Reconsideration of the efficiency of energy transduction in Paracoccus denitrificans during growth under a variety of culture conditions

1. Theoretical overall P/2e^- ratios were calculated for growth of Paracoccus denitrificans under a variety of culture conditions on the basis of a growth model and recently published schemes of electron transport and associated proton translocation. 2. Experimental overall P/2e^- ratios were calculated, using the specific rate of ATP synthesis determined in cultures grown under aerobic carbon source-limited conditions. 3. The experimental P/2e^- was equal to the theoretical P/2e^- for aerobic sulphate-limited growth with gluconate or succinate as carbon source, on the assumption that site 1 phosphorylation was completely absent. 4. The experimental and the theoretical P/2e^- were similar for growth on nitrate as terminal electron acceptor and gluconate, mannitol or succinate as carbon source, on the assumption that nitrate enters the cell via the electroneutral nitrate-nitrite antiport system. 5. The experimental and theoretical P/2e^- were similar for growth on nitrite as terminal electron acceptor and mannitol or succinate as carbon source. 6. The experimental P/2e^- was substantially lower than the theoretical P/2e^- for aerobic growth with nitrate as nitrogen source and gluconate or mannitol as carbon source. The amount of energy needed for assimilative reduction of nitrate to ammonia was calculated from this difference.Dedicated to Prof. H. G. Schlegel on the occasion of his 60th birthday     

Evidence that energy conserving electron transport pathways to nitrate and cytochrome o branch at ubiquinone in Paracoccus denitrificans

The organisation and function of electron transport pathways in Paracoccus denitrificans has been studied with both inhibitors and electrode probes for O₂ or N₂O respiration and membrane potential. Myxothiazol completely inhibits electron flow through the cytochrome bc₁ region of the electron transport chain, as judged by its effect on nitrous oxide respiration. Electron flow to oxygen via the cytochrome o oxidase was shown to be insensitive to myxothiazol in a mutant, HUUG 25, that lacks cytochrome c and in which the aa₃ oxidase is therefore inactive. Myxothiazol did not inhibit nitrate reduction. It is concluded that myxothiazol is a specific inhibitor of electron flow through the cytochrome bc₁ region and more potent than antimycin which does not give complete inhibition.As neither antimycin nor myxothiazol, nor a combination of the two, inhibits electron transport to either nitrate reductase or cytochrome o it is concluded that electron transport pathways to these enzymes do not involve the cytochrome bc₁ region but rather branch at the level of ubiquinone. Although the cytochrome o pathway branches at ubiquinone, it is associated with the generation of a protonmotive force; this is shown by measurements of membrane potential in vesicle preparations from the mutant HUUG 25.In contrast to antimycin and myxothiazol, the ubiquinone analogues 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT) and 2-n-undecyl-3-hydroxy-1,4-naphthoquinone (UHNQ) inhibit electron flow through both the cytochrome bc₁ complex and the cytochrome o pathway, although a higher titre is required in the latter case. These two inhibitors were without effect on the nitrate reductase pathway. Thus myxothiazol is the inhibitor of choice for selective and complete inhibition of cytochrome bc₁.Recently published schemes for electron transport in P. denitrificans are analysed.Non standard abbreviations S-13 2,5-dichloro-3-t-butyl-4-nitrososalicylanilide - UHNQ 2-n-undecyl-3-hydroxy-1,4-naphthoquinone - UHDBT 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole     

Electron-transport chain and coupled oxidative phosphorylation in methanol-grown Paracoccus denitrificans

Methanol dehydrogenase of Paracoccus denitrificans was shown to be very similar to the enzyme of Pseudomonas sp, M. 27. The K _m value for methanol with excess activator (ammonium ions) is 35 M. The pH optimum for enzyme activity with 2,6-dichlorophe-nolindophenol as electronacceptor was at 9.0 A CO-binding type of cytochrome c was present only in cells grown with methanol as carbon and energy source.It has been shown that methanol-oxidation involves electron-transport via cytochrome c and an a-type cytochrome to oxygen. Antimycin A did not inhibit this electron transport and 90% inhibition was obtained by 375 M potassium cyanide. Electron transport from endogenous substrates is possible via cytochrome b and possibly cytochrome o to oxygen. Potassium cyanide inhibited 90% of the electron transport via this pathway at a concentration of 1.42 mM. Measurement of respiration-driven proton translocation proved that during oxidation of methanol to formaldehyde by oxygen one mole of adenosine triphosphate is synthesized in the site 3 region of the electron transport chain. The H⁺/O value found confirmed the H⁺/site ratio of 3–4 found in heterotrophic grown cells. During electron transport from endogenous substrates to oxygen there is a possible synthesis of 3 moles of adenosine triphosphate.In heterotrophically grown cells electron transfer to oxygen follows almost only the branch of the respiratory chain containing cytochrome o. In methanol-grown cells the pathway via the a-type cytochrome seems more important.Abbreviations DCPIP 2,6-dichlorophenolindophenol - PMS phenazine methosulphate - EPR electron paramagnetic resonance - S.D. standard deviation - ATP adenosine triphosphate     

