Defective excision of pyrimidine dimers and interstrand DNA crosslinks in rad7 and rad23 mutants of Saccharomyces cerevisiae

Summary Excision of pyrimidine dimers and interstrand DNA crosslinks was examined in the deletion mutants rad7-1, rad23-1, and rad7-1 rad23-1. These mutants remove pyrimidine dimers and crosslinks much less efficiently than the RAD ⁺ strains; only 30–60% of pyrimidine dimers and 25–40% of crosslinks are removed even after prolonged incubation. The rad7 and rad23 mutations may represent defects in protein factors which increase the efficiency of the nicking enzyme complex or make chromatin more accessible to the nicking activity.     

Engineering of carbon catabolite repression in recombinant xylose fermenting<Emphasis Type="Italic"> Saccharomyces cerevisiae</Emphasis>

Two xylose-fermenting glucose-derepressed Saccharomyces cerevisiae strains were constructed in order to investigate the influence of carbon catabolite repression on xylose metabolism. S. cerevisiae CPB.CR2 (mig1, XYL1, XYL2, XKS1) and CPB.MBH2 (mig1, mig2, XYL1, XYL2, XKS1) were analysed for changes in xylose consumption rate and ethanol production rate during anaerobic batch and chemostat cultivations on a mixture of 20 g l^–1 glucose and 50 g l^–1 xylose, and their characteristics were compared to the parental strain S. cerevisiae TMB3001 (XYL1, XYL2, XKS1). Improvement of xylose utilisation was limited during batch cultivations for the constructed strains compared to the parental strain. However, a 25% and 12% increased xylose consumption rate during chemostat cultivation was achieved for CPB.CR2 and CPB.MBH2, respectively. Furthermore, during chemostat cultivations of CPB.CR2, where the cells are assumed to grow under non-repressive conditions as they sense almost no glucose, invertase activity was lower during growth on xylose and glucose than on glucose only. The 3-fold reduction in invertase activity could only be attributed to the presence of xylose, suggesting that xylose is a repressive sugar for S. cerevisiae.     

Anaerobic biosynthesis of unsaturated fatty acids in the cyanobacterium,Oscillatoria limnetica

The mechanism for synthesis of monounsaturated fatty acids under aerobic and anaerobic conditions was studied in the facultative anaerobic cyanobacterium, Oscillatoria limnetica. The hexadecenoic acid (C161) of aerobically grown O. limnetica was shown to contain both the 7 (79%) and 9 (21%) isomers, while the octadecenoic (C181) acid was entirely the 9 acid. Incorporation of [2-¹⁴C] acetate into the fatty acids under aerobic conditions resulted in synthesis of the 7 and 9 C161 and the 9 C181. Synthesis of unsaturated fatty acids in the presence of DCMU required sulfide. Anaerobic incubations in the presence of DCMU and sulfide (less than 0.003% atmospheric oxygen) resulted in a two-fold increase in monounsaturated fatty acids of both 7 and 9 C161 and 9 and 11 C181. The synthesis of these isomers is characteristic of a bacterialtype, anaerobic pathway.Abbreviations DCMU 3(3,4-dichlorophenyl)-1,1-dimethylurea - MFA monounsaturated fatty acid     

A suitable method for construction and cloning hybrid plasmids containing EcoRI-fragments of E. coli genome.

Summary A convenient procedure for the isolation of specificEcoRI-fragments ofE. coli genome and their amplification on Km-resistance plasmid vector CK 11 is described. The hybrid molecules were constructedin vitro usingEcoRI-digestion, followed by ligation. Then appropriatedE. coli strain was transformed with ligated DNA mixture and hybrid plasmids CK 11-arg ⁺, CK 11-his ⁺, CK 11-thr ⁺ and CK 11-leu ⁺ containing loci ofE. coli genome were selected by molecular cloning. The hybrid plasmids obtained consisted of oneEcoRI-fragment of initial plasmid CK 11 and one respective specific portion ofE. coli genome.     

