Multiple anion effects on photosystem II in chloroplast membranes

We investigated the activity of several anions at various sites on photosystem II, in particular those associated with the Cl^- effect (anion binding-site I) and the HCO₃ ^- effect (anion binding-site II). Chlorophyll a fluorescence changes were used to monitor partial photosystem II reactions either in the oxygen-evolving mechanism or involving endogenous quinone electron acceptors. We find that anions such as NO₃ ^-, HCO₃ ^-, HCO₂ ^-, F^-, NO₂ ^-, and acetate can, depending on conditions, bind to either anion binding-site I, anion binding-site II, or both sites simultaneously. The anions N₃ ^- and Au(CN)₂ ^- are exceptions. In their presence, oxygen-consumption reactions are enhanced. The results demonstrate that an exclusive site or mode of action of an anion on photosystem II cannot be determined by measuring the Hill reaction alone. Anion interactions with photosystem II are shown to be very complex and, therefore, caution is advisable in interpreting related experiments. Carbonic anhydrase associated with photosystem II was also investigated as a possible target for some anion effects. In Cl^--depleted thylakoids, NO₃ ^-, stimulated both electron transport and carbonic anhydrase activity at low concentrations, while higher concentrations inhibited both. However, carbonic anhydrase was more sensitive to inhibition by NO₃ ^- than was electron flow. Possible interpretations are discussed; the electron transport and carbonic anhydrase activity appear not to be functionally linked.Abbreviations ABSI Anion binding-site(s) I associated with the oxygen-evolving mechanism - ABSII Anion binding-site(s) II, which controls quinone-related reactions on the electron-acceptor side of photosystem II - OAc^- Acetate - Chl Chlorophyll - DCMU 3—(3,4-dichlorophenyl)-1,1-dimethyl urea - DCBQ 2,6-dichloro-p-benzoquinone - DMBQ 2,5-dimethyl-p-benzoquinone - Mes 2-[N-morpholino]ethanesulphonic acid - Mops 3-[N-morpholino]propanesulphonic acid - Tes N-Tris[hydroxymethyl]methyl-2-aminoethanesulphonic acid - Tricine N-Tris[hydroxymethyl]methylglycine     

The relationship of arginine groups to photosynthetic HCO 3 - uptake inUlva sp. mediated by a putative anion exchanger

The marine macroalgaUlva sp. can take up HCO ₃ ^- via a process which chemically resembles that of anion exchange in red blood cells (Drechsler et al. 1993, Planta191, 34–40). In this work we explore the possibility that high-pK amino-acid residues could be functionally involved in the binding/transport of HCO ₃ ^- . It was found that the specific arginyl-reacting agents phenylglyoxal and 2,3-butanedione inhibited photosynthesis ofUlva competitively with inorganic carbon at pH 8.2–8.4 (which is close to the pH of normal seawater), where HCO ₃ ^- was the predominant inorganic carbon form taken up. The inhibition by phenylglyoxal was irreversible at 32°C and high pH values, while that of butanedione became irreversible in the presence of borate. These interactions, as well as the protection of the irreversible phenylglyoxal-inhibition by inorganic carbon and by the membrane-impermeant agents 4,4-diisothiocyanostilbene 2,2-disulfonate and 4,4-dinitrostilbene-2,2-disulfonate indicate that arginine (and possibly also lysine) are involved in the HCO ₃ ^- uptake process, probably at the plasmalemma level. The photosynthetic affinity ofUlva to external inorganic carbon gradually decreased with increasing pH from 8.2 to 10.5, and this decrease parallels the decline in protonation of amino acids with a pK of around 10. Based on this information, as well as the inhibition studies, it is suggested that arginine and lysine residues are essential proteinaceous constituents involved in anionic inorganic carbon (HCO ₃ ^- and possibly also CO ₃ ^2- ) uptake into theUlva cells.Abbreviations AE1 anion exchanger 1 (of red blood cells) - BD 2,3-butanedione - CA carbonic anhydrase - CI inorganic carbon - DIDS 4,4-diisothiocyanostilbene-2,2-disulfonate - DNDS 4,4-dinitrostilbene-2,2-disulfonate - PG phenylglyoxal This paper is in partial fulfillment of a Ph.D. study by R. Sharkia. Supported by the Israel Academy of Sciences, grant 441/93 (to S.B.), and by the Fund for Encouragement of Research, Histadrut, Israel (to R.S.).     