The three-subunit cytochromebc 1 complex ofParacoccus denitrificans. Its physiological function,structure, and mechanism of electron transfer and energy transduction

The cytochromebc ₁ complex purified fromP. denitrificans has the same electron-transfer and energy-transducing activities, is sensitive to the same electron-transfer inhibitors, and contains cytochromesb, c ₁, iron-sulfur protein, and thermodynamically stable ubisemiquinone identical to the counterpart complexes from mitochondria. However, the bacterialbc ₁ complex consists of only three proteins, the obligate electron-transfer proteins, while the mitochondrial complexes contain six or more supernumerary poly-peptides, which have no obvious electron-transfer function. TheP. denitrificans complex is a paradigm for thebc ₁ complexes of all gram-negative bacteria. In addition, because of its simple polypeptide composition and apparently minimal damage during isolation, theP. denitrificans bc ₁ complex is an ideal system in which to study structure-function relationships requisite to energy transduction linked to electron transfer.     

Induction of crassulacean acid metabolism in Mesembryanthemum crystallinum increases reproductive success under conditions of drought and salinity stress

Summary Mesembryanthemum crystallinum L., an inducible crassulacean acid metabolism (CAM) plant, was grown for approximately 5 weeks following germination in well-watered, non-saline soil in a controlled-environment chamber. During this time, plants were characterized by C₃ photosynthetic carbon metabolism. After the initial 5 weeks, CAM was induced by a combination of high soil salinity and reduced soil water content. One group of plants was allowed to engage in CAM by being continuously exposed to normal CO₂-containing air (about 350–400 ppm). A second group of plants was deprived of ambient CO₂ each night (12 h dark period) until completion of their life cycle, thereby minimizing potential carbon gain via dark CO₂ fixation. The capacity to express CAM under conditions of drought and salinity stress markedly improved reproductive success: plants kept in normal CO₂-containing air produced about 10 times more seeds than plants kept in CO₂-free air during dark periods. Seeds from plants deprived of ambient CO₂ overnight had more negative ¹³C values than seeds from plants kept in normal air.     

Nonlinearly coupled double chain systems

We report here on numerical studies of a pair of linearly coupled mass chains with nonlinear ⁴ coupling between the two chains. General implications for double chain systems known to exist in nature such as DNA and RNA are discussed.     

Electron transport in purified glyoxysomal membranes from castor-bean endosperm

Glyoxysomes isolated from castor-bean (Ricinus communis L.) endosperm were treated with water, 0.2 M KCl, 1 M KCl, or 0.1 M Na₂CO₃. Glyoxysomal sacs, i.e. membranes which retained some visible matrix, resulted from the treatments with water and KCl. Glyoxysomal ghosts, i.e. intact membranes free of matrix, were only obtained following treatment with carbonate. The ghosts were free of activities of matrix enzymes, particularly palmitoyl-CoA oxidation, isocitrate dehydrogenase (EC 1.1.1.42) and isocitrate lyase (EC 4.1.3.1), and contained only negligible amounts of malate synthase (EC 4.1.3.2), malate dehydrogenase (EC 1.1.1.37), -hydroxyacyl-CoA dehydrogenase (EC 1.1.1.98) and catalase (EC 1.11.1.6). Distribution and appearance of membrane-associated particles in the protoplasmic and ectoplasmic faces of freeze-fracture replicas of the glyoxysomal membrane were the same in intact tissue, isolated glyoxysomes, and ghosts. Membranes purified by treatment with 0.2 M KCl or 0.1 M carbonate catalyzed the reduction of cytochrome-c when NADH or NADPH was provided as the electron donor. -Oxidation, localized in the matrix, could be linked to reduction of cytochrome-c or ferricyanide when purified membranes were combined with the matrix supernatant. Cytochrome-c could also be reduced by coupling enzyme activities in the matrix, NADP-isocitrate dehydrogenase or malate dehydrogenase, with those of the membrane. These results indicate that electrons from -oxidation, malate oxidation or isocitrate oxidation can be transferred directly to the redox components of the glyoxysomal membrane. We, therefore, conclude that any NADH and NADPH formed by enzymes in the matrix can be recycled continuously within the organelle.Abbreviations EF ectoplasmic face - ER endoplasmic reticulum - PF protoplasmic face     

