The influence of pH on the EPR and redox properties of cytochrome c oxidase in detergent solution and in phospholipid vesicles

The EPR signals of oxidized and partially reduced cytochrome oxidase have been studied at pH 6.4, 7.4, and 8.4. Isolated cytochrome oxidase in both non-ionic detergent solution and in phospholipid vesicles has been used in reductive titrations with ferrocytochrome c.The g values of the low- and high-field parts of the low-spin heme signal in oxidized cytochrome oxidase are shown to be pH dependent. In reductive titrations, low-spin heme signals at g 2.6 as well as rhombic and nearly axial high-spin heme signals are found at pH 8.4, while the only heme signals appearing at pH 6.4 are two nearly axial g 6 signals. This pH dependence is shifted in the vesicles.The g 2.6 signals formed in titrations with ferrocytochrome c at pH 8.4 correspond maximally to 0.25–0.35 heme per functional unit (aa₃) of cytochrome oxidase in detergent solution and to 0.22 heme in vesicle oxidase. The total amount of high-spin heme signals at g 6 found in partially reduced enzyme is 0.45–0.6 at pH 6.4 and 0.1–0.2 at pH 8.4. In titrations of cytochrome oxidase in detergent solution the g 1.45 and g 2 signals disappear with fewer equivalents of ferrocytochrome c added at pH 8.4 compared to pH 6.4.The results indicate that the environment of the hemes varies with the pH. One change is interpreted as cytochrome a₃ being converted from a high-spin to a low-spin form when the pH is increased. Possibly this transition is related to a change of a liganded H₂O to OH^? with a concomitant decrease of the redox potential. Oxidase in phosphatidylcholine vesicles is found to behave as if it experiences a pH, one unit lower than that of the medium.     

Structure of F1-ATPases

F₁-ATPases are large multimeric proteins that can be isolated from the membrane bound system that catalyzes the phosphorylation of ADP by inorganic phosphate in bacteria, plants, and mitochondria. They can be visualized in electron micrographs of the inner mitochondrial membranes where they appear as large protruding spheres 90 Å in diameter. The purified F₁-ATPases have a molecular weight of 320,000 to 400,000 daltons and are composed of five non-identical subunits (, , , and ). The stoichiometry of these subunits in the complex is still unknown but compositions of the type ₃₃ and ₂₂₂₂₂ were found to be consistent with some of the available experimental data. This review discusses the recent data and the experimental approaches utilized for the structural characterization of F₁-ATPases.     

The oxidation-reduction kinetics of the reaction of cytochrome c1 with non-physiological redox agents

The kinetics of the oxidation-reduction reactions of cytochrome c₁ with ascorbate, ferricyanide, triphenanthrolinecobalt(III) and N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) have been examined using the stopped-flow technique. The reduction of ferricytochrome c₁ by ascorbic acid is investigated as a function of pH. It is shown that at neutral and alkaline pH the reduction of the protein is mainly performed by the doubly deprotonated form of ascorbate. From the ionic-strength-dependence studies of the reactions of cytochrome c₁ with ascorbate, ferricyanide and triphenanthrolinecobalt(III), it is demonstrated that the reaction rate is governed by electrostatic interactions. The second-order rate constants for the reaction of cytochrome c₁ with ascorbate, ferricyanide, TMPD and triphenanthrolinecobalt(III) are 1.4·10⁴, 3.2·10³, 3.8·10⁴ and 1.3·10⁸ M^?1·s^?1 (pH 7.0, I = 0, 10°C), respectively. Application of the Debye-Hückel theory to the the ionic-strength-dependence studies of these redox reactions of cytochrome c₁ yielded for ferrocytochrome c₁ and ferricytochrome c₁ a net charge of ?5 and ?4, respectively. The latter value is close to that of ?3 for the oxidized enzyme, calculated from the amino acid sequence of the protein. This implies that not a local charge on the surface of the protein, but the overall net charge of cytochrome c₁ governs the reaction rate with small redox molecules.     

Tolerance Limit of the Sugarbeet to Heterodera schachtii

Field and greenhouse experiments showed that yield losses of sugarbeet, Beta vulgaris, did not occur in soil infested with fewer than eight Heterodera schachtii eggs/g soil. However, larger population densities greatly reduced sugarbeet yield. In the field experiment, the yield in microplots inoculated with more than 64 eggs/g soil was less than 20% of yields in uninoculated microplots. Nevertheless, tolerance limits of 4 and 1.8 eggs/g soil, in greenhouse and field microplots, respectively, were derived by fitting the data with the equation y =m + (l - m)z^P-T. Maximum rates of multiplication of 55 and more than 300, and equilibrium densities of 340 and 130 eggs/g soil, were estimated in greenhouse and field microplot tests, respectively.     

Urea hydrolysis in some tea soils

The influence of application of hexachlorocyclohexane (HCH=gamma BHC), to a submerged tropical field soil at rates equivalent to recommended field rates (1–2.5kg a.i./ha) and twice this level, upon the rhizosphere soil nitrogenase, nitrogen fixers, and soil redox potential (Eh) was investigated. The rhizosphere soil from HCH-treated field exhibited significantly higher nitrogenase activity than that from untreated fields. HCH retarded the drop in redox potential of the field soil upto 80 days after transplantation under submerged conditions. Populations of nitrogen-fixingAzospirillum sp. and Azotobacter, to a greater extent, and anaerobic organisms, to a lesser extent, were stimulated in HCH-treated soils. Results indicate the stimulation of heterotrophic nitrogen-fixing bacteria by HCH in submerged paddy soils.     

