Proton n.m.r. of ferredoxin from Clostridium pasteurianum

Proton magnetic resonance spectra at 500 MHz are reported for the oxidized and reduced forms of the 2[4Fe-4S]-ferredoxin from Clostridium pasteurianum. The reduced protein showed additional peaks in the 10–60 ppm region, which were previously unobserved, and there were significant differences between oxidized and reduced states in the whole region. The electron exchange rate in partially reduced ferredoxin is slow on the n.m.r. time scale when reduced with sodium dithionite, but fast when zinc reduced methyl viologen is used as reducing agent. We explain the difference between fast and slow exchange as being due to the different chemical properties of the two reducing agents.     

The role of slow potassium current in phenomena of voltage-dependent action of 4-aminopyridine in amphibian myelinated nerve fibres

The mechanism underlying the voltage-dependent action of 4-aminopyridine (4-AP) is investigated in experiments on amphibian myelinated nerve fibres (Rana ridibunda Pallas) by way of extracellular recording of electrical activity and using activators of potassium current (potassium-free solution and nitric oxide NO) and inhibitors of sodium current (tetrodotoxin). Measurement of action potential (AP) areas was used to evaluate the extent of general membrane depolarization during the activity of nerve fibres. Tetrodotoxin-induced decrease in general membrane depolarization (when the action potential amplitude was reduced by less than 20%) leads to an increase in the duration of depolarizing after-potential (DAP). This supports the dependence of time course of DAP in the presence of 4-AP on ratio of fast and slow potassium channels. In the absence of 4-AP, potassium-free solution and NO increase the potassium current through fast potassium channels (decreasing AP duration, reducing DAP and sometimes producing fast hyperpolarizing after-potential (HAP) after shortened AP), and in the presence of 4-AP these activators increase potassium current through unblocked slow potassium channels (making the development of slow HAP induced by 4-AP more rapid). The increase of slow HAP induced by 4-AP under the influence of potassium-free solution with NO supports the idea that slow HAP is due to activation of slow potassium channels and argues against the notion of removal of block of fast potassium channels. All analyzed phenomena of voltage-dependent action of 4-AP in amphibian myelinated nerve fibers can be accounted for by the activation of slow potassium current produced by membrane depolarization and a decrease of the amount of fast potassium channels involved in the membrane repolarization.     

Esterases of laboratory-reared and field-collected cotton boll weevils,Anthonomus grandis Boh.: Polymorphism of adult esterases and formal genetics of esterase II

The esterases of the cotton boll weevil were separated by polyacrylamide gel electrophoresis into four major regions. These were named Est I–IV in order of migration from anode to origin. Polymorphism was observed in all regions. The Est II region was shown to consist of no more than two bands (fast and slow). The inheritance of the fast and slow bands of Est II was demonstrated to be controlled by codominant autosomal alleles. Analysis of the gene frequency of the Est II region showed that one field population was consistent with the Hardy-Weinberg law (P=0.995), while a second field population was not at equilibrium (P<0.001).This work was supported in part by a Faculty Research Grant from Memphis State University.     

Internal pH of human neutrophil lysosomes

We focus this report on the relationship between signal II fast and slow during a flash sequence for Tris-washed chloroplasts at different pH-values. The pH influences both the redox state and spectral form of signal II slow in dark-adapted chloroplasts. At pH 6.0, signal II slow is oxidized and does not influence the kinetics of signal II fast equally formed on each flash. At pH 8.5, signal II slow in mainly reduced in the dark and the first flash produces signal II slow and no signal fast. Signal II fast appears on the following flashes only. Signals II fast and slow are connected to the same center and signal II fast is observed only if signal II slow is oxidized.     