Cyanamide mediated syntheses under plausible primitive earth conditions

Summary When an aqueous solution (pH 7.0) of deoxythymidine 5-phosphate, 4-amino-5-imidazolecarboxamide and cyanamide was dried and heated for 18 h at 60°C, P¹, P²-dideoxythymidine 5-pyrophosphate (I) was formed in a 58% yield. Oligonucleotides were not detected in the reaction product. Under conditions employed in the above reaction, (I) was shown to be stable. In prebiotic polymerization reactions employing deoxythymidine 5-triphosphate as the polymerizing species, (I) could therefore function as a primer and minimize the formation of cyclic nucleotides.Abbreviations dT deoxythymidine - dTMP deoxythymidine 5-phosphate - dTppT P¹, P²-dideoxythymidine 5-pyrophosphate - dTTP deoxythymidine 5-triphosphate - AICA 4-amino-5-imidazolecarboxamide     

Increased plasma α (1 → 3)-l-fucosyltransferase activities in patients with hepatocellular carcinoma

Alpha(1 3)-l-fucosyltransferase (1,3FT) activity was determined in plasma of patients with chronic liver diseases, namely, chronic hepatitis (CH), liver cirrhosis (LC) and hepatocellular carcinoma (HCC). The plasma 1,3FT activity was significantly higher (p<0.01) in chronic liver diseases than that in normal controls. The enzyme activity in plasma of patients with HCC was also significantly higher than that in LC (p<0.05) or that in CH (p<0.01). However, no significant difference was observed in the enzyme activity between LC and CH. Plasma 1,3FT activity in patients with HCC was not significantly changed before and after transcatheter arterial embolization. In addition, the enzyme activity in the homogenate of the cirrhotic liver tissue was higher than that in the preparation of the hepatoma tissue in the same patient. These results suggest that the increased plasma 1,3FT activity in patients with HCC reflects mainly the enzyme activity of cirrhotic liver tissue, not that of hepatoma tissue. The significance of the elevated levels of plasma 1,3FT and its decreased hepatoma tissue activity in patients with HCC, compared with that in LC, remains to be clarified.     

Kinetics of the flash-induced electrochromic absorbance change in the presence of background illumination. Turnover rate of the electron transport. II. Higher plant leaves

The flash-induced electrochromic absorbance change (A ₅₁₅) was measured in leaves of higher plants in the absence and presence of continuous monochromatic background illumination of different intensities and wavelengths. The variation of the amplitude of A ₅₁₅ in background light was used to estimate the steady-state turnover time of the electron transport. In red light we obtained about 5 msec which was accounted for by the turnover of the linear electron transport. With far red background illumination or in the presence of the photosystem 2 inhibitor, DCMU, the steady-state turnover time tentatively assigned to photosystem 1 cyclic electron transport was much larger (100 msec).Increasing the intensity of background illumination with far red light gradually diminished the slow rise of A ₅₁₅ in parallel with suppression of the initial rise generated by photosystem 1. At high intensities of the red light, however, while A ₅₁₅ was attenuated, the slow rise was not eliminated and its proportion relative to the initial rise did not vary appreciably.     

The loss of carbon-20 in C19-gibberellin biosynthesis in a cell-free system from Pisum sativum L.