Interaction between photosynthetic and respiratory electron-transfer chains in the membranes of Anabaena variabilis

The rate of CO₂- and p-benzoquione-dependent photosynthetic O₂ evolution by Anabaena variabilis cells remained unaltered and the rate of O₂ uptake observed after switching off the light (endogenous respiration) was enhanced by a factor of 6–8 when the O₂ concentration was increased from 200 to 400 M. Photosystem-I-linked O₂ uptake and respiration of the cells incubated with ascorbate and N,N,NN-tetramethyl-p-phenylenediamine was not appreciable influenced by the O₂ concentration. 2-Iodo-6-isopropyl-3-methyl-2,4,4-trinitrodiphenyl ether, blocking electron transfer at the plastoquinone level, suppressed O₂ evolution and had no influence on endogenous respiration. 2-n-Heptyl-4-hydroxyquinoline-N-oxide, an inhibitor of electron transfer between photosystems II and I, as well as the cytochrome-oxidase inhibitors N ₃ ^- , CN^- and NH₂OH, caused a 35–50% retardation of endogenous respiration and blocked photosynthetic O₂ evolution. The molar ratio of cytochromes b₆, f, c-553, aa₃ and photosystem-I reaction centers in the isolated membranes equalled approx. 2:1:2:0.7:2. It is inferred that endogenous respiration of A. variabilis cells is inhibited by the light-induced electron flow through both photosystems at the level of the plastoquinone-plastocyanin-oxidoreductase complex.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DNP-INT 2-iodo-6-isopropyl-3-methyl-2,4,4-trinitrodiphenyl ether - Hepes 4-(2-hydroxyethyl)-1-piperazine ethansulfonic acid - TMPD N,N,NN-tetramethyl-p-phenylenediamine     

The effect of glucose on chloride uptake by Chlorella

Active Cl^- uptake by Chlorella fusca was examined by using ³⁶Cl as a label. Under light/air conditions chloride influx from a 2.4·10^-5 M solution was 4.0±0.04 nmol m^-2s^-1. After 70±10 min a stationary 380±40 fold accumulation was reached. In dark/air and dark/argon influx and accumulation were reduced to 25±6%, respectively, 5±1.5% of the light/air control. Cl^- uptake had a broad optimum around pH 7 and showed saturation kinetics with a K _M of 1.25·10^-5 M and a v _max of 7.0 nmol m^-2s^-1 in light/air. Br^- inhibited Cl^- uptake strongly, J^-, ClO ₄ ^- , SO ₄ ^2- , and NO ₃ ^- had no inhibitory effect. Inhibitor studies with carbonyl cyanide m-chlorophenylhydrazone and N,N-dicyclohexylcarbodiimide resulted in a good correlation between Cl^- uptake and ATP level. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea and darkness reduced transport activity without affecting the ATP level.The magnitudes of the pH gradient and the membrane potential across the cell membrane were determined and/or estimated under different conditions. It could be shown that in Chlorella Cl^- transport cannot proceed via secondary active H⁺/Cl^- cotransport. In addition, 2H⁺/Cl^- cotransport seems unlikely for energetic reasons. On the basis of the results of this and the following study, a primary active ATP-driven Cl^-/OH^- exchange pump is proposed.Abbreviations CCCP carbonyl cyanide m-chlorophenylhyd razone - DCCD N,N-dicyclohexylcarbodiimide - DCMU 3-(3.4-dichlorophenyl)-1.1-dimethylurea - DMO 5,5-dimethyloxazolidine-2,4-dione - Hepes N-2-hydroxyethylpiperazine-N ethane-sulfonic acid - POPOP 1.4-bis-2-(4-methyl-5-phenyloxazolyl)-benzene - PPO 2.5-diphenyloxazole To whom correspondence should be addressed     

Electron transport and respiration in Beggiatoa and Vitreoscilla

Seven strains of bacteria belonging to the Beggiatoa-Vitreoscilla group were studied for their respiratory activity and for the presence of electron transport conponents. All strains tested oxidized [1-¹⁴C] and [2-¹⁴C] acetate to ¹⁴CO₂ at relatively high rates. All strains tested were N,N,N,N-tetramethylphenylenediamine (TMPD)-oxidase positive and contained spectra representing a-type and carbon monoxide-binding cytochromes. Most of the strains also contained spectra representing c-type and b-type cytochromes. Beggiatoa alba B18LD contained b-type, a-type, c-type and CO-binding cytochromes, the latter two being located in the 144,000 x g soluble fraction. B. alba also contained ubiquinone-8 as its only detectable quinone.Non-standard abbreviations BSS basal salts solution - BH Beggiatoa heterotrophic medium - BSO Beggiatoa sulfide oxidation medium - TMPD N,N,N,N-tetramethylphenylenediamine - Q8 ubiquinone-8     

