The use of Light Green and Orange II as quantitative protein stains, and their combination with the Feulgen method for the simultaneous determination of protein and DNA

The protein dyes Light Green and Orange II were studied separately and in combination with the Feulgen-Pararosanilin(SO2) and -Thionin(SO2) method for the simultaneous determination of DNA and protein. - With polyacrylamide modelfilms the pH dependency, specificity and stoichiometry of Light Green and Orange II have been investigated. The results of both staining methods with different biological objects have been compared. - In addition, the Feulgen-Thionin(SO2) method was studied with model films with respect to its specificity and stoichiometry. In biological objects it has been compared with the Feulgen-Pararosanilin(SO2) method. - When combining the Light Green staining with the Feulgen-Pararosanilin(SO2) procedure and the Orange II staining with Feulgen-Thionin(SO2), both Feulgen-DNA stainings, which were first applied, proved to be unaffected by the following protein staining procedure. When the Feulgen procedure was carried out without the dye, followed by Light Green staining, the latter became reduced when a sulfite water rinse was included but was unaffected when a running tap water rinse was used. In the case of the Orange II staining a serious reduction in dye binding capacity was found in both situations. - When the Feulgen-Pararosanilin(SO2) Light Green procedure was carried out on isolated nuclei with all dyes present, a decrease of protein dye binding was observed, similar to that found with the well-known Feulgen-Pararosanilin(SO2) Naphthol Yellow S combination. It is concluded that in spite of this reduction the latter two combinations can be used for the cytophotometric analysis of DNA and protein in the same object.     

Purification and identification of the violaxanthin deepoxidase as a 43 kDa protein

Violaxanthin deepoxidase (VDE) has been purified from spinach (Spinacia oleracea) leaves. The purification included differential sonication of thylakoid membranes, differential (NH₄)₂SO₄ fractionation, gel filtration chromatography and finally either hydrophobic interaction chromatography or anion exchange chromatography. A total purification of more than 5000-fold compared to the original thylakoids enabled the identification of a 43 kDa protein as the VDE, in contrast to earlier reported molecular weight of 54–60 kDa. A detailed comparison was made for the VDE activity and polypeptide pattern for the different fractions throughout the purification and the best correlation was always found for the 43 kDa protein. The highest specific activity obtained was 256 mol g^–1 s^–1 protein, which is at least 10-fold higher than reported earlier. We estimate that there is 1 VDE molecule per 20–100 electron transport chains. The 43 kDa protein was N-terminally sequenced, after protection of cysteine residues with -mercaptoethanol and iodoacetamid, and a unique sequence of 20 amino acids was obtained. The amino acid composition of the protein revealed a high abundance of charged and polar amino acids and remarkably, 11 cysteine residues. Two other proteins (39.5 kDa and 40 kDa) copurifying with VDE were also N-terminally sequenced. The N-terminal part of the 39.5 kDa protein showed complete sequence identity both with the N-terminal part of cyt b ₆ and an internal sequence of polyphenol oxidase.Abbreviations DMSO dimethylsulfoxid - HIC hydrophobic interaction chromatography - MGDG monogalactosyl diacylglycerol - VDE violaxanthin deepoxidase A preliminary report of these results was presented at the Xth Int. Congress on Photosynthesis, Montpellier, France, 1995.     

Junctional uvomorulin/E-cadherin and phosphotyrosine-modified protein content are correlated with paracellular permeability in Madin-Darby canine kidney (MDCK) epithelia

Strains I and II of Madin-Darby canine kidney (MDCK) cells, which differ markedly in transepithelial resistance (R _t ) and paracellular permeability, have been used to investigate whether differences in the cellular content of uvomorulin/E-cadherin and phosphotyrosine may be correlated with junctional properties. Using immunocytochemistry, the strain I tight epithelia showed significantly stronger uvomorulin staining at regions of cell-cell contact compared with strain II leaky MDCK epithelia. In contrast, strain I MDCK cells showed a relatively faint phosphotyrosine staining, distributed evenly throughout the cytoplasm, while strain II MDCK cells displayed intense staining for phosphotyrosine residues in the junctional region and the lateral cell membrane with additional labelling of the cytoplasm. Exposure to vanadate in conjunction with H₂O₂ (which are potent inhibitors of protein tyrosine phosphatases) resulted in a dramatic increase in phosphotyrosine staining at the intercellular area and, concomitantly, induced changes in cell morphology, a significant decrease in R _t , increase in paracellular inulin permeability, and time-dependent disappearance of uvomorulin from the cell-cell contact sites. Moreover, the effects of vanadate/H₂O₂ treatment were more dramatic in strain II compared with strain I cells, consistent with greater generation of tyrosine-modified protein in strain II cells. An inverse relationship was demonstrated between membrane-associated uvomorulin/E-cadherin and cellular phosphotyrosine content, which varied between the two strains of MDCK cells and when phosphotyrosine was directly manipulated. These data support the hypothesis that regulation of paracellular permeability may result from specific tyrosine phosphorylation of protein components of the junctional complex.     