The fate of the carbon-20 atom in gibberellin (GA) biosynthesis was studied in a cell-free system from Pisum sativum. This carbon atom is lost at the aldehyde stage of oxidation when C₂₀-GAs are converted to C₁₉-GAs. Gibberellin A₁₂ labeled with ¹⁴C at C-20 was prepared from [3-¹⁴C]mevalonic acid with a cell-free system from Cucurbita maxima and incubated with the pea system. Analysis of the gas and aqueous phases showed that ¹⁴CO₂ was formed at the same rate and in nearly equivalent amounts as ¹⁴C-labeled C₁₉-GAs whereas [¹⁴C]formic acid and [¹⁴C]formaldehyde were not detectable. The possibility that C-20 had been lost as formic acid which had then been converted to CO₂ was investigated by control incubations with [¹⁴C]formic acid. The rate of release of ¹⁴CO₂ from [¹⁴C]formic acid was only one fiftieth of the rate of ¹⁴CO₂ release from [¹⁴C]GA₁₂ as the substrate. We conclude that in the formation of C₁₉-GAs from C₂₀-GAs, the C-20 is removed directly as CO₂.Abbreviations GA_n Gibberellin A_n     

Secretion of a heat-stable fungalβ-glucanase from transgenic,suspension-cultured barley cells

Transgenic plant cell cultures have a potential for production and secretion of important proteins and peptides. To assess the possibilities of using a stable barley suspension culture for secretion of heterologous proteins in active form, we expressed the cDNA of the thermostable-glucanase (EGI) ofTrichoderma reesei in barley suspension cells. The cDNA coding for EGI and its signal sequence was placed under the control of the CaMV 35S promoter and the construction was transferred to the cells by particle bombardment. Stably transformed lines were obtained by selecting for a cotransformed antibiotic resistance marker. The expression of EGI cDNA led to accumulation of EGI in the culture medium, as shown by analysis with EGI-specific antibodies. Enzymatic assays confirmed that the EGI secreted by the suspension cells retained its activity and thermostable character. Furthermore, it was shown that the enzyme produced by the transgenic suspension culture could be used for degradation of soluble-glucans during mashing.     

Over-expression of the gene (bglBC1) from Bacillus circulans encoding an endo-β-(1→ 3),(1→ 4)-glucanase useful for the preparation of oligosaccharides from barley β-glucan

An endo--(13),(14)-glucanase gene (bglBC1) from Bacillus circulans ATCC21367 was modified by substituting its native promoter with a strong promoter, BJ27X, to increase expression of the gene when cloned into B. subtilis RM125 and B. megaterium ATCC14945. A 771-bp endo--(13),(14)-glucanase open reading frame was inserted into a new shuttle plasmid, pBLC771, by ligating the ORF and pBE1, the latter of which contained the strong promoter, BJ27X. B. subtilis, transformed with the recombinant plasmid pBLC771, produced an extracellular endo--(13),(14)-glucanase that was 130 times (7176 mU ml^–1) more active than that of the gene donor cells (55 mU ml^–1), while the enzyme from the transformed B. megaterium was 7 times (378 mU ml^–1) more active than that of the gene donor cells. M _r of the enzyme was 28 kDa, with proteolytic processing of the enzyme being observed only in B. subtilis cells. The major products of water-soluble -glucan hydrolyzed by over-produced endo--(13),(14)-glucanase were tri- and tetra-oligosaccharides which can be developed as useful products such as anti-hypercholesterolemic, anti-hypertriglyceridemic, and anti-hyperglycemic agents.     

Seven members of the (1→3)-β-glucanase gene family in barley (Hordeum vulgare) are clustered on the long arm of chromosome 3 (3HL)

Members of the (13)--glucan glucanohydrolase (EC 3.2.1.39) gene family have been mapped on the barley genome using three doubled haploid populations and seven wheat-barley addition lines. Specific probes or polymerase chain reaction (PCR) primers were generated for the seven barley (13)--glucanase genes for which cDNA or genomic clones are currently available. The seven genes are all located on the long arm of chromosome 3 (3HL), and genes encoding isoenzymes GI, GII, GIII, GIV, GV and GVII (ABG2) are clustered in a region less than 20 cM in length. The region is flanked by the RFLP marker MWG2099 on the proximal side and the Barley Yellow Mosaic Virus (BYMV) resistance gene ym4 at the distal end. The gene encoding isoenzyme GVI lies approximately 50 cM outside this cluster, towards the centromere. With the exception of the gene encoding isoenzyme GIV, all of the (13)--glucanase genes are represented by single copies on the barley genome. The probe for the isoenzyme GIV gene hybridized with four DNA bands during Southern blot analysis, only one of which could be incorporated into the consensus linkage map.     