Relation between the anion exchange protein in kidney medullary collecting duct cells and red cell band 3

Summary A membrane protein that is immunochemically similar to the red cell anion exchange protein, band 3, has been identified on the basolateral face of the outer medullary collecting duct (MCD) cells in rabbit kidney. In freshly prepared separated rabbit MCD cells, M.L. Zeidel, P. Silva and J.L. Seifter (J. Clin. Invest. 77:1682–1688, 1986) found that Cl^–/HCO ₃ ^- exchange was inhibited by the stilbene anion exchange inhibitor, DIDS (4,4-diisothiocyano-2,2-disulfonic stilbene), with aK ₁ similar to that for the red cell. We have measured the binding affinities of a fluorescent stilbene inhibitor, DBDS (4,4-dibenzamido-2,2-disulfonic stilbene), to MCD cells in 28.5 mM citrate and have characterized both a high-affinity site (K ₁ ^s =93±24 mM) and a lower affinity site (K ₂ ^s =430±260 nM), which are closely similar to values for the red cell of 110±51 nM for the high-affinity site and 980±200 nM for the lower affinity site (A.S. Verkman, J.A. Dix & A.K. Solomon,J. Gen. Physiol. 81:421–449, 1983). When Cl^– replaces citrate in the buffer, the two sites collapse into a single one withK ₁ ^s =1500±400 nM, similar to the singleK ₁ ^s =1200±200 nM in the red cell (J.A. Dix, A.S. Verkman & A.K. Solomon,J. Membrane Biol. 89:211–223, 1986). The kinetics of DBDS binding to MCD cells at 0.25 M^–1 are characterized by a fast process, =0.14±0.03 sec, similar to =0.12±0.03 sec in the red cell. These similarities show that the physical chemical characteristics of stilbene inhibitor binding to MCD cell band 3 closely resemble those for red cell band 3, which suggests that the molecular structure is highly conserved.     

Characterization of novel mono-O-acetylated GM3s containing 9-O-acetyl sialic acid and 6-O-acetyl galactose in equine erythrocytes

Novel mono-O-acetylated GM3s, one containing 9-O-acetylN-glycolyl neuraminic acid and another containing 6-O-acetyl galactose, were isolated as a mixture from equine erythrocytes, and the structures were characterized by one- and two-dimensional proton nuclear magnetic resonance (NMR) and fast atom bombardment-mass spectrometry (FAB-MS). The position of theO-acetyl residue was identified by the downfield shift of the methylene protons at C-9 ofN-glycolyl neuraminic acid (9-O-Ac GM3) and C-6 of galactose (6-O-Ac GM3) in the NMR spectrum, in comparison to the respective non-acetylated counterparts. To confirm the presence of 6-O-Ac GM3, theO-acetylated GM3 mixture was desialylated withArthrobacter neuraminidase, giving 6-O-acetyl galactosyl glucosylceramide, the structure of which was estimated by NMR and FAB-MS, together with non-acetylated lactosylceramide with a ratio of 1:1. Abbreviations: Ac, acetyl; Gc, glycolyl; NeuGc,N-Gc neuraminic acid; GM3 (Gc), GM3 containing NeuGc (II³NeuGc-LacCer); 4-O-Ac GM3 (Gc), GM3 containing 4-O-Ac NeuGc; 9-O-Ac GM3 (Gc), GM3 containing 9-O-Ac NeuGc; 6-O-Ac GM3 (Gc), GM3 containing 6-O-Ac Gal; 1D-NMR, one-dimensional nuclear magnetic resonance spectrometry; 2D-COSY, two-dimensional chemical shift-correlated spectrometry; FAB-MS, fast atom bombardment-mass spectrometry; GLC, gas-layer chromatography; GC-MS, gas chromatography-mass spectrometry; TLC, thin-layer chromatography; Ggl, ganglioside; Cer, ceramide; CMH, monohexosylceramide; LacCer, lactosylceramide; 6-O-Ac LacCer, LacCer containing 6-O-Ac Gal; Me₂SO-d₆,²H₆-dimethylsufloxide; CMW, chloroform-methanol-water; Nomenclature and abbreviations of glycosphingolipids follow the system of Svennerholm (J Neurochem [1963]10: 613–23) and those recommended by the IUPAC-IUB Nomenclature Commission (Lipids [1977]12: 455–68).     