The effect of bisulfite modification on the template activity of DNA for DNA polymerase I

Cytosine residues of poly(C) and heat-denatured calf thymus DNA were transformed into 5,6-dihydrouracil-6-sulfonate (U(SO⁻₃)) residues by treatment with bisulfite. The poly(U(SO⁻₃)₂, C₃) and poly(U(SO⁻₃)₉, C₁) prepared did not form inter-base binding with either poly(A) or poly(I) as judged by the absence of hypochromicity in ultraviolet absorbance. U(SO⁻₃) residues in the DNA inactivated it to serve as template for E.coli DNA polymerase I, while the template activity was restored by conversion of the U(SO⁻₃) residues into U.     

Ultrastructural localization of the maltose-binding protein within the cell envelope of Escherichia coli

Logarithmically growing cells of Escherichia coli were fixed with glutaraldehyde and incubated with antimaltose-binding protein F_ab coupled to horseradish peroxidase (molecular weight of the complex 80,000). The position of this complex within the cell envelope was determined by reacting with diaminobenzidine-H₂O₂, staining with osmium tetroxide and processing for thin section electron microscopy. The following observations were made: (i) induction of the maltose-binding protein resulted in swelling and staining of the outer membrane; (ii) the swelling and staining was more prominent in short cells, less prominent or absent in long cells; (iii) rare examples exhibited granular staining in the space between the plasma membrane and the peptidoglycan layer. These stainings were observable mainly in pole caps; (iv) a mutant lacking the receptor for phage showed altered staining pattern. Treatment of glutaraldehyde-fixed cells with EDTA-lysozyme prevented the specific labelling of the maltose-binding protein.Lists of Non Common Abbreviations MBP maltose-binding protein - MBP-F_ab)-HRPO F_ab fragments against maltose-binding, protein coupled to horseradish peroxidase - IgG immunoglobulin - PBS pnosphate buffered saline     

Extended map for the phaseolin linkage group ofPhaseolus vulgaris L

Summary The linkage relationship of 11 bean (Phaseolus vulgaris) seed proteins (including phaseolin), 9 enzyme loci, and theP locus were analyzed in backcross and F₂ progenies by use of the software package Mapmaker. The progenies were obtained by crossing the breeding line XR-235-1 and the cultivar Calima. Allelic differences for seed protein loci were detected with SDS-PAGE and those for enzyme loci with starch gel electrophoresis and activity stains. The seed coat color of Calima is a red/beige mottled pattern and that of XR-235-1 is white. Segregation at theP locus was followed by recording the phenotype of the BC₁S₁ and F₃ seed. A linkage group comprising ca. 90 cM was detected with the following gene order:Est-2 — 11 —Pha — 8 — (Spe/Spg) — 24 — P — 9 — (Spa/Spv) — 16 —Spba — 22 —Mdh-1. In addition, another linkage group was detected: (Spd/Spf/Sph) — 5 -Spca. Therefore, the seed proteins appear to be organized in clusters in the bean genome.Florida Agricultural Experiment Station, Journal Series No. R-01131     

Comparison of two bacterial azoreductases acquired during adaptation to growth on azo dyes