Oxygen-dependent xylitol metabolism in Pichia stipitis

Pichia stipitis CBS 6054 was cultivated in chemostat cultures under aerobic and oxygen-limited conditions with xylitol alone, a mixture of xylitol and glucose and a mixture of xylitol and xylose. Xylitol metabolism was strictly respiratory and no ethanol was formed. Simultaneous feeding of xylitol and glucose and xylitol and xylose to oxygen-limited xylitol-pregrown cells resulted in ethanol formation. In vitro both pyruvate decarboxylase activity and alcohol dehydrogenase activity were present in cells metabolising xylitol under oxygen-limited conditions; however, this did not result in ethanol formation. Glucose, xylose and xylitol utilisation, respectively, were compared under anaerobic conditions with regard to growth rate, carbon source and oxygenation level during pre-cultivation. Irrespective of pre-growth conditions, xylitol was not metabolised under anaerobic conditions, whereas ethanol was formed from both xylose and glucose. Anaerobic xylose utilisation required induction of a xylose-utilising metabolic pathway during pre-cultivation. Received: 23 February 1999 / Received last revision: 20 July 1999 / Accepted: 1 August 1999     

Genetic analysis of carbon isotope discrimination and agronomic characters in a bread wheat cross

Carbon isotope discrimination () has been suggested as a selection criterion to improve transpiration efficiency (W) in bread wheat (Triticum aestivum L.). Cultivars Chinese Spring with low A (high W) and Yecora Rojo with high (low W) were crossed to develop F₁, F₂, BC₁, and BC₂ populations for genetic analysis of and other agronomic characters under well-watered (wet) and water-stressed (dry) field conditions. Significant variation was observed among the generations for only under the wet environment. Generation x irrigation interactions were not significant for . Generation means analysis indicated that additive gene action is of primary importance in the expression of under nonstress conditions. Dominance gene action was also detected for , and the direction of dominance was toward higher values of . The broad-sense and the narrow-sense heritabilities for were 61 % and 57% under the wet conditions, but were 48% and 12% under the draughted conditions, respectively. The narrow-sense heritabilities for grain yield, above-ground dry matter, and harvest index were 36%, 39%, and 60% under the wet conditions and 21%, 44%, and 20% under dry conditions, respectively. The significant additive genetic variation and moderate estimate of the narrow-sense heritability observed for indicated that selection under wet environments should be effective in changing in spring bread wheat.     

Positional specificity in the incorporation of isomeric cis- and trans-octadecenoic acids into glycerolipids of cultured soya cells

Heterotrophically grown cell suspension cultures of soya (Glycine max L.) were incubated with two different mixed substrates consisting of positional isomers of either cis-[1-¹⁴C]octadecenoic acids (8 to 15) or trans-[1-¹⁴C]octadecenoic acids (8 to 16), each with known composition. With both substrates, about one-fourth of the radioactivity supplied was incorporated into the diacylglycerophosphocholines, while another one-fourth of the radioactivity was almost equally distributed between diacylglycerophos-phoethanolamines and triacylglycerols. All the positional isomers of cis-and trans-octadecenoic acids supplied to the cells were readily incorporated into various classes of glycerolipids. None of the octadecenoic acids was isomerized, elongated or desaturated during incubation. From the cis-octadecenoic acids, only the naturally occurring 9-isomer (oleic acid) was preferentially incorporated into position 2 of diacylglycerophosphocholines, diacylglycerophospho-ethanolamines, and triacyglycerols; all the other isomers exhibited a strong affinity for position 1 of the glycerophospholipids and positions 1 and 3 of the triacylglycerols. From the trans-octadecenoic acids, only the 9-isomer (elaidic acid) was preferentially incorporated into position 2 of diacylglycerophospho-cholines and triacylglycerols; all the other isomers preferred position 1 and positions 1 and 3, respectively, of these lipids. In diacylglycerophospho-ethanolamines, however, each of the trans-octadecenoic acids, including the 9-isomer, exhibited a strong affinity for position 1. Apparently, the enzymes involved in the incorporation of exogenous monounsaturated fatty acids into membrane lipids of plant cells can recognize the preferred substrate in a mixture of closely related isomers.     