Study of CO2 exchange processes,resistances to carbon dioxide and chlorophyll content during leaf ontogenesis in poplar

Net photosynthetic (P _N) and dark respiration (R _D) rate, stomatal (rs′) and internal (ri′) resistances to carbon dioxide were measured by gas exchange methods on leaves of different ages, expressed in leaf plastochron index units (LPI) for a fast growing poplar cultivar Unal 2. Although the optimal leaf age differs slightly for the different gas exchange parameters, leaf ontogeny is reflected in the same way in these different parameters. MaximalP _N and minimalrs′ and ri′ values were found at LPI between 6 and 10. Chlorophyll concentrations were lowest at LPI lower than 10 although an increase in two steps was found, when leaf age increases up to maturity.     

NMR redox studies of Desulfovibrio vulgaris Cytochrome c3. Electron transfer mechanisms

The 300-MHz proton NMR spectra of the tetrahaem cytochrome c3 from Desulfovibrio vulgaris were examined while varying the pH and the redox potential. The analysis of the complete NMR reoxidation pattern was done taking into account all the 16 redox states that can be present in the redox titration of a tetra-redox-center molecule. A network of saturation transfer experiments performed at different oxidation stages, between the fully reduced and the fully oxidized states, allowed the observation of different resonances for some of the haem methyl groups. In the present experimental conditions, some of the haems show a fast intramolecular electron exchange rate, but the intermolecular electron exchange is always slow. In intermediate reoxidation stages, large shifts of the resonances of some haem methyl groups were observed upon changing the pH. These shifts are discussed in terms of a pH dependence of the haem midpoint redox potentials. The physiological relevance of this pH dependence is discussed.     

Anaerobic reaction of ascorbate oxidase with ascorbate

Ascorbate oxidase is fully reduced by 4 mol of ascorbate in the absence of air, as monitored by optical and electron paramagnetic resonance spectra. At less than stoichiometric ascorbate concentration there is a slow equilibration between the 605-and 330-nm absorption bands: The 605-nm chromophore is first reduced, then its color reappears while the 330-nm absorption band decreases. Upon reoxidation with air the process takes place in the opposite direction. Intramolecular rather than intermolecular electron exchange appears to be responsible for this process. The reduced protein is about twice as fluorescent as the oxidized protein. The fluorescence quenching in the oxidized protein is related to the 330-nm absorption band rather than to the 605-nm band as previously reported for laccase.     

Doubling the catabolic reducing power (NADH) output of Escherichia coli fermentation for production of reduced products

Homofermentative production of reduced products requires additional reducing power output (NADH) from glucose catabolism. Anaerobic expression of the pyruvate dehydrogenase complex (PDH, encoded by aceEF‐lpd, a normal aerobic operon) is able to provide the additional NADH required for production of reduced products in Escherichia coli fermentation. The multiple promoters (pflBp(1–7)) of pyruvate formate lyase (pflB) were evaluated for anaerobic expression of the aceEF‐lpd operon. Four chromosomal constructs, pflBp(1–7)‐aceEF‐lpd, pflBp(1–6)‐aceEF‐lpd, pflBp(6,7)‐aceEF‐lpd, and pflBp6‐aceEF‐lpd efficiently expressed the PDH complex in anaerobically grown cells. Doubling the reducing power output was achieved when glucose was oxidized to acetyl‐CoA through glycolysis and pyruvate oxidation by the anaerobically expressed PDH complex (glucose →2 acetyl‐CoA + 4 NADH). This additional reducing power output can be used for production of reduced products in anaerobic E. coli fermentation. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

1H, 13C, and 15N assignment of the oxidized and reduced forms of T. brucei glutathione peroxidase-type tryparedoxin peroxidase

The cysteine-homologues of glutathione peroxidases in Trypanosoma brucei catalyze the trypanothione/tryparedoxin-dependent reduction of hydroperoxides. We report the ¹H, ¹³C, and ¹⁵N assignment of the oxidized and reduced form of the enzyme by NMR. Major changes between these two forms were only observed for residues close to the catalytic site.     

Sulfate reduction in Catharanthus roseus (L.)