Chemical equilibrium and E value of Zn (ZnE) in inceptisols and entisols of tropical india

Summary The chemical equilibrium and E value for Zn was calculated in 9 soils of Haryana (India) which represented mainly entisol and inceptisols. The rate of isotopic exchange between⁶⁵Zn and native soil Zn was quite rapid and radioactivity reduced to about 0.2 per cent of initial activity after 3 days but total Zn concentration in soil solution did not decrease. In 5 out of 9 soils equilibrium was established in 2 to 3 days. In typic ustochrepts having high clay content (3,4) equilibrium was attained in two days. In typic udipsamments (2), typic camborthids (8) and aquic vertic ustichrepts (9) it took 3 days to set equilibrium. Typic ustochrepts (5) took 5 days for equilibrium whereas typic ustipssaments (6) and typic camborthids of high O.C. did not attain equilibrium even in 7 days. This indicated that the chemistry and availability of Zn in soil would depend on soil types. When Zn_E was estimated by applying activity after equilibrium with carrier Zn and by applying activity with carrier Zn before equilibrium was set, there was no agreement in Zn_E in two methods. Increasing Zn_E with increasing Zn dose was observed by both methods only in alkaline typic ustochrept (4). In some soils higher E values than added amounts were observed, whereas, in soil 8 negative E values were obtained. The E values become erratic and are over estimated where complex reactions take place due to high pH, high O.C. and high complex forming elements.     

西南岩溶区广西生态安全及资源利用效率

构建了基于生态足迹理论的生态安全综合评价模型,然后基于生态安全评价指标,把数据驱动下动态计量经济学的单位根检验和与协整分析模型引进到生态安全与资源利用效率之间的动态均衡关系的研究中来。以西南岩溶区广西为例,首先采用生态足迹理论方法来测算1990～2003年广西生态安全的生态足迹、生态承载力、生态盈亏及生态压力指数,然后采用动态计量的协整分析方法对广西区域资源利用效率与生态安全状况诸指标之间的长期关系进行了协整分析。结果显示,广西生态足迹呈不断增加趋势,生态承载力呈不断下降趋势,出现了严重的生态赤字,生态足迹压力增幅明显,从临界安全状态发展到不安全状态;资源利用效率不高与生态安全指标之间呈现一种长期稳定的趋势,这表明岩溶区广西的资源利用效率不高,生态安全形势不容乐观,需要及时采取应对策略与措施加以调控。     

Computation of pH evolution versus ionic products concentration in a fermentation broth

An algorithm developed for pH computation has been tested to calculate the theoretical pH changes in a culture medium during the course of a fermentation. A divergence between the computed pH value and the value measured with the electrode allows us to highlight the presence of undetected ionic products. The calculation with the algorithm by means of a computer requires only the knowledge of the ionic properties of the substrates and detected products and existing thermodynamic constants. (c) 1993 Wiley & Sons, Inc.     

糙皮侧耳(平菇)疏水蛋白的分离纯化及其界面自组装特性研究

从糙皮侧耳Pleurotus ostreatus Pm039菌丝体中分离纯化到一种疏水蛋白并命名为Po.HYD1,SDS-PAGE显示其分子量约15kDa。Po.HYD1具有高度的表面活性,100μg/mL浓度下能够降低水表面张力至25.5mN/m。在1~100μg/mL浓度范围内存在6μg/mL和24μg/mL两个关键浓度,说明了不同浓度范围内自组装条件的改变。水接触角测定证明了Po.HYD1自组装膜的包被能够逆转固体表面的可湿润性。原子力显微镜分析揭示了Po.HYD1在云母表面形成厚度4.2±0.1nm"小杆层";在高定向热裂解石墨表面形成厚度3.2~3.8nm吸附层;在剧烈振荡诱导下的水溶液中形成形状相似、取向一致但体积大小不等的"耳型"颗粒。     

FAD oxidizes the ERO1-PDI electron transfer chain: the role of membrane integrity

The molecular steps of the electron transfer in the endoplasmic reticulum from the secreted proteins during their oxidation are relatively unknown. We present here that flavine adenine dinucleotide (FAD) is a powerful oxidizer of the oxidoreductase system, Ero1 and PDI, besides the proteins of rat liver microsomes and HepG2 hepatoma cells. Inhibition of FAD transport hindered the action of FAD. Microsomal membrane integrity was mandatory for all FAD-related oxidation steps downstream of Ero1. The PDI inhibitor bacitracin could inhibit FAD-mediated oxidation of microsomal proteins and PDI, but did not hinder the FAD-driven oxidation of Ero1. Our data demonstrated that Ero1 can utilize FAD as an electron acceptor and that FAD-driven protein oxidation goes through the Ero1-PDI pathway and requires the integrity of the endoplasmic reticulum membrane. Our findings prompt further studies to elucidate the membrane-dependent steps of PDI oxidation and the role of FAD in redox folding.