Selection for utilization of carboxy-Orange I [1-(4-carboxyphenylazo)-4-naphthol] in the chemostat yielded Pseudomonas strain K24 which was unable to grow on carboxy-Orange II [1-(4-carboxyphenylazo)-2naphthol] while selection for growth on carboxy-Orange II had previously led to strain KF 46 which did not utilize carboxy-Orange I. Orange I azoreductase of strain K24, the key enzyme of dye degradation, was purified 80-fold with 17% yield to electrophoretic homogeneity and compared to the previously purified Orange II azoreductase of strain KF46. Common properties of the two enzymes were their monomeric structure, their specificity for NADPH and NADH as cosubstrates, the range of their K _m values for substrates and cosubstrates as well as their reactivity towards a series of substrate analogs. They differed from each other with respect to molecular weight (21,000 and 30,000) and in the absolute requirement of Orange I azoreductase for a hydroxy group in the 4 position of the naphthol ring of the substrate molecule as compared to the requirement for substrates with a 2-naphthol moiety by Orange II azoreductase. The pure enzymes did not exhibit immunological cross-reaction with each other. Crude extracts of strains K24 and KF46 and of azoreductase-negative strains isolated at different stages of the adaptation experiments, however, contained material which cross-reacted (CRM) with both anti Orange I azoreductase serum and anti Orange II azoreductase serum. The CRM may represent a common precursor protein of the azoreductases in strains K24 and KF46.Abbreviations Orange I 1-(4-sulfophenylazo)-4-naphthol - carboxy-Orange I 1-(4-carboxy phenylazo)-4-naphthol - Orange II 1-(4-sulfophenylazo)-2-naphthol - carboxy-Orange II 1-(4-carboxyphenylazo)-2-naphthol - SDS sodium dodecyl sulfate - DCAB 4,4-dicarboxyazobenzene - CRM cross reacting material - anti OrIar serum antiserum against Orange I azoreductase - anti OrIIar serum antiserum against Orange II azoreductase Enzymes Orange I azoreductase or NAD(P)H 1-(4-sulfophenylazo)-4-naphthol oxidoreductase (EC 1.6.6-) - Orange II azoreductase or NAD(P)H 1-(4-sulfophenylazo)-2-naphthol oxidoreductase (EC 1.6.6-)     

Spectroscopic studies of the multiple binding modes of a trimethine-bridged cyanine dye with DNA

The interaction between DNA and a benzothiazole-quinoline cyanine dye with a trimethine bridge (TO-PRO-3) results in the formation of three noncovalent complexes. Unbound TO-PRO-3 has an absorption maximum (λ_max) of 632 nm, while the bound dyes (with calf thymus DNA) have electronic transitions with λ_max = 514nm (complex I), 584nm (complex II) and 642 nm (complex III). The blue shifts in the electronic transitions and the bisignate shape of the circular dichroism bands indicate that TO-PRO-3 aggregates with DNA. Complex I has a high dye:base pair stoichiometry, which does not depend on base sequence or base modifications. The bound dyes exhibit strong interdye coupling, based on studies with a short oligonucleotide and on enhanced resonance scattering. From thermal dissociation studies, the complex is weakly associated with DNA. Studies with poly(dGdC)₂ and poly(dIdC)₂ and competitive binding with distamycin demonstrate that complex II is bound in the minor groove. This complex stabilizes the helix against dissociation. For complex III, the slightly red-shifted electronic transition and the stoichiometry are most consistent with intercalation. Using poly(dAdT)₂, the complexes have the following dye mole fractions (X_dye): X_dye = 0.65 (complex I), 0.425 (complex II) and 0.34 (complex III).     

Oxidation and removal of industrial textile dyes by a novel peroxidase extracted from post-harvest lentil (Lens culinaris L.) stubble

The degradation and removal of a series of dyes used in the textile industry for polyester/wool (PES/WO) blends and present in effluents, such as Green, Ash-Grey, Black, Navy Blue, Red and Yellow Domalan, and Orange and Red Bemacid, by catalytic action, in the presence of H₂O₂, of extracts of a novel peroxidase from postharvest lentil stubble was investigated. The extracts of this peroxidase (LSP) were effective in degrading these lastgeneration textile dyes, especially Green Domalan, Orange Bemacid, Grey and Black Domalan. A sensitivity study was carried out for Green Domalan biodegradation to determine the effects of process parameters such as pH, H₂O₂, enzyme and dye concentrations, contact and centrifugation times, and temperature. Standard ecotoxicity studies performed with Vibrio fischeri revealed that the dye solutions treated with peroxidase and H₂O₂ were less ecotoxic than the untreated ones.     