Cell homogenates from Catharanthus roseus (L.) G. Don. grown S-autotrophically on sulfate in the dark are capable of reducing adenylysulfate (APS) to cysteine. This reduction required a particulate protein fraction from the cell extract and reduced ferredoxin as the electron donor. The protein fraction (MW 700,000±50,000) was found to contain Fd:NADP⁺ reductase, glutathione reductase and an unspecific dithiol reductase, and APS-sulfotransferase and thiosulfonate reductase activity. Resolution into these individual enzyme activities led to a non-restorable loss of the APS reducing activity. It was observed that a slow gradual decay of the APS reducing activity was accompanied by a likewise slow generation of a ferredoxin-dependent sulfite reductase.Enzymes and abbreviations APS Adenosine 5-phosphosulfate - APS-kinase E.C.2.7.1.25 - ATP-sulfurylase E.C.2.7.7.4 - Fd ferredoxin - Fd-NADP⁺-reductase E.C.1.6.7.1. - Glutathione reductase E.C.1.6.4.2. - G6P Glucose 6-phosphate - G6PDH glucose 6-phosphate dehydrogenase, E.C.1.1.49 - GSSG oxidized glutathione - GSSO₃H S-sulfoglutathione - MVH reduced methylviologen - OASS O-acetylserine sulfhydrylase-E.C. 4.2.99.8 - Sulfite reductase E.C.1.8.1.2     

Phosphorus Sequestration in Lake Sediment with Iron Mine Tailings

Internal phosphorus loading can lead to eutrophication in lakes when anoxic sediments release bioavailable phosphorus into the water column. In laboratory experiments, iron mine tailings helped to sequester phosphorus in sediment from a eutrophic lake. Phosphorus release from the sediments after extraction with distilled water or 0.02 N H ₂ SO ₄ was significantly reduced when mine tailings were added (1:1 w/w), even when the system was anaerobic (～ 1 mg O ₂ /L). The degree of sequestration was enhanced when glucose (1% w/w) was added to stimulate the growth of microorganisms, suggesting that the process was microbially mediated. We suggest that oxidized iron in the mine tailings served as an electron sink for microbial respiration via dissimilatory Fe³⁺ reduction. The reduced iron released into solution sequestered phosphorus, either as it re-oxidized and formed hydrous ferric oxide complexes containing phosphorus (HFO-P), or through precipitation. Since mine tailings are inexpensive, they may prove useful for preventing phosphorus from entering surface waters, as well as reducing internal phosphorus loading.     

Reaction centers fromRhodopseudomonas sphaeroides in reconstituted phospholipid vesicles. II. Light-induced proton translocation

Unidirectional light-dependent proton translocation was demonstrated in a suspension of reconstituted reaction center (RC) vesicles supplemented with cytochromec and 2,3-dimethoxy-5-methyl-1,4-benzoquinone (UQ₀), a lipid-and water-soluble quinone. Proton translocation was detected only at alkaline pH. The pH dependence can be accounted for by the slow redox reaction between the reduced quinone (UQ₀H₂) and oxidized cytochromec. This conclusion is based on (i) the pH dependence of partial reactions of the reconstituted proton translocation cycle, measured either optically or electrometrically and (ii) titration studies with cytochromec and UQ₀. At 250 and 25 µM UQ₀ and cytochromec, respectively, maximal proton translocation was observed at pH 9.6. This pH optimum can be extended to a more acidic pH by increasing the concentration of the soluble redox mediators in the reconstituted cyclic electron transfer chain. At the alkaline side of the pH optimum, proton translocation appears to be limited by electron transfer from the endogenous primary to the secondary quinone within the RCs. The light intensity limits the reconstituted proton pump at the optimal pH. The results are discussed in the context of a reaction scheme for the cyclic redox reactions and the associated proton translocation events.Abbreviations RC reaction center - UQ₀/UQ₀H₂ oxidized and reduced form of 2,3-dimethoxy-5-methyl-1,4-benzoquinone - D/D⁺ reduced and oxidized form of the primary electron donor of the RCs - CCCP carbonylcyanide-trichloromethoxy phenylhydrazone - UQ_A/UQ _A ^– oxidized and semiquinone form of the primary electron acceptor of the RCs - UQ_B/UQ _B ^– /UQ_BH₂ oxidized, semiquinone, and reduced form of the secondary electron acceptor of the RCs - LDAO lauryldimethylamine-N-oxide During the course of this study K.J.H. was supported by a grant from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.). This research was supported by grants from the National Institutes of Health (EY-02084) and from the Office of Naval Research (ONR-NOOO 14-79-C 0798) to M. Montal.     