Superoxide production by thylakoids during chilling and its implication in the susceptibility of plants to chilling-induced photoinhibition

Factors influencing the rate of superoxide (O ₂ ^- ) production by thylakoids were investigated to determine if increased production of the radical was related to injury induced by chilling at a moderate photon flux density (PFD). Plants used were Spinacia oleracea L., Cucumis sativus L. and Nerium oleander L. grown at either 200° C or 45° C. Superoxide production was determined by electron-spin-resonance spectroscopy of the (O ₂ ^- )-dependent rate of oxidation of 2-ethyl-1-hydroxy-2,5,5-trimethyl-3-oxazolidine (OXANOH) to the corresponding oxazolidinoxyl radical, OXANO ·. For all plants, the steady-state rate of O ₂ ^- production by thylakoids, incubated at 25° C and 350 mol photon · m^–2 · s^–1 (moderate PFD) with added ferredoxin and NADP, was between 7.5 and 12.5 mol · (mg chlorophyll)^–1 · h^–1. Incubation at 5° C and a moderate PFD, decreased the rate of O ₂ ^- production 40% and 15% by thylakoids from S. oleracea and 20° C-grown N. oleander, chillinginsensitive plants, but increased the rate by 56% and 5% by thylakoids from C. sativus and 45° C-grown N. oleander, chilling-sensitive plants. For all plants, the addition of either ferredoxin or methyl viologen increased the rate of O ₂ ^- -production at 25° C by 75–100%. With these electron acceptors, lowering the temperature to 5° C caused only a slight decrease in O ₂ ^- production. In the absence of added electron acceptors, thylakoids produced O ₂ ^- at a rate which was about 45% greater than that when ferredoxin and NADP were present. The addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea reduced O ₂ ^- production under all conditions tested. The results show that the rate of O ₂ ^- production increases in thylakoids when the rate of electron transfer to NADP is reduced. This could explain differences in the susceptibility of thylakoids from chilling-sensitive and chilling-insensitive plants to chilling at a moderate PFD, and is consistent with the proposal that O ₂ ^- production is involved in the injury leading to the inhibition of photosynthesis induced under these conditions.Abbreviations Chl chlorophyll - DCMU 3-(3,4-dichlorophen-yl)-1,1-dimethylurea - Fd ferredoxin - MV methyl viologen - 20°oleander Nerium oleander grown at 20° C - 45°-oleander N. oleander grown at 45° C - OXANOH 2-ethyl-1-hydroxy-2,5,5-tri-methyl-3-oxazolidine - PFD photon flux density (photon fluence rate) - TEMED tetramethyl ethylenediamine We would like to thank R.T. Furbank, R.S.B.S., Australian National University, Canberra, A.C.T., and C.B. Osmond, now of Duke University, Durham, N.C., USA, for the gift of ferredoxin, R.A.J.H. was supported by a Commonwealth Postgraduate Research Award.     

Active transport of proline into washed cells of Rhodopseudomonas sphaeroides f. sp. denitrificans grown with nitrate

Washed cells of Rhodopseudomonas sphaeroides f. sp. denitrificans, prepared from cultures grown anaerobically in light with NO ₃ ^- as the terminal acceptor, readily incorporated [¹⁴C]-proline both in light and in the dark. The proline uptake was coupled to the reduction of either NO ₃ ^- , NO ₂ ^- , N₂O or O₂. Light stimulated the accumulation of proline in these cells. The addition of NO ₃ ^- to washed cells in light decreased the K _m for proline from 40 M to 5.7 M. Proline transport was inhibited by antimycin A, 2-n-heptyl-4-hydroxyquinoline-N-oxide both in light and in the dark with nitrate indicating that electron transfer from both denitrification and photosynthesis are involved in this uptake. Inhibition by carbonyl cyanide-m-chlorophenyl hydrazone and 2.4-dinitrophenol indicate that proline transport is energy dependent. The H⁺/proline stoichiometry increased from 1 to 2.5 when the external pH was increased from 6.0 to 8.0. Under these conditions pro increased but p decreased markedly above pH 7.0.Abbreviations TPP⁺ Tetraphenylphosphonium bromide - EDTA ethylenediamine-tetra-acetic acid - CCCP carbonyl cyanide-m-chlorophenyl hydrazone - DNP 2,4-dinitrophenol - HOQNO 2-n-heptyl-4-hydroxyquinoline-N-oxide - DBMIB dibromo-methyl-isopropyl-p-benzoquinone - DCCD N,N-dicyclohexylcarbodiimide     

Observations on the reduction of non-activated carboxylates by Clostridium formicoaceticum with carbon monoxide or formate and the influence of various viologens