Desaturation of oxygenated fatty acids in Lesquerella and other oil seeds

Species of the genus Lesquerella, within the Brassicaceae family, have seed oils containing hydroxy fatty acids. In most Lesquerella species, either lesquerolic (14-hydroxy-eicosa-11-enoic), auricolic (14-hydroxy-eicosa-11,17-dienoic) or densipolic (12-hydroxy-octadeca-9,15-dienoic) acid dominates in the seed oils. Incubations of developing seed from Lesquerella species with 1-¹⁴C-fatty acids were conducted in order to study the biosynthetic pathways of these hydroxylated fatty acids. [¹⁴C]Oleic (octadeca-9-enoic) acid, but not [¹⁴C]linoleic (octadeca-9,12-dienoic) acid, was converted into the hydroxy fatty acid, ricinoleic (12-hydroxy-octadeca-9-enoic) acid, which was rapidly desaturated to densipolic (12-hydroxy-octadeca-9,15-dienoic) acid. In addition, [¹⁴C] ricinoleic acid added to Lesquerella seeds was efficiently desaturated at the 15 carbon. A pathway for the biosynthesis of the various hydroxylated fatty acids in Lesquerella seeds is proposed. The demonstration of desaturation at position 15 of a fatty acid with a hydroxy group at position 12 in Lesquerella prompted a comparison of the substrate recognition of the desaturases from Lesquerella and linseed. It was demonstrated that developing linseed also was able to desaturate ricinoleate at position 15 into densipolic acid. In addition, the linseed 15 desaturase was able to desaturate vernolic (12,13-epoxy-octadeca-9-enoic) acid and safflower microsomal 12 desaturase was able to desaturate 9-hydroxy-stearate. Thus, hydroxy and epoxy groups may substitute for double bonds in substrate recognition for oil-seed 12 and 15 desaturases.Abbreviations GLC gas-liquid chromatography - lysoPC palmitoyl-lysophosphatidylcholine - PC phosphatidylcholine This work was supported by grants from Stifteisen Svensk Oljeväxtforskning, Skanska Lantmännen Foundation, Swedish Farmers Foundation for Agricultural research, The Swedish Natural Science Research Council and The Swedish Council for Forestry and Agricultural Research. Nicki Engeseth was supported by the National Science Foundation under a grant award in 1992.     

Thermodynamic compensation in microbial thermal death

Sixty eight Arrhenius plots of thermal death in six mesophilic yeast species, tested at various concentrations of NaCl, lacked an isokinetic temperature. Nevertheless the H ^#/S ^# plot was apparently linear with a slope corresponding to 314° K. It was concluded that linear thermodynamic compensation of thermal death is non-existent in heterogeneous groups of yeasts and is unlikely to occur in heterogeneous groups of other organisms and that H ^#/S ^# plots lack sensitivity for the detection of non-linearity over narrow temperature ranges.However, the H ^# and S ^# parameters of thermal death displayed non-linear compensation in such a way that the extrapolated Arrhenius plots of death attained nearly identical values near the respective maximum temperatures for growth.Linear thermodynamic compensation occurred in each of the six strains, when stationary populations of the same strain were tested at various NaCl concentrations. On the other hand, exponential populations of each of the strains, tested in the same way, lacked an isokinetic temperature of thermal death.The significance of linear and non-linear thermodynamic compensation in biological rate processes is discussed.     