Phosphorylation of smooth muscle myosin by type II Ca2+/calmodulin-dependent protein kinase

Brain type II Ca²⁺/calmodulin-dependent protein kinase was found to phoshorylate smooth muscle myosin, incorporating maximally 2 mol of phosphoryl per mol of myosin, exclusively on the 20,000 dalton light chain subunit. After maximal phosphorylation of myosin or the isolated 20,000 dalton light chain subunit by myosin light chain kinase, the addition of type II Ca²⁺/calmodulin-dependent protein kinase led to no further incorporation indicating the two kinases phosphorylated a common site. This conclusion was supported by two dimensional mapping of tryptic digests of myosin phosphorylated by the two kinases. By phosphoamino acid analysis the phosphorylated residue was identified as a serine. The phosphorylation by type II Ca ²⁺/calmodulin-dependent protein kinase of myosin resulted in enhancement of its actin-activated Mg²⁺-ATPase activity. Taken together, these data strongly support the conclusion that type II Ca²⁺/calmodulin-dependent protein kinase phosphorylates the same amino acid residue on the 20,000 dalton light chain subunit of smooth muscle myosin as is phosphorylated by myosin light chain kinase and suggest an alternative mechanism for the regulation of actin-myosin interaction.Abbreviations SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis - EGTA Ethylene Glycol Bis (-amino-ethyl ether)-N,N,N,N-Tetraacetic Acid - DTT Dithiothreitol - LC₂₀ Gizzard Smooth Muscle Phosphorylatable 20 kDa Myosin Light Chain - LC₁₇ Gizzard Smooth Muscle, 17 kDa Myosin Light Chain - H Chain Gizzard Smooth Muscle 200 kDa Myosin Heavy Chain - TPCK L-1-Tosylamido-2-Phenylethyl Chloromethyl Ketone - MOPS 3-(N-morpholino) Propanesulfonic Acid     

Evaluation of monitoring approaches and effects of culture conditions on recombinant protein production in baculovirus-infected insect cells

The baculovirus infection process ofSpodoptera frugiperda (Sf9) insect cells in oxygen-controlled bioreactors in serum-free medium was investigated using a recombinantAutographa californica (AcNPV) virus expressing -galactosidase enzyme as a model system. A variety of monitoring techniques including trypan blue exclusion, fluorescent dye staining, oxygen uptake rate (OUR) measurements, and glucose consumption were applied to infected cells to determine the best way of evaluating cell integrity and assessing the course of baculovirus infection. The metabolism of newly-infected cells increased 90% during the first 24 hours, but as infection proceeded, and cells gradually succumbed to the baculovirus infection, the cytopathic effect of the baculovirus on the cells became evident. Oxygen and glucose uptake rate measurements appeared to more accurately assess the condition of infected cells than conventional trypan blue staining, which tended to overestimate cell viability in the mid stages of infection. The optimal harvest time varied, depending on which technique — SDS-PAGE, chromogenic (ONPG) or fluorometric (C₁₂FDG) — was used to monitor -galactosidase production. Specific -galactosidase production was found to be insensitive to a wide range of culture dissolved oxygen tensions, whereas resuspending cells in fresh medium prior to infection increased volumetric productivity approximately two-fold (800,000 units -galactosidase/ml) compared to cultures infected in batch mode and allowed successful infections to occur at higher cell densities.Abbreviations ONPG ortho-phenyl 2--D-galactopyranoside - OUR oxygen uptake rate (-mol O₂/liter/hour) - q_glucose specific glucose uptake rate (mg glucose/10⁶cell/hour) - q_glutamine specific glutamine uptake rate (mg glutamine/10⁶cell/hour) - qO₂ specific oxygen uptake rate (-mol O₂/10⁶cell/hour) - MOI virus multiplicity of infection (viral plaque forming units/cell)     

Half-sandwich Ru[9]aneS3 complexes structurally similar to antitumor-active organometallic piano-stool compounds: Preparation, structural characterization and in vitro cytotoxic activity

