Copper effect on cytochrome b of photosystem II under photoinhibitory conditions

Toxic Cu (II) effect on cytochrome b ₅₅₉ under aerobic photoinhibitory conditions was examined in two different photosystem II (PSII) membrane preparations active in oxygen evolution. The preparations differ in the content of cytochrome b ₅₅₉ redox potential forms. Difference absorption spectra showed that the presence of Cu (II) induced the oxidation of the high-potential form of cytochrome b ₅₅₉ in the dark. Addition of hydroquinone reduced the total oxidized high-potential form of cytochrome b ₅₅₉ present in Cu (II)-treated PSII membranes indicating that no conversion to the low-potential form took place. Spectroscopic determinations of cytochrome b ₅₅₉ during photoinhibitory treatment showed slower kinetics of Cu (II) effect on cytochrome b ₅₅₉ in comparison with the rapid loss of oxygen evolution activity in the same conditions. This result indicates that cytochrome b ₅₅₉ is affected after PSII centres are photoinhibited. The high-potential form was more sensitive to toxic Cu (II) action than the low-potential form under illumination at pH 6.0. The content of the high-potential form of cytochrome b ₅₅₉ was completely lost; however, the low-potential content was unaffected in these conditions. This loss did not involve cytochrome protein degradation. The results are discussed in terms of different binding properties of the heme iron to the protonated or unprotonated histidine ligand in the high-potential and low-potential forms of cytochrome b ₅₅₉, respectively.     

Expression of Bradyrhizobium japonicum cbb3 terminal oxidase under denitrifying conditions is subjected to redox control

Bradyrhizobium japonicum utilizes cytochrome cbb ₃ oxidase encoded by the fixNOQP operon to support microaerobic respiration under free-living and symbiotic conditions. It has been previously shown that, under denitrifying conditions, inactivation of the cycA gene encoding cytochrome c ₅₅₀, the electron donor to the Cu-containing nitrite reductase, reduces cbb ₃ expression. In order to establish the role of c ₅₅₀ in electron transport to the cbb ₃ oxidase, in this work, we have analyzed cbb ₃ expression and activity in the cycA mutant grown under microaerobic or denitrifying conditions. Under denitrifying conditions, mutation of cycA had a negative effect on cytochrome c oxidase activity, heme c (FixP and FixO) and heme b cytochromes as well as expression of a fixP '–' lacZ fusion. Similarly, cbb ₃ oxidase was expressed very weakly in a napC mutant lacking the c -type cytochrome, which transfers electrons to the NapAB structural subunit of the periplasmic nitrate reductase. These results suggest that a change in the electron flow through the denitrification pathway may affect the cellular redox state, leading to alterations in cbb ₃ expression. In fact, levels of fixP '–' lacZ expression were largely dependent on the oxidized or reduced nature of the carbon source in the medium. Maximal expression observed in cells grown under denitrifying conditions with an oxidized carbon source required the regulatory protein RegR.     

The cytochrome bd terminal oxidase of Azotobacter vinelandii: Low temperature photodissociation spectrophotometry reveals reactivity of cytochromes b595 and d with both carbon monoxide and oxygen

Abstract Cytochromes d and b ₅₉₅ were studied by low temperature photodissociation of CO-ligated Azotobacter vinelandii membranes. White light or He-Ne laser irradiation revealed 436 and 594–597 nm absorption bands to be due to Fe¹¹ cytochrome b ₅₉₅. Oxy-cytochrome d (648 nm) was formed when the CO adduct was photolysed in the presence of oxygen. This was followed by ligand recombination (presumably oxygen) to the high-spin cytochrome b ₅₉₅, with a distinctive shift to shorter wavelengths of the α-band of the cytochrome, and a decrease in the oxygenated form. All spectral changes were light-reversible. We demonstrate the light-reversible binding of CO to both cytochromes b ₅₉₅ and d , and suggest migration of oxygen from cytochrome d to cytochrome b ₅₉₅ at a haem-haem binuclear centre during the oxidase reaction.     

Role of cytochrome b562 in the archaeal aerobic respiratory chain of Sulfolobus sp. strain 7

Abstract The role of cytochrome b ₅₆₂, a fragile constituent of the respiratory terminal oxidase supercomplex of the thermoacidophilic archaeon, Sulfolobus sp. strain 7, was investigated spectroscopically in the membrane-bound state. Cytochrome b ₅₆₂ did not react with CO or cyanide in the membrane-bound state, while it was irreversibly modified to a CO-reactive form ( b ₅₆₂) upon solubilization in the presence of cholate and LiCl. Cyanide titration analyses with the succinate-reduced membrane suggested that cytochrome b ₅₆₂ was upstream of both the ' g _y= 1.89' Rieske FeS cluster and the a -type cytochromes. These results show that the b -type cytochrome functions as an intermediate electron transmitter in the terminal oxidase supercomplex.     