Anti-u.v. activity of lignin biopolymers on Euglena gracilis

Sulphur-free lignin biopolymer and its oxidized and reduced derivatives have been prepared and their inhibitory activity against u.v.-induced mutagenesis in Euglena gracilis was evaluated. The structure- and dose-dependent anti-u.v. activity of lignins was observed at concentrations higher than 250gml^–1. The oxidized lignin showed the most antimutagenic activity, followed by the reduced lignin and the unmodified lignin had the least antimutagenic activity.     

The inheritance of a urease-null trait in soybeans

Summary Four soybean seed urease nulls (lacking both the activity and antigen of the embryo-specific urease) were intermated and the F₁ and F₂ seed examined for urease activity. Both generations were without urease activity, and the nulls were therefore considered noncomplementing. In crosses of each null line to cultivars homozygous for the allelic, codominantly inherited urease slow or fast isozyme, the F₁ seed expressed the embryo-specific urease isozyme of the urease-expressing parent. A 3 1 segregation for presence and absence of urease was observed in progeny from F₁ and heterozygous F₂ plants. The F₂ and F₃ from fastXnull combinations revealed that urease-positive seed were all phenotypically urease fast, while the same seed from slowXnull combinations showed a segregation of one seed containing a fast urease, either exclusively or in a heterozygous state with the slow isozyme, for every 69 phenotypic slows. Data pooled from F₂ plants which segregate for both the presence (Sun) and absence (Sun) of urease and for the fast (Eu1-b) or slow (Eu1-a) urease allele indicate that the null lesion (Sun) is linked to Eu1 by approximately one map unit. The evidence is consistent with two models: (1) sun is an allele at the embryo-specific urease isozyme locus (Eu1) and that a high degree of exchange (and/or conversion) within the locus results in a 1% recombination frequency between the null trait and urease allozyme; (2) sun is at a distinct locus which is separated by one map unit from the embryo-specific urease isozyme locus (Eu1) upon which it acts in the cis position. Polyadenylated embryo RNA from one of the null lines, PI 229324, exhibited no urease template activity in vitro. Thus, the lack of urease antigen is due to lack of accumulation of translatable urease mRNA. The availability of soybeans lacking seed urease should be extremely useful to breeders as a trait for linkage studies and to geneticists as a transformation marker.Portions of this work were funded by the Illinois and Missouri Agricultural Experiment Stations, the SOHIO-University of Illinois Center of Excellence in Crop Molecular Genetics and Genetic Engineering and by grants PCM-8219652 from the National Science Foundation and USDA/SEA-CRCR-1-1374 from the USDA Competitive Grants Office     

Studies on fast and slow growing Rhizobium spp. nodulating Cqjanus cajan and Cicer arietinum

