Evidence for a significant contribution by peroxidase-mediated O2 uptake to root respiration of Brachypodium pinnatum

This study describes the O₂ uptake characteristics of intact roots of Brachypodium pinnatum. In the presence of 25 mM salicylhydroxamic acid (SHAM), concentrations of KCN below 3.5 νM had no effect on the rate of root respiration, whereas in the absence of 25 mM SHAM a significant inhibition of approx. 18% was observed. This indicates that an O₂-consuming reaction, not associated with the cytochrome pathway, the alternative pathway or the “residual component”, operates in the absence of any inhibitors in roots of B. pinnatum. We demonstrate here that this fourth O₂-consuming reaction is mediated by a peroxidase. A peroxidase which catalyzed O₂ reduction in the presence of NADH was readily washed from the roots of B. pinnatum. This peroxidase was stimulated by 5 mM SHAM, whereas ascorbic acid, catalase, catechol, gentisic acid, low concentrations potassium cyanide (3.5 μM), sodium azide, sodium sulfide, superoxide dismutase and high concentrations SHAM (25 mM) inhibited this reaction. Except for high concentrations of SHAM and concentrations of KCN higher than approx. 3.5 μM, these effectors could not be used to inhibit the peroxidase-mediated O₂ uptake in intact roots of B. pinnatum. Concentrations of SHAM below 10 mM stimulated O₂ uptake up to 15% of the control rate, depending on concentration, whereas 25 mM SHAM inhibited O₂ uptake by 35%. The stimulation at low concentrations resulted from a SHAM-stimulated peroxidase activity, whereas 25 mM SHAM completely inhibited both the peroxidase-mediated O₂ uptake and the activity of the alternative pathway. A method is presented for determining the relative contributions of each of the four O₂-consuming reactions, i.e. the cytochrome pathway, the alternative pathway, the “residual component” and the peroxidase-mediated O₂ uptake. The peroxidase-mediated O₂ uptake contributed 21% to the total rate of oxygen uptake in roots of B. pinnatum, the cytochrome pathway contributed 41%, the alternative pathway 14% and the “residual component” 24%.     

微电泳U—50488对大鼠疑核呼吸相关单位电活动的影响

本工作观察了微电泳给予 U-50488对大鼠延髓疑核呼吸相关单位自发放电活动的影响。在96个单位中,呈抑制效应的51个,兴奋效应的11个,先抑制后兴奋的3个,先兴奋后抑制的6个,其余25个单位的自发放电活动无明显变化。在16个 U-50488产生抑制效应的单位中,12个单位的抑制效应能被纳洛酮对抗。2个 U-50488 表现为兴奋效应的单位,纳洛酮也显出拮抗效应。U-50488产生抑制效应持续的时间明显长于兴奋效应持续的时间。本工作的结果提示,k 受体可能也参与呼吸调控作用,并且介导 U-50488抑制效应的阿片受体亚型可能不同于介导兴奋效应的阿片受体亚型。     

Size and settling behaviour in male and female cypris larvae of the parasitic barnacle Sacculina carcini Thompson (Crustacea: Cirripedia: Rhizocephala)

Twenty-one broods from different externae of Sacculina carcini Thompson were cultured to the cypris stage. The size of the cyprids was measured and the larvae subjected to settling upon unparasitized crabs (Garcinus maenas (L.)) and small juvenile externae. The cyprids occur in two sizes that may appear singly or together in the same brood. Small cyprids are of the female sex that settle upon crabs and are infective, while large cyprids are of the male sex and only settle upon juvenile externae. These results are in agreement with other well-studied rhizocephalans.     

Pyrobaculum gen. nov., a new genus of neutrophilic,rod-shaped archaebacteria from continental solfataras growing optimally at 100°C

Seven members of a new group of rod-shaped hyperthermophilic neutrophilic archaebacteria were isolated from boiling neutral to alkaline solfataric waters from the Azores, Iceland, and Italy. The organisms are strict anaerobes, growing optimally at 100°C. The cells are motile due to peritrichous or bipolar polytrichous flagellation. The isolates grow facultatively chemolithoautotrophically or obligately heterotrophically. Molecular hydrogen or complex organic substances are used as electron donors. During heterotrophic growth, elemental sulfur, thiosulfate, sulfite, l(-)cystine and oxidized glutathione may serve as electron acceptors depending on the individual strain. Elemental sulfur is strictly required as an electron acceptor for autotrophic growth. The G+C content of the DNA is around 46 mol%. The isolates represent a new genus which we have named Pyrobaculum (the fire stick). Two species are described: the facultatively autotrophic Pyrobaculum islandicum (DSM 4184), which is the type species, and the obligately heterotrophic Pyrobaculum organotrophum (DSM 4185).     