Crude extracts or supernatants of broken cells of Clostridium formicoaceticum reduce unbranched, branched, saturated and unsaturated carboxylates at the expense of carbon monoxide to the corresponding alcohols. The presence of viologens with redox potentials varying from E ₀=-295 to-650 mV decreased the rate of propionate reduction. The more the propionate reduction was diminished the more formate was formed from carbon monoxide. The lowest propionate reduction and highest formate formation was observed with methylviologen. The carbon-carbon double bond of E-2-methyl-butenoate was only hydrogenated when a viologen was present. Formate as electron donor led only in the presence of viologens to the formation of propanol from propionate. The reduction of propionate at the expense of a reduced viologen can be followed in cuvettes. With respect to propionate Michaelis Menten behavior was observed. Experiments are described which lead to the assumption that the carboxylates are reduced in a non-activated form. That would be new type of biological reduction.Non-standard abbreviations glc Gas liquid chromatography - HPLC high performance liquid chromatography - RP reverse phase; Mediators (the figures in parenthesis of the mediators are redox potentials E ₀ in mV) - CAV²⁺ carbamoylmethylviologen, 1,1-carbamoyl-4,4-dipyridinium dication (E ₀=-296 mV) - BV²⁺ benzylviologen, 1,1-dibenzyl-4,4-dipyridinium dication (E ₀=-360 mV) - MV methylviologen, 1,1-dimethyl-4,4-dipyridinium-dication (E ₀=-444 mV) - DMDQ²⁺ dimethyldiquat, 4,4-dimethyl-2,2-dipyridino-1,1-ethylendication (E ₀=-514 mV) - TMV²⁺ tetramethylviologen, 1,1,4,4-tetramethyl-4,4-dipyridinium dication (E ₀=-550 mV) - PDQ²⁺ propyldiquat, 2,2-dipyridino-1,1-propenyl dication (E ₀=-550 mV) - DMPDQ²⁺ dimethylpropyldiquat, 4,4-dimethyl-2,2-dipyridino-1,1-propenyl dication (E ₀=-656 mV) - PN productivity number=mmol product (obtained by the uptake of one pair of electrons) x (biocatalyst (dry weight) kg)^-1×h^-1     

Characterization of esophageal desalination in the seawater eel,Anguilla japonica

To characterize mechanisms of esophageal desalination, osmotic water permeability and ion fluxes were measured in the isolated esophagus of the seawater eel. The osmotic permeability coefficient in the seawater eel esophagus was 2·10^-4 cm·s^-1. This value was much lower than those in tight epithelial, although the eel esophagus is a leaky epithelium with a tissue resistance of 77 ohm·cm^-2. When the esophagus was bathed in normal Ringer solutions on both sides no net ion and water fluxes were observed. However, when mucosal NaCl concentration was increased by a factor of 3, Na⁺ und Cl^- ions were transferred from mucosa to serosa (desalination). If only Na⁺ or Cl^- concentration in the mucosal fluid was increased by a factor of 3, net Na⁺ and Cl^- fluxes were reduced to 30–40%, indicating that 60–70% of the net Na⁺ and Cl^- fluxes are coupled mutually. The coupled NaCl transport seems to be effective in desalting the luminal high NaCl. The remaining 30–40% of the total Na⁺ and Cl^- fluxes seems to be due to a simple diffusion, because these components are independent of each other and follow their electrochemical gradients, and also because these fluxes remain even after treatment with NaCN or ouabain. A half of the coupled NaCl transport could be explained by a Na⁺/H⁺–Cl^-/HCO ₃ ^- double exchanger on the apical membrane of the esophageal epithelium, because mucosal amiloride and 4.4-diisothiocyanatostilbene-2,2-disulphonic acid inhibited the net Na⁺ and Cl^- fluxes by approximately 30%. The other half of the coupled NaCl transport, which follows their electrochemical gradients, still remains to be explained.Abbreviations DIDS 4,4-diisothiocyanatostilbene-2,2-disulphonic acid - NMDG N-methyl-d-glucosamine - P _Cl Cl^- permeability coefficient - PD transepithelial potential difference - P _Na Na⁺ permeability coefficient - P _osm osinotic permeability coefficient - TALH thick ascending limb of Henle's loop     

Interactions of glycolate-, HCO 3 - -, Cl--, and H+-balance of Scenedesmus obliquus