The nitrite oxidizing system of Nitrobacter winogradskyi

Abstract Cytochrome components which participate in the oxidation of nitrite in Nitrobacter winogradskyi have been highly purified and their properties studied in detail. Cytochrome a ₁ c ₁ is an iron-sulphur molybdoenzyme which has haems a and c and acts as a nitrite-cytochrome c oxidoreductase. Cytochrome c -550 is homologous to eukaryotic cytochrome c and acts as the electron mediator between cytochrome a ₁ c ₁ and aa ₃-type cytochrome c oxidase. The oxidase is composed of two kinds of subunits, has two molecules of haem a and two atoms of copper in the molecule, and oxidizes actively eukaryotic ferrocytochrome c as well as its own ferrocytochrome c -550. Further, a flavoenzyme has been obtained which has transhydrogenase activity and catalyses reduction of NADP⁺ with benzylviologen radical. This enzyme may be responsible for production of NADPH in N. winogradskyi . The electron transfer against redox potential from NO₂⁻ to cythochrome c could be pushed through prompt removal by cytochrome aa ₃ of H⁺ formed by the dehydrogenation of NO₂⁻+ H₂O. As cytochrome c in anaerobically kept cell-free extracts is rapidly reduced on addition of NO₂⁻, a membrane potential does not seem necessary for the reduction of cytochrome c by cytochrome a ₁ c ₁ with NO₂⁻ in vivo.     

Role of Bradyrhizobium japonicum cytochrome c550 in nitrite and nitrate respiration

Bradyrhizobium japonicum cytochrome c ₅₅₀, encoded by cycA , has been previously suggested to play a role in denitrification, the respiratory reduction of nitrate to dinitrogen. However, the exact role of this cytochrome in the denitrification process is unknown. This study shows that cytochrome c ₅₅₀ is involved in electron transfer to the copper-containing nitrite reductase of B. japonicum , as revealed by the inability of a cycA mutant strain to consume nitrite and, consequently, to grow under denitrifying conditions with nitrite as the electron acceptor. Mutation of cycA had no apparent effect on methylviologen-dependent nitrite reductase activity. However, succinate-dependent nitrite reduction was largely inhibited, suggesting that c ₅₅₀ is the in vivo electron donor to copper-containing nitrite reductase. In addition, this study demonstrates that a cytochrome c ₅₅₀ mutation has a negative effect on expression of the periplasmic nitrate reductase. This phenotype can be rescued by extending the growth period of the cells. A model is proposed whereby a mutation in cycA reduces expression of the cbb ₃-type oxidase, affecting oxygen consumption rate by the cells and consequently preventing maximal expression of the periplasmic nitrate reductase during the first days of the growth period.     

CaMKII associates with CaV1.2 L-type calcium channels via selected β subunits to enhance regulatory phosphorylation

Calcium/calmodulin-dependent kinase II (CaMKII) facilitates L-type calcium channel (LTCC) activity physiologically, but may exacerbate LTCC-dependent pathophysiology. We previously showed that CaMKII forms stable complexes with voltage-gated calcium channel (VGCC) β_1b or β_2a subunits, but not with the β₃ or β₄ subunits ( Grueter et al. 2008 ). CaMKII-dependent facilitation of Ca_V1.2 LTCCs requires Thr498 phosphorylation in the β_2a subunit ( Grueter et al. 2006 ), but the relationship of this modulation to CaMKII interactions with LTCC subunits is unknown. Here we show that CaMKII co-immunoprecipitates with forebrain LTCCs that contain Ca_V1.2α₁ and β₁ or β₂ subunits, but is not detected in LTCC complexes containing β₄ subunits. CaMKIIα can be specifically tethered to the I/II linker of Ca_V1.2 α₁ subunits in vitro by the β_1b or β_2a subunits. Efficient targeting of CaMKIIα to the full-length Ca_V1.2α₁ subunit in transfected HEK293 cells requires CaMKII binding to the β_2a subunit. Moreover, disruption of CaMKII binding substantially reduced phosphorylation of β_2a at Thr498 within the LTCC complex, without altering overall phosphorylation of Ca_V1.2α₁ and β subunits. These findings demonstrate a biochemical mechanism underlying LTCC facilitation by CaMKII.     

