylamino2-phenyl-3-(2H) pyridazinone), SANDOZ 9789 (4-chloro-5 (methylamino)-2-(α, α, α-trifluoro-m-tolyl-3-(2H) pyridazinone) and SANDOZ 6706 (4-chloro-5-(methylamino)-2-(α, α, α-trifluoro-m-tolyl-3-(2H) pyridazinone) inhibited photosystem II electron transport inChlorella protothecoides, when the herbicides were added to the assay medium. The inhibitory eficiency varied with the algal species and the nature of substitution of pyridazinones. Using 3 algal systemsviz., Chlorella, Scenedesmus andAnacystis, the I₅₀ value of for the inhibition of photosynthesis of 3 substituted pyridazinones (SANDOZ 9785, SANDOZ 6706 and SANDOZ 9789) were determined. SANDOZ 9789 was found to be the weakest inhibitor of photosystem II electron transport (H₂O→ benzoquinone) as compared to SANDOZ 9785 and SANDOZ 6706. In general, the order of inhibition could be given as SANDOZ 6706 >- SANDOZ 9785 > SANDOZ 9789. The I₅₀ value of photosynthetic particles obtained fromChlorella cells was similar to that of whole cells, suggesting that the cell wall ofChlorella did not act as a barrier for the herbicide action. Studies on the light intensity dependence of SANDOZ 9785 inhibition of electron transport (H₂O→ benzoquinone) showed that the light-dependent portion of the curve was more sensitive than the light independent portion of the curve. It is suggested that the site of action was on the reducing side of photosystem II.     

Evidence for two sites of inhibition of photosynthetic electron transport by dibromothymoquinone

Two sites in the photosynthetic electron transport chain of spinach chloroplasts are sensitive to inhibition by the plastoquinone antagonist dibromothymoquinone (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone). This compound imposes maximal inhibition on reactions involving electron transport from water to a terminal acceptor such as ferricyanide at concentrations of about 1 μm. At concentrations of about 10 μm, dibromothymoquinone also inhibits electron transport reactions catalyzed by photosystem II in the presence of p-phenylenediimines or p-benzoquinones. This inhibition is observed in both untreated and $\text{KCN}\text{Hg}$-inhibited chloroplast preparations. Thiol incubation of chloroplasts exposed to dibromothymoquinone relieves inhibition at both sites. This reversal of inhibition is, however, different for the two sites. Restoration of ferricyanide reduction, which is blocked by 1 μm dibromothymoquinone, required high thiol/inhibitor ratios and incubation times with thiol of up to 3 min. The reversal of inhibition of p-phenylenediimine reduction by photosystem II, on the other hand, requires a thiol/inhibitor ratio of 1, and incubation times as short as 5 s. Addition of bovine serum albumin to absorb dibromothymoquinone results in a partial restoration of photosystem II reactions, but ferricyanide reduction, which requires photosystem II and photosystem I, cannot be restored by this procedure.     

Facilitated water transport in cyanobacterium Synechococcus sp. PCC 7942 studied by phycobilisome-sensitized chlorophyll a fluorescence

Water transport across plant cell membranes is difficult to measure. We present here a model assay, based on chlorophyll (Chl) a fluorometry, with which net water transport across the cell membrane of freshwater cyanobacterium Synechococcus sp. PCC7942 (S7942) can be followed kinetically with millisecond-time resolution. In cyanobacteria, the phycobilisome (PBS)-sensitized Chl a fluorescence increases when cells expand (e.g., in hypo-osmotic suspension) and decreases when cells contract (e.g., in hyper-osmotic suspension). The osmotically-induced Chl a fluorescence changes are proportional to the reciprocal of the suspension osmolality (ΔF ∝ Osm⁻¹; Papageorgiou GC and Alygizaki-Zorba A (1997) Biochim Biophys Acta 1335: 1–4). In our model assay, S7942 cells were loaded with NaCl (passively penetrating solute) and shrunk in hyper-osmotic glycine betaine (nonpenetrating solute). Upon injecting these cells into hypo-osmotic medium, the PBS-sensitized Chl a fluorescence rose to a maximum due to the osmotically-driven water uptake. The rise of Chl a fluorescence (water uptake) was partially inhibited by HgCl₂, at micromolar concentrations. Arrhenius plots of the water uptake rates gave activation energies of E_A=4.9 kcal mol⁻¹, in the absence of HgCl₂, and E_A=11.9 kcal mol⁻¹ in its presence. These results satisfy the usual criteria for facilitated water transport through protein water pores of plasma membranes (aquaporins), namely sensitivity to Hg²⁺ ions and low activation energy.