The preparation, structural characterization, and chemical behavior in aqueous solution of a series of new Ru[9]aneS₃ half-sandwich complexes of the type [Ru([9]aneS₃)Cl(NN)][CF₃SO₃] and [Ru([9]aneS₃)(dmso-S)(NN)][CF₃SO₃]₂ (5–15, NN = substituted bpy or 2 × 1-methylimidazole) are described. The X-ray structures of [Ru([9]aneS₃)Cl(3,3′-H₂dcbpy)][CF₃SO₃] (9) (3,3′-H₂dcbpy = 3,3′-dicarboxy-2,2′-bipyridine), [Ru([9]aneS₃)Cl(4,4′-dmobpy)][CF₃SO₃] (13) (4,4′-dmobpy = 4,4′-dimethoxy-2,2′-bipyridine), and [Ru([9]aneS₃)Cl(1-MeIm)₂][CF₃SO₃] (15) (1-MeIm = 1-methylimidazole) were also determined. The new compounds are structurally similar to anticancer-active organometallic half-sandwich complexes of formula [Ru(η⁶-arene)Cl(NN)][PF₆]. Three chloro compounds (5, 9, 15) were tested in vitro for cytotoxic activity against two human cancer cell lines in comparison with the previously described [Ru([9]aneS₃)Cl(en)][CF₃SO₃] (1, en = ethylenediamine), [Ru([9]aneS₃)Cl(bpy)][CF₃SO₃] (2), and with their common dmso precursor [Ru([9]aneS₃)Cl(dmso-S)₂][CF₃SO₃] (3). Only the ethylenediamine complex 1 showed some antiproliferative activity, ca. one order of magnitude lower than the reference organometallic half-sandwich compound RM175 that contains biphenyl instead of [9]aneS₃. This compound was further tested against a panel of human cancer cell lines (including one resistant to cisplatin).     

Protoplasmaresistenz von Meeresalgen und Meeresanthophyten gegen Schwermetallsalze

Zusammenfassung Es wurde die Resistenz des Plasmas von 14 Phaeophyceen, 32 Rhodophyceen, 17 Chlorophyceen und 3 Meeres-Phanerogamen durch Einlegen der Thalli 48 Stunden lang in konzentrationsabgestuften Lösungsreihen von MnSO₄, ZnSO₄, Cr₂(SO₄)₃, VOSO₄ und CuSO₄ (10^–6–1 Mol/L) ermittelt.Diese Resistenzgrenzen in den verschiedenen Lösungen von Mangan-, Zink-, Chrom-, Vanadyl- und Kupfersulfat ergeben für die einzelnen Meeresalgen charakteristische Resistenzkombinationen, die der Ausdruck konstitutioneller Unterschiede des Plasmas der einzelnen Algen sind. Die Resistenzkombinationen der untersuchten Meeresalgen decken sich im allgemeinen mit denen von Anthophyten.Alle von uns untersuchten Algen und Meeres-Phanerogamen sind durch eine hohe Resistenz gegen Mangansulfat, die Meeres-Phanerogamen auch gegen Zinksulfat ausgezeichnet.Das Protoplasma der meisten untersuchten Phaeophyceen und der drei Meeres-Phanerogamen weist gegen Mangansulfat die höchste Resistenz auf (1 Mol/L), während das Protoplasma der Rhodophyceen die geringste Resistenz besitzt (0,1–10^–2 Mol/L). Das Protoplasma der Chlorophyceen nimmt hinsichtlich der Resistenzgrenzen gegenüber Mangansulfat eine Stellung zwischen Phaeophyceen und Rhodophyceen ein.Die Resistenzgrenzen des Plasmas der untersuchten Meeresalgen gegen Zink-, Chrom-, Vanadyl- und Kupfersulfat liegen in niederen, hypotonischen Konzentrationen.Es ist zu vermuten, daß die Widerstandsfähigkeit gegen hypertonische Konzentrationen —wie bei uns in MnSO₄ — darauf beruht, daß auf dem Plasma eine irreversibel koagulierte Oberflächenschicht erzeugt wird, welche den Salzen ein weiteres Eindringen ins Binnenplasma verwehrt. Da bei niedrigeren, hypotonischen Konzentrationen — wie bei uns in ZnSO₄, Cr₂(SO₄)₃, VOSO₄ und CuSO₄ — diese Möglichkeit nicht gegeben ist, stirbt das Plasma ab, falls es nicht resistent ist.