Phospholipase Cγ1 in Bovine Rod Outer Segments: Immunolocalization and Light-Dependent Binding to Membranes

Abstract: We have investigated the isozymes of a phosphoinositide-specific phospholipase C (PLC) in bovine retina using several monoclonal antisera to PLCβ₁, γ₁, and δ₁. Immunoblot analysis showed that all three isozymes were present in the retina. Immunocytochemical localization in frozen bovine retina sections showed that PLCγ₁ was present in the photoreceptor cell layer, outer plexiform cell layer, inner plexiform cell layer, and ganglion cell layer. Immunoreaction within the photoreceptor cell layer was dependent on dark/light adaptation state of retinas. Immunoblot analysis of rod outer segments (ROS) with monoclonal or polyclonal antibodies to PLCγ₁ showed the presence of an immunoreactive band of 140 kDa. ROS prepared from retinas light-adapted in vitro had more PLCγ₁ on immunoblots than ROS from dark-adapted retinas. PLC enzyme activity in ROS from light-adapted retinas was 69 and 46% higher than ROS from dark-adapted retinas, when assayed in the presence and absence of ATP, respectively. This increase in enzyme activity was observed at [Ca²⁺]_free between 0.32 and 100 µ M . These results demonstrate the presence of PLCγ₁ in bovine ROS and show that ROS prepared from light-adapted retinas are enriched in this isozyme, suggesting that light may promote the binding of this isozyme to bleached ROS membranes.     

31P NMR Relaxation Does Not Affect the Quantitation of Changes in Phosphocreatine, Inorganic Phosphate, and ATP Measured In Vivo During Complete Ischemia in Swine Brain

Abstract: Ischemia-induced changes in ³¹P NMR relaxation were examined in 16 piglets. NMR spectra were acquired under control conditions and during complete cerebral ischemia induced via cardiac arrest. Changes in T ₁ were assessed directly in six animals during control conditions and after 30–45 min of complete ischemia when changes in brain P₁ levels had reached a plateau. The T ₁ for P₁ did not change, i.e., 2.3 ± 0.5 s during control conditions versus 2.4 ± 1.0 s during ischemia. To evaluate phosphocreatine and ATP, two types of spectra, with a long (25-s) or short (1-s) interpulse delay time, were collected during the first 10 min of ischemia (n = 10). Both types of spectra showed the same time course of changes in phosphocreatine and ATP levels, implying that the T ₁ relaxation times do not change during ischemia. There were no changes in the linewidths of phosphocreatine, ATP, or P₁ during ischemia, implying that the T *₂ values remain constant. Our results suggest that the ³¹P T ₁ and T *₂ for phosphocreatine, P_i, and ATP do not change during ischemia, and therefore changes in ³¹P NMR peak intensity accurately reflect changes in metabolite concentrations.     

Mapping and disruption of the cybB gene coding for cytochrome b561 in Escherichia coli

Abstract The cybB gene on a plasmid encoding cytochrome b ₅₆₁ in Escherichia coli was disrupted by insertion of Km^rl determinant DNA. The cromosomal cybB gene was replaced by the inactivated cybB gene on the plasmid by homologous recombination using λ phage lysogenization and heat-induction. The replacement was confirmed by Southern and Western blotting analyses. Deficiency on the cybB gene product did not affect the growth properties of the cells, and the oxidase activities of the cells dependent on various substrates were similar to those of the parental strain. Cytochrome b ₅₆₁ is concluded to be expressed in E. coli , but may not play a major role in cell growth. In the genetic map of E. coli , the cybB gene was determined by conjugational and transductional crosses to be at 31 min between trg and terC .     

Molecular Determinants of General Anesthetic Action: Role of GABAA Receptor Structure

Abstract: Using receptors expressed from mouse brain mRNA in Xenopus oocytes, we found that enhancement of type A γ-aminobutyric acid (GABA_A) receptor-gated Cl⁻ channel response is a common action of structurally diverse anesthetics, suggesting that the GABA_A receptor plays an important role in anesthesia. To determine if GABA_A receptor subunit composition influences actions of anesthetics, we expressed subunit cRNAs in Xenopus oocytes and measured effects of enflurane on GABA-activated Cl⁻ currents. Potentiation of GABA-activated currents by enflurane was dependent on the composition of GABA_A receptor protein subunits; the order of sensitivity was α₁β₁ > α₁β₁γ_2s=α₁β₁γ_2L > total mRNA. The results suggest that anesthetics with simple structures may act on the GABA_A receptor protein complex to modulate the Cl⁻ channel activity and provide a molecular explanation for the synergistic clinical interactions between benzodiazepines and general anesthetics.     