Combination of induced autolysis and sodium hypochlorite oxidation for the production of Saccharomyces cerevisiae (1 → 3)-β-D-glucan

(1 3)--D-Glucans have received much attention with respect to their biological functions. A novel method to extract (1 3)--D-glucan from Saccharomyces cerevisiae cell wall is proposed in present work, which is based on the combination of induced autolysis and subsequent oxidation of the autolysed cell by sodium hypochlorite to remove undesirable substances. Influences of temperature, pH value and organic solvent on S. cerevisiae FL 1 autolysis were investigated. Results indicated that each factor had its significant effect on induced autolysis and the optimal conditions were 52 °C, pH 5.5 and 1.5% (v/v) ethyl acetate. The kinetic behaviour of the yeast autolytic process under the optimized conditions was further studied. After 36 h of autolysis, 42.0% (w/w) cellular substances were released while the cell wall nearly remained intact. Finally, an ideal glucan yield as high as 22.9% (w/w) was obtained when S. cerevisiae FL 1 was treated by the novel method.     

Secretion of vascular coating components by xylem parenchyma cells of tomatoes infected withVerticillium albo-atrum

Summary Massive infusion of conidia ofVerticillium albo-atrum into the xylem of tomato induces a cell wall coating response in resistant and susceptible near-isolines. In the early stages two types of coating material develop in the xylem vessels. The first, designated type A, is formed in association with xylem parenchyma cells that lack secondary walls; the localized accumulation of type A coating in the in the adjacent intercellular spaces, primary walls (i.e., pit membranes) and vessels occurs in conjunction with localized development of apposition wall layers within the parenchyma cells. Type B coating is initially formed in association with xylem parenchyma cells with secondary walls; the localized accumulation of typeB coating in the adjacent intercellular spaces, primary walls (i.e., pit membranes) and vessels occurs in conjunction with development of protective layers within the parenchyma cells. Most vessels are surrounded by a number of parenchyma cells including both cell types; therefore, in most vessels the coatings are mixed in later stages of development (i.e.,> 48 hours). The formation of both types of coating is stopped by the application of L--aminooxy--phenylpropionate, a specific inhibitor of phenylpropanoid synthesis. Histochemically, type A coating resembles lignin and type B, suberin. The data suggest that the coating response is due, wholly or in part to hypersecretion and/or chemical modification of normal cell wall components, induced by the pathogen.     

The elimination ofO-linked glycans from glycoproteins under non-reducing conditions

A simple procedure is described for the elimination ofO-linked glycans from bovine submaxillary mucin under non-reducing conditions, using triethylamine in aqueous hydrazine. The glycans were isolated as the hydrazones, which were converted to the reducing glycans by exchange with acetone in neutral aqueous solution. The glycan alditols obtained after reduction corresponded to those obtained by the reductive -elimination ofO-glycans.     

Molecular cloning and nucleotide sequence of the mutT mutator of Escherichia coli that causes A:T to C:G transversion

Summary The Escherichia coli mutator gene mutT, which causes A:TC:G transversion, was cloned in pBR 322. mutT ⁺ plasmids carry a 0.9 kb PvuII DNA fragment derived from the E. coli chromosome. Specific labelling of plasmid-encoded proteins by the maxicell method revealed that mutT codes for a polypeptide of about 15,000 daltons. The protein was overproduced when the mutT gene was placed under the control of the lac regulatory region on a multicopy runaway plasmid. The nucleotide sequence of the mutT gene was determined by the dideoxy method.Abbreviations Ap ampicillin - IPTG isopropyl--d-thiogalactopyranoside - kb kilobase pair(s) - kDa kilodalton(s) - SDS sodium dodecyl sulphate - Tc tetracycline     

Design of specific antagonists of the fibrinogen receptor

The aim of this work was to elucidate, usingmolecular modeling, structural and electrostaticcommon parameters of several selective fibrinogenreceptor antagonists. From this theoretical pattern,we are currently designing and synthesizing originalnon-peptidic and selective carbohydrate-basedantagonists of the fibrinogen receptor.     

Layered structure of trichoderma cell walls

Trichoderma pseudokoningii mycelium was treated sequentially with the following enzymes; (1) lysing enzyme and laminarinase, (2) pronase, and (3) chitinase. (1) Removed the outermost amorphous glucan layer and revealed a striated layer which was removed by (2). Microfibrils were exposed and removed by (3). The wall thus has a layered structure comprising glucan, protein and/or glycoprotein, and chitin.Glucan was prepared and subjected to methylation and hydrolysis. 1,3,5,6-Tetra-O-acetyl-2,4-di-O-methyl-D-glucose was identified suggesting that the 1–3 and 1–6 glucans are cross-linked at intervals.