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Protoplasmic resistances of marine algae and marine anthophytes to heavy metal salts

Summary Plasmatic resistances of 14Phaeophyceae, 32Rhodophyceae, 17Chlorophyceae, and 3 marine phanerogams were determined by immersion of thalli for 48 hours into concentration-graded solution series of MnSO₄, ZnSO₄, Cr₂(SO₄)₃, VOSO₄, and CuSO₄ (10^–6–1 mol/l).These limits of resistance in various solutions of manganese, zinc, chromium, vanadyl, and copper sulfates yield resistance combinations characteristic for the algal species, which are an expression of constitutional differences in the plasma of the algae tested. Resistance combinations of the marine algae investigated generally coincide with those of anthophytes. All the algae and marine phanerogams investigated by us are characterized by high resistance to manganese sulfate, the marine phanerogams also to zinc sulfate.The protoplasm of most of thePhaeophyceae investigated and of the three marine phanerogams shows the highest resistance to manganese sulfate (1 mol/l), whereas the protoplasm of theRhodophyceae is least resistant (10^–1–10^–2 mol/l). The protoplasm ofChlorophyceae occupies an intermediate position betweenPhaeophyceae andRhodophyceae in respect to the resistance limits.Plasmatic resistance limits to zinc, chromium, vanadyl, and copper sulfates of the marine algae investigated correspond to low, hypotonic concentrations.It may be guessed that the resistance to hypertonic concentrations—as, in our case, of MnSO₄—is due to the formation of an irreversibly coagulated surface layer, which prohibits the salts from entering the ground plasm. Since this possibility is absent in lower, hypotonic concentrations (as, in our experiments, in ZnSO₄, Cr₂(SO₄)₃, VOSO₄, and CuSO₄) the protoplasm is killed, provided it is not hardy.

     

Fetuin: an acute phase protein in cattle

Summary Fetuin is a plasma protein present in high concentrations during fetal development in animals of the order Artiodactyla. Its role is not known. The human homologue of fetuin — ₂HS glycoprotein — has been shown to be a negative acute phase protein in adult plasma. In the present study, the concentration of fetuin was measured in the serum of healthy cattle (Bovis bovis) and in animals with various injuries and inflammatory disorders. The levels were decreased by 30% in pregnancy but increased up to 10-fold in some trauma cases. A significant negative correlation between the concentrations of fetuin and albumin has also been found. Thus, fetuin appears to be a positive acute phase protein in cattle.Abbreviations ₂HS ₂HS glycoprotein - AP acute phase     

Separation and partial characterization of two ribonucleic Acid polymerases from pea seedlings

Two DNA-dependent RNA polymerases (ribonucleoside triphosphate:RNA nucleotidyl transferase, EC 2.7.7.6) have been isolated from pea (Pisum sativum) seedlings. The enzymes were solubilized by sonication in high salt buffer and were separated by chromatography on diethylaminoethyl cellulose using a linear salt gradient. Polymerase I eluted at 0.10 m (NH₄)₂SO₄, accounted for about 10% of the recovered activity and was completely insensitive to α-amanitin. Polymerase II eluted at 0.14 m (NH₄)₂SO₄, accounted for the remaining 90% of recovered activity and was strongly inhibited by α-amanitin. Both enzymes preferred denatured to native DNA as template, both showed an absolute requirement of divalent cation, and both were sensitive to the ionic strength of the assay medium. The developing pea seedling seems a promising system for studies of possible changes in relative activities and roles of multiple RNA polymerases during eukaryotic development.     

Light and the maintenance of photosynthetic competence in leaves of Populus balsamifera L. during short-term exposures to high concentrations of sulfur dioxide