Two tertiary branched tetraamines, N4-aminopropylnorspermidine and N4-aminopropylsperimidine, are the major polyamines in thermophilic eubacteria, Thermoleophilum

Abstract Two novel polyamines were found as major polyamines of Thermoleophilum album and Thermoleophilum minutum , which are Gram-negative eubacteria obligate for thermophily and n -alkane substrates. They were identified as tertiary branched tetraamines, N⁴-aminopropylnorspermidine ( tris (3-aminopropyl)amine) [NH₂(CH₂)₃N-((CH₂)₃NH₂)(CH₂)₃NH₂ or N(CH₂CH₂CH₂NH₂)₃] and N⁴-aminopropylspermidine [NH₂(CH₂)₃N((CH₂)₃NH₂)(CH₂)₄NH₂] by high-performance liquid chromatography and gas chromatography-mass spectrometry.     

Monoclonal Antibodies to the Human γ2 Subunit of the GABAA/Benzodiazepine Receptors

Abstract: The large intracellular loop (IL) of the γ₂ subunit of the cloned human γ-aminobutyric acid_A (GABA_A) receptor (γ₂IL) was expressed in bacteria as glutathione- S -transferase and staphylococcal protein A fusion proteins. Mice were immunized with the fusion proteins (one protein per animal), and monoclonal antibodies were obtained. Six monoclonal antibodies reacted with the γ₂IL moiety of the fusion proteins. Three of these monoclonal antibodies also immunoprecipitated a high proportion of the GABA_A/benzodiazepine receptors from bovine and rat brain and reacted with a wide 44,000–49,000-M_r peptide band in immunoblots of affinity-purified GABA_A receptors. These monoclonal antibodies are valuable reagents for the molecular characterization of the GABA_A receptors in various brain regions.     

Chlorate resistant mutants of Pseudomonas stutzeri affected in respiratory and assimilatory nitrate utilization and expression of cytochrome cd1

Abstract Chlorate-resistant mutants were generated by random insertion of the transposon Tn5 into genomic DNA of Pseudomonas stutzeri ZoBell strain and selected for loss of nitrate respiration (Nar phenotype). The mutants were differentiated by restriction-fragment analysis, by assaying for nitrate assimilation and for molybdenum co-factor activity, and by the amount of respiratory nitrate reductase. Two mutants, lacking both nitrate respiration and nitrate assimilation, over-produced an inactive nitrate reductase but synthesized in the presence of nitrate only a reduced amount of respiratory nitrite reductase (cytochrome cd ₁). Expression of cytochrome cd ₁ in these mutants was specifically induced by nitrate, suggesting a sensor system for this substrate.     

The Subunit Composition of a GABAA/Benzodiazepine Receptor from Rat Cerebellum

Abstract: The pentameric subunit composition of a large population (36%) of the cerebellar granule cell GABA_A receptors that show diazepam (or clonazepam)-insensitive [³H]Ro 15-4513 binding has been determined by immunoprecipitation with subunit-specific antibodies. These receptors have α₆, α₁, γ_2S, γ_2L, and β₂ or β₃ subunits colocalizing in the same receptor complex.     

Glutamate Uptake Stimulates Na+,K+-ATPase Activity in Astrocytes via Activation of a Distinct Subunit Highly Sensitive to Ouabain

Abstract: The excitatory amino acid glutamate was previously shown to stimulate aerobic glycolysis in astrocytes by a mechanism involving its uptake through an Na⁺-dependent transporter. Evidence had been provided that Na⁺,K⁺-ATPase might be involved in this process. We have now measured the activity of Na⁺,K⁺-ATPase in cultured astrocytes, using ouabain-sensitive ⁸⁶Rb uptake as an index. l -Glutamate increases glial Na⁺,K⁺-ATPase activity in a concentration-dependent manner with an EC₅₀ = 67 µ M . Both l - and d -aspartate, but not d -glutamate, produce a similar response, an observation that is consistent with an uptake-related effect rather than a receptor-mediated one. Under basal conditions, concentration-dependent inhibition of Na⁺,K⁺-ATPase activity in astrocytes by ouabain indicates the presence of a single catalytic site with a low affinity for ouabain ( K _0.5 = 113 µ M ), compatible with the presence of an α₁ isozyme. On stimulation with glutamate, however, most of the increased activity is inhibited by low concentrations of ouabain ( K _0.5 = 20 n M ), thus revealing a high-affinity site akin to the α₂ isozyme. These results suggest that astrocytes possess a glutamate-sensitive isoform of Na⁺,K⁺-ATPase that can be mobilized in response to increased neuronal activity.     