Fast and slow growing Rhizobium spp. isolated from Cajanus cajan and Cicer arietinum were compared in terms of colony characteristics, utilisation of carbon sources, acid production, symbiotic effectiveness and nodulating competitiveness. Fast growing isolates from C. cajan and C. arietinum formed 3–6 mm diameter colonies on yeast-extract mannitol agar after 4 days and were unlike the slow growers which produced colonies of c. 1 mm diameter after 7–10 days at 28 °C. The fast growing Rhizobium spp. from C. cajan utilised a wider range of carbon sources than the slow growing isolates from this host. Fast and slow growing strains from C. arietinum were able to utilise most of the carbon sources tested suggesting that the slow growers possessed glycolytic pathways similar to those in other fast growing species of Rhizobium. In culture, slow growing isolates from C. cajan produced a near-neutral to alkaline reaction (pH 66·7-5) whereas the fast growers from this host and both fast and slow growing isolates from C. arietinum produced an acidic reaction (pH 4·4–5·6). These data are discussed in the context of Norris' (1965) evolutionary concept of the Leguminosae. Under glassshouse conditions, fast and slow growing strains isolated from C. cajan and C. arietinum were equally effective on their respective hosts. In competition with slow growing rhizobia, half of the fast growers formed more than 70% of the nodules on C. cajan grown in sand. In all but one instance similar results were obtained when plants were grown in soil. With C. arietinum grown in sand or soil, all fast growing isolates from this host formed more than 85% of the nodules in competition with slow growing strains.     

Evolutionary genetics of Metridium senile. I. Kinetic differences in phosphoglucose isomerase allozymes

Populations of the sea anemone Metridium senile from the northeast coast of the United States exhibit a one-locus, two-allele polymorphism for phosphoglucose isomerase. No additional hidden variation is exposed by changes in pH, gel pore size, or heat denaturation. The allozymes are similar in pH optimum, sensitivity of K _m to pH, and sensitivity of K _m and V _max to temperature. In other respects they are functionally different, with the fast allozyme having a 3.5-fold higher specific activity and a slightly higher K _m of fructose-6-phosphate than the slow form. In these respects, heterozygotes produce a mixture of enzymes that appears to function roughly as the sum of its component parts. Comparisons of V _max/K _m ratios reveal significant differences among genotypes, with the fast form having higher values at all temperatures than the slow form and heterozygotes falling intermediate. In addition, there is a significant difference among genotypes in sensitivity of this parameter to temperature, with the fast homozygote and heterozygote displaying greater sensitivity than the slow homozygote. Temperature is probably an important selective agent in maintaining this polymorphism.Supported by Grant T-4 from the Health Research and Services Foundation, NSF DEB77-14442, NIH GM25809, and NIH GM28024.     

Digestion of insulin derivatives with subtilisin: a kinetic study.

Native, denatured, performic acid-oxidized or S-sulfo insulin and S-sulfo or performic acid-oxidized A- and B-chains were digested with subtilisin type Carsberg. The proteolysis was followed by measuring the uptake of alkali through autotitration. The kinetic study shows the existence of 2 first-order reaction classes which differ markedly in rate constant. The number of bonds split with fast and with slow reactions has been calculated. Only one of a total of 12 cleavable bonds in native insulin is opened by fast reaction. In the denatured protein the number of bonds split by the fast reaction increases to 4 and in the oxidized and S-sulfo protein 3 bonds are cleaved, while the slow cleavable bonds number 2 and 7, respectively, The kinetic study of the proteolysis of S-sulfo A-chain and of oxidized or S-sulfo B-chain shows that two bonds are split in A-chain with the fast and slow reactions, while in B-chain only one of the six cleavable bonds is susceptible to fast attack.     