Leaves of Populus balsamifera grown under full natural sunlight were treated with 0, 1, or 2 l SO₂·1^-1 air under one of four different photon flux densities (PFD). When the SO₂ exposures took place in darkness or at 300 mol photons·m^-2·s^-1, sulfate accumulated to the levels predicted by measurements of stomatal conductance during SO₂ exposure. Under conditions of higher PFD (750 and 1550 mol·m^-2·s^-1), however, the predicted levels of accumulated sulfate were substantially higher than those obtained from anion chromatography of the leaf extracts. Light-and CO₂-saturated capacity as well as the photon yield of photosynthetic O₂ evolution were reduced with increasing concentration of SO₂. At 2 l SO₂·1^-1 air, the greatest reductions in both photosynthetic, capacity and photon yield occurred when the leaves were exposed to SO₂ in the dark, and increasingly smaller reductions in each occurred with increasing PFD during SO₂ exposure. This indicates that the inhibition of photosynthesis resulting from SO₂ exposure was reduced when the exposure occurred under conditions of higher light. The ratio F _v/F _M (variable/maximum fluorescence emission) for photosyntem II (PSII), a measure of the photochemical efficiency of PSII, remained unaffected by exposure of leaves to SO₂ in the dark and exhibited only moderate reductions with increasing PFD during the exposure, indicating that PSII was not a primary site of damage by SO₂. Pretreatment of leaves with SO₂ in the dark, however, increased the susceptibility of PSII to photoinhibition, as such pretreated leaves exhibited much greater reductions inF _V/F _M when transferred to moderate or high light in air than comparable control leaves.Abbreviations and symbols A₁₂₀₀ photosynthetic capacity (CO₂-saturated rate of O₂ evolution at 1200 mol photons·m^-2·s^-1) - F_o instantaneous fluorescence emission - F_M maximum fluorescence emission - F_V variable fluorescence emission - PFD photon flux density (400–700 nm) - PSII photosystem II     

Inhibition of photosynthesis,acidification and stimulation of zeaxanthin formation in leaves by sulfur dioxide and reversal of these effects

Leaves of Pelargonium zonale L. and Spinacia oleracea L. were fumigated with high concentrations of SO₂ for very short periods of time with the aim of first producing acute symptoms of damage and then observing repair. The response of different photosynthetic parameters to SO₂ was monitored during and after fumigation. The following results were obtained: (1) Inhibition of CO₂ assimilation in the light was accompanied by increased reduction of the quinone acceptor, Q_A, of photosystem II and by increased oxidation of the electrondonor pigment P₇₀₀ of photosystem I. Increased control of photosystem II activity in the SO₂-inhibited state was also indicated by increased light scattering and by increased non-photochemical quenching of chlorophyll fluorescence. Both are indicators of chloroplast energization. Apparently, SO₂ did not decrease but rather increased energization of the chloroplast thylakoid system by light. (2) Accumulation of dihydroxyacetone phosphate, fructose-1,6-phosphate and ribulose-1,5-phosphate and a decrease of 3-phosphoglycerate and hexosephosphate indicated that SO₂ inhibited enzymes of the Calvin cycle. (3) Stimulated postillumination CO₂ evolution suggested that when photosynthesis declined respiration increased to provide energy for repair reactions. (4) Increased leaf absorbance at 505 nm indicated increased stimulation of zeaxanthin formation in thylakoid membranes under the influence of SO₂. A similar increase in 505-nm absorbance could be induced by high concentrations of CO₂. In darkened leaves, SO₂ did not produce changes in 505-nm absorbance. (5) While zeaxanthin formation was stimulated, changes in the fluorescence of the pH-indicating dye pyranine, which had been fed to the leaves, indicated acidification of the cytoplasm of leaf cells by SO₂. Maximum acid production by SO₂ required light. In contrast, cytoplasmic acidification of leaf cells by CO₂ was similar in the light and in the dark. (6) Since zeaxanthin formation is known to depend on the acidification of the thylakoid lumen, SO₂-dependent zeaxanthin formation indicated SO₂-dependent acidification of the thylakoid lumen as the indirect result of cytoplasmic acidification by SO₂. (7) Inhibition of photosynthesis and other effects of SO₂ were fully reversible in the light. Detoxification of SO₂ and reactivation of the photosynthetic apparatus were slow or absent in the dark. Light had a dual effect on the action of SO₂. Transiently, it first increased the extent of inhibition of assimilation, but, finally, it reversed inhibition. Sulfur dioxide was inhibitory as a consequence of the chemical reactivity of its hydration products rather than as a result of cellular acidification by the produced acid. The initial acidification was followed by an appreciable alkalisation demonstrating the action of the pH-stat mechanism. (8) The data are discussed in relation to SO₂ toxicity under field conditions when plants are chronically exposed to polluted air.Abbreviations Chl chlorophyll - DHAP dihydroxyacetone phosphate - FBP fructose-1,6-bisphosphate - F6P fructoce-6-phosphate - F, Fm, Fm, Fo, Fo chlorophyll fluorescence levels - PGA 3-phosphoglycerate - P₇₀₀ primary donor of photosystem I - Q_A primary quinone acceptor of photosystem II - q_p photochemical quenching of chlorophyll fluorescence - NPQ non-photochemical quenching of chlorophyll fluorescence - RuBP ribulose-1,5-bisphosphate Dedicated to Professor O.L. Lange on the occasion of his 65th birthdayOn leave from the Centre for Multidisciplinary Sciences, University of Belgrade, YugoslaviaThis work was supported by the Deutsche Forschungsgemeinschaft within the Sonderforschungsbereich 251 of the University of Würzburg. S. V.-J. acknowledges support by the Leibniz program of the Deutsche Forschungsgemeinschaft and by the Fonds for Science of the Republic of Serbia (contract no. 8604). We are grateful to Drs. Z.-H. Yin, U. Takahama and K.-J. Dietz (Julius-von-Sachs-Institut für Biowissenschaften, Universität Würzburg, FRG) for cooperation and helpful discussions.     