Excretion and absorption of glycolate by young cells of Scenedesmus obliquus (Turp.) Krüger strain D₃ grown synchronously with 2% CO₂ was compared after no pretreatment with air (CO₂-adapted) or after a 2 h adaptation to normal air (0.03% CO₂) (air-adapted). At 21% O₂, excretion occurred only from CO₂-adapted cells at high pH (pH 8.0). Under conditions where no excretion occurred, external glycolate (0.2 mM) was taken up by both air-and CO₂-adapted cells at a much faster rate at pH 5 than at pH 8. The uptake was accompanied by an apparent stoichiometric uptake of H⁺. CO₂-adapted algae exhibited high uptake rates that were even higher in the dark than in the light. Air-adapted algae showed high uptake rates in the light but only minimal uptake in the dark. The uptake rate was decreased to about 1/3 with 5% CO₂, except with CO₂-adapted cells in the light, in which a slight stimulation occurred. Cl^- ions inhibited glycolate uptake by air-adapted cells in the light; conversely, light-stimulated Cl^- uptake of these cells was inhibited by glycolate. A hypothesis is discussed according to which the internal pH regulates the uptake and release of Cl^-, HCO ₃ ^- , and glycolate.Abbreviations DCMU 3-(3,4 dichlorophenyl)-1, 1-dimethyl urea - FCCP carbonyl cyanide p-trifluoro-methoxyphenylhydrazone - HEPES 2-(4-(2-hydroxyethyl)-piperazinyl) ethanesulfonic acid - HPMS -hydroxypyridinemethanesulfonate - MES 2-morpholinoethanesulfonic acid - PCV packed cell volume     

Inhibition of nitrate reduction by light and oxygen in Rhodobacter sphaeroides forma sp. denitrificans

Light inhibited each step of the denitrification process in whole cells of Rhodobacter sphaeroides forma sp. denitrificans. This inhibition by light was prevented in the presence of exogenous electron donors like N,N,N,N-tetramethyl-p-phenylenediamine (TMPD) plus ascorbate or in the presence of an uncoupler (carbonyl cyanide m-chlorophenylhydrazone). Addition of myxothiazol restored the inhibition by light in uncoupled cells. Measurements of light-induced absorbance changes under these conditions showed that this inhibition is due, for the steps of reduction of nitrite to dinitrogen, to the photooxidation of cytochromes c ₁ plus c ₂ and not due to the photoinduced membrane potential. Moreover, the presence of oxygen inhibited almost all of the reduction of nitrate and nitrous oxide but only 70% of the reduction of nitrite to nitrous oxide. These inhibitions were overcome in the presence of TMPD plus ascorbate. This implies that the inhibition in presence of oxygen was due to a diversion of the reducing power from the denitrifying chain to the respiratory chain. It was concluded from this series of experiments that the reduction of nitrate to nitrite is inhibited when the ubiquinone pool is partly oxidized and that nitrite and nitrous oxide reductions are inhibited when cytochromes c ₁ plus c ₂ are oxidized by photosynthesis or respiration.Abbreviations R Rhodobacter - TMPD N,N,N,N-tetramethyl-p-phenylenediamine - HOQNO 2-n-heptyl-4-hydroxyquinoline N-oxide - CCCP carbonyl cyanide m-chlorophenylhydrazone - cytochrome c ₁ cytochrome c ₂ plus cytochrome c ₁     

Germinal Excision and Reinsertion Frequencies of the Mobile Element Ds Transposed from Two Unlinked T-DNA Loci in Tomato

Acceptor sites of unlinked transposed Ds element from two T-DNA loci in tomato were mapped. Experimental data obtained from TC₁ progeny testing were employed for estimation of germinal excision frequency (GEF) of Ds element and frequency of its reinsertion (FR). The donor T-DNAs 1481J and 1601D, containing a 35S:NPT transformation marker, a 35S:BAR or nos:BAR excision marker conferring phosphinothricine resistance and a Ds element in the 5 untranslated leader of the nos (or 35S): BAR gene, were located on chromosome 7 and 8, respectively. Ds transposition was induced by 105121 T-DNA carrying stabilized Ac (sAc) which provides a source of transposase and 2:GUS marker conferring -glucuronidase activity. Tomato plants harbouring the Ds in 1481J or 1601D locus and sAc were crossed and F₁D, were crossed individually as seed parents to wild-type plants to generate TC₁ progenies. TC₁ seed was germinated on phosphinothricine (Basta)-containing medium, and individual seedlings carrying a transposed Ds and lacking sAc were identified by PCR (to detect the Ds) on phosphinothricine resistant individuals that lacked -glucuronidase activity. From segregation ratio in TC₁ the germinal excision and reinsertion frequencies of the Ds element were estimated for individual F₁ plants. A total of 14560 TC₁ seedlings of 1481J and 16195 TC₁ seedlings of 1601D was analyzed. We observed high variation between individual plants as regards both GEF and FR despite of donor locus (1481J or 1601D), however, the average germinal excision frequencies as well as average frequencies of reinsertion were very similar for both donor loci: GEF_1481J = 24 %, GEF_1501D = 25 %, FR_1481J = 42 %, FR_1601D = 46 %.     