Genetic regulation of liver alcohol dehydrogenase in Peromyscus

Data from genetic crosses of Peromyscus maniculatus and P. polionotus suggest that electrophoretic variants of liver alcohol dehydrogenase are coded by alleles at a single locus. These alleles, designated Adh ^F , Adh ^S , and Adh ^N , determine, respectively, the fast, slow, and not detectable (null) ADH electrophoretic phenotypes. Heterozygotes (Adh ^F /Adh ^S ) exhibit three bands on zymograms, suggesting a dimeric subunit structure for the enzyme. However, Adh ^F /Adh ^N and Adh ^S /Adh ^N animals exhibit a single band, suggesting that the Adh ^N allele does not produce a polypeptide subunit capable of dimerizing into an active molecule. Fast and slow electrophoretic phenotypes exhibit multiple bands which can be converted into single major fast and slow bands, respectively, upon treatment with oxidized or reduced NAD. Addition of NAD also stabilizes both the fast and slow enzyme to heat inactivation at 60 C for at least 30 min.This work was supported by Predoctoral Fellowship AA-05067 from the National Institute of Alcohol Abuse and Alcoholism to K. G. B. and South Carolina Commission on Alcohol and Drug Abuse Grant 7607. Also, partial support was provided by NIH Grant CA-16184.     

Reaction center and UQH2:cytc 2 oxidoreductase act as independent enzymes inRps. sphaeroides

Turnover of the ubiquinol oxidizing site of the UQH₂:cyt c₂ oxidoreductase (b/c ₁ complex) ofRps. sphaeroides can be assayed by measuring the rate of reduction of cytb ₅₆₁ in the presence of antimycin (AA). Oxidation of ubiquinol is a second-order process, with a value ofk ₂ of about 3 × 10⁵ M^–1. The reaction shows saturation at high quinol concentrations, with an apparentK _m of about 6–8 mM (with respect to the concentration of quinol in the membrane). When the quinone pool is oxidized before illumination, reduction of the complex shows a substantial lag (about 1 ms) after a flash, indicating that the quinol produced as a result of the photochemical reactions is not immediately available to the complex. We have suggested that the lag may be due to several factors, including the leaving time of the quinol from the reaction center, the diffusion time to the complex, and the time for the head group to cross the membrane. We have suggested aminimal value for the diffusion coefficient of ubiquinone in the membrane (assuming that the lag is due entirely to diffusion) of about 10^–9 cm^–2 sec^–1. The lag is reduced to about 100 µsec when the pool is significantly reduced, showing that quinol from the pool is more rapidly available to the complex than that from the reaction center. With the pool oxidized, similar kinetics are seen when the reduction of cytb ₅₆₁ occurs through the AA-sensitive site (with reactions at the quinol oxidizing site blocked by myxothiazol). These results show that there is no preferential reaction pathway for transfer of reducing equivalents from reaction center tob/c ₁ complex. Oxidation of cytb ₅₆₁ through the AA-sensitive site can be assayed from the slow phase of the carotenoid electrochromic change, and by comparison with the kinetics of cytb ₅₆₁. As long as the quinone pool is significantly oxidized, the reaction is not rate-determining for the electrogenic process. On reduction of the pool below 1 quinone per complex, a slowing of the electrogenic process occurs, which could reflect a dependence on the concentration of quinone. If the process is second-order, the rate constant must be about 2–5 times greater than that for quinol oxidation, since the effect on rate is relatively small compared with the effect seen at the quinol oxidizing site when the quinol concentration is changed over theE _h range where the first few quinols are produced on reductive titration. When the quinone pool is extracted (experiments in collaboration with G. Venturoli and B. A. Melandri), the slowing of the electrochromic change on reduction of the pool is not enhanced; we assume that this is due to the fact that a minimum of one quinone per active complex is produced by turnover of the quinol oxidizing site. Two lines of research lead us to revise our previous estimate for the minimal value of the quinone diffusion coefficient. These relate to the relation between the diffusion coefficient and the rate constants for processes involving the quinones: (a) The estimated rate constant for reaction of quinone at the AA-site approaches the calculated diffusion limited rate constant, implying an improbably efficient reaction. (b) From a preliminary set of experiments, the activation energy determined by measuring the variation of the rate constant for quinol oxidation with temperature, is about 8 kcal mol^–1. Although we do not know the contribution of entropic terms to the pre-exponential factor, the result is consistent with a considerably larger value for the diffusion coefficient than that previously suggested.