Evidence for the participation of disulfide and sulfhydril groups in the specific binding of [3H]prazosin in cerebral cortex

The tritiated ₁ antagonist prazosin [³H]PRZ binds specifically and with high affinity to postsynaptic adrenoceptors in membrane preparations from cerebral cortex. Since adrenoceptors are of protein nature, it was of interest of investigate the possible role of disulfide (—SS—) and sulfhydril (—SH) groups in the binding of [³H]PRZ. Pretreatment of the membranes with the disulfide and sulfhydryl reactivesdl0Dithiothreitol,l-Dithiothreitol, Dithioerythritol or 5,5-Dithiobis-(2-nitrobenzoic acid) (DTNB), alone or in combination with the alkylating agent N-Methylmaleimide (NMM), decreased specific [³HPRZ binding, with minor changes in the non-specific counts. Saturation experiments revealed that all these reagents reduced the affinity of the binding site for [³H]PRZ, as judged by theK _d 25°C, but only the alkylating agent NMM and the oxydizing reagent DTNB produced in addition to the increase inK _d, a decrease of the maximum binding capacity (B _max). The present results provide evidence for a participation of—SS—and/or—SH groups in the recognition site of the ₁-adrenoceptor of cerebral cortex.     

The effect of sulfide on the blue-green algae of hot springs II. Yellowstone National Park

In the Mammoth Springs (Yellowstone National Park) waters with near neutral pH and soluble sulfide (H₂S, HS^–, S^2–) of over 1–2 mg/liter (30–60M) are characterized by substrate covers of phototrophic bacteria (Chloroflexus and aChlorobium-like unicell) above 50C and by a blue-green alga (Spirulina labyrinthiformis) below this temperature.Synechococcus. Mastigocladus, and other blue-green algae typical of most hot springs of western North America are excluded, apparently by sulfide. The sulfide-adaptedSpirulina photosynthesized at maximum rates at 45C and at approximately 300 to 700Ein/m²/sec of visible radiation. Sulfide (0.6–1.2 mM) severely poisoned photosynthesis of nonadapted populations, but those continuously exposed to over 30M tolerated at least 1 mM without inhibition. A normal¹⁴C-HCO₃ photoincorporation rate was sustained with 0.6–1 mM sulfide in the presence of DCMU (7M) or NH₂OH (0.2 mM), although both of these photosystem II inhibitors prevented photoincorporation without sulfide. Other sulfur-containing compounds (S₂O₃ ^2– SO₃ ^2–, S₂O₄ ^2– thioglycolic acid cysteine) were unable to relieve DCMU inhibition. The lowering of the photoincorporation rate by preferentially irradiating photosystem I was also relieved by sulfide. The most tenable explanation of these results is that sulfide is used as a photo-reductant of CO₂, at least when photosystem II is inhibited. It is suggested that in some blue-green algae photosystem II is poisoned by a low sulfide concentration, thus making these algae sulfidedependent if they are to continue photosynthesizing in a sulfide environment. Presumably a sulfidecytochrome reductase enzyme system must be synthesized for sulfide to be used as a photo-reductant.