Properties of the inner membrane anion channel in intact mitochondria

The mitochondrial inner membrane possesses an anion channel (IMAC) which mediates the electrophoretic transport of a wide variety of anions and is believed to be an important component of the volume homeostatic mechanism. IMAC is regulated by matrix Mg²⁺ (IC₅₀=38 µM at pH 7.4) and by matrix H⁺ (pIC₅₀=7.7). Moreover, inhibition by Mg²⁺ is pH-dependent. IMAC is also reversibly inhibited by many cationic amphiphilic drugs, including propranolol, and irreversibly inhibited byN,N-dicyclohexylcarbodiimide. Mercurials have two effects on its activity: (1) they increase the IC₅₀ values for Mg²⁺, H⁺, and propranolol, and (2) they inhibit transport. The most potent inhibitor of IMAC is tributyltin, which blocks anion uniport in liver mitochondria at about 1 nmol/mg. The inhibitory dose is increased by mercurials; however, this effect appears to be unrelated to the other mercurial effects. IMAC also appears to be present in plant mitochondria; however, it is insensitive to inhibition by Mg²⁺, mercurials, andN,N-dicyclohexylcarbodiimide. Some inhibitors of the adenine nucleotide translocase also inhibit IMAC, including Cibacron Blue, agaric acid, and palmitoyl CoA; however, atractyloside has no effect.     

Na/H exchange in cultured epithelial cells from fish gills

Using primary cultures of gill pavement cells from freshwater rainbow trout, a method is described for achieving confluent monolayers of the cells on glass coverslips. A continuous record of intracellular pH was obtained by loading the cells with the pH-sensitive flourescent dye 2,7-bis(2-carboxyethyl)-5(6)-carboxyfluorescein and mounting the coverslips in the flowthrough cuvette of a spectrofluorimeter. Experiments were performed in HEPES-buffered media nominally free of HCO₃. Resting intracellular pH (7.43 at extracellular pH=7.70) was insensitive to the removal of Cl^– or the application of 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (0.1 mmol·l^–1), but fell by about 0.3 units when Na⁺ was removed or in the presence of amiloride (0.2 mmol·l^–1). Exposure to elevated ammonia (ammonia prepulse; 30 mmol·l^–1 as NH₄Cl for 6–9 min) produced an increase in intracellular pH (to about 8.1) followed by a slow decay, and washout of the pulse caused intracellular pH to fall to about 6.5. Intracellular non-HCO ₃ ^– buffer capacity was about 13.4 slykes. Rapid recovery of intracellular pH from intracellular acidosis induced by ammonia prepulse was inhibited more than 80% in Na⁺-free conditions or in the presence of amiloride (0.2 mmol·l^–1). Neither bafilomycin A₁ (3 mol·l^–1) nor Cl removal altered the intracellular pH recovery rate. The K _m for Na⁺ of the intracellular pH recovery mechanism was 8.3 mmol·l^–1, and the rate constant at V _max was 0.008·s^–1 (equivalent to 5.60 mmol H⁺·l^–1 cell water·min^–1), which was achieved at external Na⁺ levels from 25 to 140 mmol·l^–1. We conclude that intracellular pH in cultured gill pavement cells in HEPES-buffered, HCO ₃ ^– -free media, both at rest and during acidosis, is regulated by a Na⁺/H⁺ antiport and not by anion-dependent mechanisms or a vacuolar H⁺-ATPase.Abbreviations BCECF 2,7-bis(2-carboxyethyl)-5(6)-carboxy-fluorescein - BCECF/AM 2,7-bis(2-carboxyethyl)-5(6)-carboxy-fluorescein, acetoxymethylester - Cholin-Cl choline chloride - DMSO dimethyl sulfoxide - EDTA ethylene diamine tetra-acetic acid - FBS foetal bovine serum - H ⁺ -ATPase Proton-dependent adenosine triphosphatase - HEPES N-[2-hydroxyethyl]piperazine-N[2-ethanesulfonic acid] - pH _i intracellular pH - pH _e extracellular pH - PBS phosphate-buffered saline - SITS 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid     

Electron transport-linked proton translocation at nitrite reduction inCampylobacter sputorum subspeciesbubulus