Isolation of an Escherichia coli mutant defective in cytochrome biosynthesis

Abstract A pleiotropic mutant of Escherichia coli affected in cytochrome biosynthesis was detected by anaerobic screening on a solid medium containing triphenyltetrazolium. When grown anaerobically on glycerol, nitrate and Casamino acids, this mutant exhibited a level of soluble cytochrome c ₅₅₂ which was ten times higher than that found in wild-type cells. The level of membrane-bound cytochrome b and the activity of nitrate reductase were about half the normal level. The mutant grew aerobically on succinate or d,l -lactate at a greatly reduced rate. The mutation impairing the growth ability at the locus sox (succinate oxidation) is also responsible for the deficiency of cytochrome b , nitrate reductase and formate dehydrogenase. Mapping by transduction placed sox at 86.7 min on the chromosome, very close to the glnA locus. Genetic analysis also indicated that the elevated level of cytochrome c ₅₅₂ was the result of a separate mutation, the location of which is yet to be determined.     

LEAF INFECTION OF COTTON BY XANTHOMONAS MALVACEARUM (E.F.SM.) DOWSON

Xanthomonas malvacearum spread more rapidly along vascular tissue than into mesophyll when inoculated to the main veins of susceptible cotton leaves. The extent of spread varied with the concentrations of inocula, tissue age and cotton variety.
Increasing concentrations of inocula accelerated the initial spread of disease.
Bacteria spread more rapidly in young leaves than in old—increasing age greatly decreased disease in the mesophyll. The initial invasion was quicker in young leaves of young plants than in young leaves of old plants.
Three types of behaviour, according to the host's reaction, distinguish Knight's resistance factors: ( a ) where X. malvacearum spread extensively along veins and into mesophyll of plants containing factors B₃ and B₅; ( b ) where it was restricted to the point of inoculation in plants containing B ₄, B₉ and combinations with B _6m; and ( c ) where it spread along veins but not appreciably into mesophyll in varieties containing B ₂ and B ₂ B ₃.
From this and four other different types of tests, factors B ₂ and B ₃ seem to increase mesophyll resistance but only B ₂ gives appreciable vascular resistance. Further, the vascular bundles in varieties with B ₂ seem to be surrounded by an additional 'barrier' which resists X. malvacearum.     

Differential Effects of 4'-Chlorodiazepam on Expressed Human GABAA Receptors

Abstract: The interactions of the atypical benzodiazepine 4'-chlorodiazepam (Ro 5-4864) with functionally expressed human GABA_A receptor cDNAs were determined. Cotransfection of human α₂, β₁, and γ₂ subunits was capable of reconstituting a 4'-chlorodiazepam recognition site as revealed by a dose-dependent potentiation of t -[³⁵S]butylbicyclophosphorothionate ([³⁵S]TBPS) binding to the GABA-activated chloride channel. This site is found on GABA_A receptor complexes containing sites for GABA agonist-like benzodiazepines and neuroactive steroids. The importance of the α subunit was further demonstrated as substitution of either α₁ or α₃ for the α₂ subunit did not reconstitute a 4'-chlorodiazepam recognition site that was capable of modulating [³⁵S]TBPS binding under the same experimental conditions. The 4'-chlorodiazepam modulatory site was shown to be distinct from the benzodiazepine site, but the phenylquinolines PK 8165 and PK 9084 produced effects similar to 4'-chlorodiazepam, consistent with the previous analysis of the 4'-chlorodiazepam site in brain homogenates. Further analysis of the subunit requirements revealed that coexpression of α₂ and β₁ alone reconstituted a 4'-chlorodiazepam recognition site. It is interesting, however, that the 4'-chlorodiazepam site was found to inhibit [³⁵S]TBPS binding to the GABA-activated chloride channel. Thus, the 4'-chlorodiazepam site may be reconstituted with only the α and β polypeptides.