Control of mitochondrial respiration in muscle

Summary Control of mitochondrial respiration depends on ADP availability to the F₁ATPase. An electrochemical gradient of ADP and ATP across the mitochondrial inner membrane is maintained by the adenine nucleotide translocase which provides ADP to the matrix for ATP synthesis and ATP for energy-dependent processes in the cytosol. Mitochondrial respiration is responsive to the cytosolic phosphorylation potential, ATP/ADP · P_i which is in apparent equilibrium with the first two sites in the electron transport chain. Conventional measures of free adenine nucleotides is a confounding issue in determining cytosolic and mitochondrial phosphorylation potentials. The advent of phosphorus-31 nuclear magnetic resonance (P-31 NMR) allows the determination of intracellular free concentrations of ATP, creatine-P and Pi in perfused muscle in situ. In the glucose-perfused heart, there is an absence of correlation between the cytosolic phosphorylation potential as determined by P-31 NMR and cardiac oxygen consumption over a range of work loads. These data suggest that contractile work leads to increased generation of mitochondrial NADH so that ATP production keeps pace with myosin ATPase activity. The mechanism of increased ATP synthesis is referred to as stimulusre-sponse-metabolism coupling. In muscle, increased contractility is a result of interventions which increase cytosolic free Ca²⁺ concentrations. The Ca^2- signal thus generated increases glycogen breakdown and myosin ATPase in the cytosol. This signal is concomitantly transmitted to the mitochondria which respond to small increases in matrix Ca²⁺ by activation of Ca²⁺-sensitive dehydrogenases. The Ca²⁺-activated dehydrogenase activities are key rate-controlling enzymes in tricarboxylic acid cycle flux, and their activation by Ca^2- leads to increased pyridine nucleotide reduction and oxidative phosphorylation. These observations which have been consistent in preparations both in vitro and in situ do not obviate a role for ADP control of muscle respiration, but do explain, in part, the lack of dramatic fluctuations in the cytosolic phosphorylation potential over a large range of contractile activities.     

Isolation of the sulphur reductase and reconstitution of the sulphur respiration of Wolinella succinogenes

Wolinella succinogenes can grow at the expense of sulphur reduction by formate. The enzymes involved in the catalysis of this catabolic reaction have been investigated. From the results the following conclusions are drawn: 1. The enzyme isolated as a sulphide dehydrogenase from the cytoplasmic membrane of W. succinogenes is the functional sulphur reductase that operates in the electron transport from formate to sulphur. 2. The enzyme (M_r 200,000) consists essentially of one type of subunit with the M_r 85,000 and contains equal amounts of free iron and sulphide (120 mol/g protein), but no heme. It represents the first functional sulphur reductase ever isolated. 3. The electron transport chain catalyzing sulphur reduction by formate consists merely of formate dehydrogenase and sulphur reductase. A lipophilic quinone which mediates the transfer of electrons between enzymes in other chains, is apparently not involved. This is the first known example of a phosphorylative electron transport chain that operates without a quinone. 4. The same formate dehydrogenase appears to operate in the electron transport both with sulphur and with fumarate as the terminal electron acceptor in W. succinogenes.Abbreviations DMN 2,3-Dimethyl-1,4-naphthoquinone - DTT dithiothreitol - MK menaquinone (vitamin K₂) - PMSF phenylmethane sulfonylfluoride - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]-glycine - Tea triethanolamine - Hepes 4-(2-hydroxyethyl)-1-piperazineethane sulfonate Dedicated to Professor F. Schneider (Philipps-Universität Marburg) on the occasion of his 60th birthday     

Investigations on microbial sulfur respiration

The reduction of elemental or sulfane sulfur to hydrogen sulfide by eubacteria was investigated. Spirillum 5175 had the most active sulfur oxidoreductase. It could be cultivated with fumarate (F), elemental sulfur (S) or nitrate (N) as electron acceptor. Maximum activity was found for Spirillum 5175_S but activity was also present in Spirillum 5175_F and Spirillum 5175_N, i.e. the sulfur oxidoreductase is a constitutive enzyme. It was localized in the membrane, and no activity was found in the cytoplasm in contrast to Desulfovibrio baculatus. Different procedures were applied for the measurement of the sulfur oxidoreductase activity. In the manometric assay hydrogenase was coupled to the sulfur oxidoreductase, and the uptake of dihydrogen was measured in the presence of elemental sulfur. Alternatively, H₂S was assayed directly or was trapped in 12% NaOH and determined by the methylene blue procedure. Using ³⁵S sulfur and ³⁵S-labelled compounds both the substrate and H₂S could be measured. A further increase in sensitivity was achieved using phenosafranin. It was reduced photochemically, and served as the electron donor to the sulfur oxidoreductase, i.e. no hydrogenase was required. This was an important result in view of the fact that not all sulfur-reducing bacteria contain hydrogenase. However, in those cases the hydrogenase isolated from Clostridium pasteurianum could be coupled to the sulfur oxidoreductase. Among the different forms of elemental sulfur Janek sulfur gave the best results in terms of activity and reproducibility. The reduction of elemental sulfur to hydrogen sulfide had a pH optimum at pH 8.7–8.9. There was always a lag-phase which was pH-dependent. During this period the turbidity of the solution changed. Addition of thiols, such as GSH, shortened the lag-phase and caused an increase in activity of the sulfur oxidoreductase. In the presence of p-chloromercuribenzenesulfonic acid the reaction rate decreased significantly. Comparable reaction rates and activity values of the sulfur oxidoreductase in Spirillum 5175_F were obtained with organic trisulfides, RS-S-SR. In contrast to elemental sulfur RS-S-SR are well-defined chemical compounds suitable for quantitative and mechanistic investigations. Labelling the central sulfur of RS-S-SR with ³⁵S gave a satisfactory recovery of the total radioactivity in form of (³⁵S) H₂S in our assay. Trisulfides were shown to be formed as reactive intermediates in bacteria. This process required the sulfur transferase rhodanese which was present in Spirillum 5175, or other sulfur-reducing eubacteria.Abbreviations EPR Electron Paramagnetic Resonance - A Absorbance - PCMS p-chloromercuribenzenesulfonic acid - Sp. 5175_F Splrillum 5175 grown with fumarate - Sp. 5175_S with sulfur - Sp. 5175_N with nitrate - SCE Standard Calomel Electrode