Campylobacter sputorum subspeciesbubulus contains a membrane-bound nitrite reductase which catalyses the six-electron reduction of nitrite to ammonia. Formate andL-lactate are used as hydrogen donors. Cells ofC. sputorum grown with nitrate or nitrite contain cytochromes of theb-andc-type and a carbon monoxide-binding cytochromec. In addition, a special membrane-bound carbon monoxide-binding pigment is found. Nitrite reduction with formate orL-lactate as a hydrogen donor is strongly inhibited by 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO). Nitrite reduction by bacterial suspensions with lactate as a hydrogen donor is strongly inhibited by carbonylcyanide-m-chlorophenyl-hydrazone (CCCP) whereas nitrite reduction with formate as a hydrogen donor is not inhibited at all. H⁺/O values and H⁺/NO ₂ ^- values were measured with ascorbate + N,N,N,N-tetramethyl-p-phenylenediamine (TMPD), formate (in the absence and presence of carbonic anhydrase) andL-lactate as a hydrogen donor. The results are summarized in a scheme for electron transport from formate or lactate to oxygen or nitrite which shows a periplasmic orientation of formate dehydrogenase and nitrite reductase and a cytoplasmic orientation of lactate dehydrogenase and oxygen reduction, and which shows proton translocation with a H⁺/2e value of 2.0. The H⁺/O and H⁺/NO ₂ ^- values predicted by this scheme are in good agreement with the experimental values.Abbreviations CCCP carbonylcyanide-m-chlorophenylhydrazone - HQNO 2-n-heptyl-4-hydroxyquinoline-N-oxide - MTPP⁺ methyltriphenylphosphonium cation - TMPD N,N,N,N-tetramethyl-p-phenylenediamine; H⁺/O (H⁺/NO ₂ ^- ), number of protons liberated in the outer bulk phase at the reduction of one atom O (one ion NO ₂ ^- ); H⁺/2e (q⁺/2e), number of protons (charges) translocated across the cytoplasmic membrane during flow of two electrons to an acceptor     

Some characteristics of anion transport at the tonoplast of oat roots,determined from the effects of anions on pyrophosphatedependent proton transport

The effects of anions on inorganicpyrophosphate-dependent H⁺-transport in isolated tonoplast vesicles from oat (Avena sativa L.) roots were determined. Both fluorescent and radioactive probes were used to measure formation of pH gradients and membrane potential in the vesicles. Pyrophosphate hydrolysis by the H⁺-translocating pyrophosphatase was unaffected by anions. Nonetheless, some anions (Cl^-, Br^- and NO₃-) stimulated H⁺-transport while others (malate, and iminodiacetate) did not. These differential effects were abolished when the membrane potential was clamped at zero mV using potassium and valinomycin. Stimulation of H⁺-transport by Cl^- showed saturation kinetics whereas that by NO₃- consisted of both a saturable component and a linear phase. For Cl^- and NO₃-, the saturable phase had a K _m of about 2 mol·m^-3. The anions that stimulated H⁺-transport also dissipated the membrane potential (.) generated by the pyrophosphatase. It is suggested that the stimulatory anions cross the tonoplast in response to the positive generated by the pyrophosphatase, causing dissipation of and stimulation of pH, as expected by the chemiosmotic hypothesis. The work is discussed in relation to recent studies of the effects of anions on ATP-dependent H⁺-transport at the tonoplast, and its relevance to anion accumulation in the vacuole in vivo is considered.Abbreviations and symools BTP 1,3-bis[tris(hydroxymethyl)-methylamino]-propane - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - Hepes 4-(2-hydroxyethyl)-1-piperazine ethanesulphonic acid - IDA iminodiacetate - membrane potential - pH pH gradient - PPase inorganic pyrophosphatase - PPi morganic pyrophosphate     

In addition to the ARS core, the ARS box is necessary for autonomously replicating sequences

Summary Autonomously replicating sequences (ARSs) were cloned from nuclear and mitochondrial DNA of D. melanogaster using YIp5, which is composed of pBR322 and the yeast ura3 gene, as the cloning vector and YNN27, a Ura^- yeast strain as the recipient. The nucleotide sequences of six ARSs, two from nuclear bulk, two from the nuclear 1.688 satellite, and two from mitochondorial DNA, were determined. The relationship between the transformation frequency and the inclusion of the ARS core, 5 _T ^A TT-TAT _A ^G TTT _T ^A 3, of these fragments was analysed. All the ARSs contained an ARS core or a single base change of it. However, not all the fragments that contained a single base change of the ARS core were able to transform the recipient cells, suggesting that certain bases in the ARS core were not exchangeable. It is suggested by transformation experiments with subfragments that in addition to an ARS core, an ARS box which is located within 25 bp upstream of the ARS core and whose sequence is composed of 5TNT _G ^A AA 3, is necessary for autonomous replication.