Effects of cannabinoids on the activities of mouse brain lipases

Cannabinoids were found to augment phospholipase activities and modify lipid levels of mouse brain synaptosomes, myelin and mitochondria. Delta-1-tetrahydrocannabinol (¹-THC) and several of its metabolites induced a dose-dependent (0.32–16 M) stimulation of phospholipase A₂ (PLA₂) activity resulting in the increased release of free arachidonic acid from exogenous [1-¹⁴C]phosphatidylcholine (PC). The potencies of the cannabinoids in modulating PLA₂ activity were approximately of the order: 7-OH-¹-THC > ¹-THC > 7-oxo-¹-THC > ¹-THC-7oic acid = 6 OH-¹-THC 6-OH-¹-THC. The hydrolysis of phosphatidylinositol (PI) by synaptosomal phospholipase C (PLC) was enhanced significantly by ¹-THC and promoted diacylglyceride levels by greater than 100 percent compared to control values. In contrast, arachidonate was the major product resulting from phospholipase activities of a 20,000g pellet. Synaptosomal diacylglyceride lipase activity was inhibited by ¹-THC. [1-¹⁴C]Arachidonic acid was readily incorporated into subcellular membrane phospholipids and after exposure to cannabinoids led to diminished phosphoglyceride levels and concomitant increases in released neutral lipid products. These data suggest that cannabinoids control phospholipid turnover and metabolism in mouse brain preparations by the activation of phospholipases and, through this mechanism, may exert some of their effects.     

Expression of Bacillus thuringiensis full-length and N-terminally truncated vip184 gene in an acrystalliferous strain of subspecies kurstaki

Vip3A is an 89-kDa protein secreted by Bacillus thuringiensis during vegetative growth. The 3.5 kb full-length vip184 gene was cloned from a wild-type isolate of B. thuringiensis, and the vip184S gene was constructed by deletion of the putative signal peptide encoding sequence. Both genes were expressed in the acrystalliferous strain Cry^–B of B. thuringiensis. Vip184 protein was observed mainly in the centrifuged pellets of B. thuringiensis Cry^–B(pHPT3), which contains the vip184 gene, and was less abundant in the concentrated supernatant. However, Vip184S proteins were not detected in the concentrated supernatant, but only in the pellets of Cry^–B(pHPT3S), which contains vip184S gene. This indicated that Vip184S proteins were not secreted into the culture medium and that the putative signal peptides were essential for the secretion of Vip184. The toxicity of Cry^–B(pHPT3) and Cry^–B(pHPT3S) were demonstrated against the neonate larvae of Spodoptera exigua and S. litura. Pellets and concentrated supernatant of Cry^–B(pHPT3) showed high activity against S. exigua and S. litura, but the Cry^–B(pHPT3S) strain was not toxic to either because of the deletion of N-terminal putative signal peptides. Therefore, this may suggest that the putative signal peptides are required for lethality.     

SCM2, a tryptophan permease in Saccharomyces cerevisiae, is important for cell growth

SCM2, a novel gene encoding a yeast tryptophan permease, was cloned as a high-copy-number suppressor of cse2-1. The cse2-1 mutation causes cold sensitivity, temperature sensitivity and chromosome missegregation. However, only the cold-sensitive phenotype of cse2-1 cells is suppressed by SCM2 at high copy. SCM2 is located on the left arm of yeast chromosome XV, adjacent to SUP3 and encodes a 65 kDa protein that is highly homologous to known amino acid permeases. Four out of five disrupted scm2 alleles (scm21-4) cause slow growth, whereas one disrupted allele (scm25) is lethal. Cells with both the scm21 and trp1-101 mutations exhibit a synthetic cold-sensitive phenotype and grow much more slowly at the permissive temperature than cells with a single scm21 or trp1-101 mutation. A region of the predicted SCM2 protein is identical to the partial sequence recently reported for the yeast tryptophan permease TAP2, indicating that SCM2 and TAP2 probably encode the same protein.     

The utilization of metabolic inhibitors for shifting product formation from xylitol to ethanol in pentose fermentations using Candida tropicalis

Summary Xylose, glucose and xylose/glucose mixtures were fermented with Candida tropicalis ATCC 32113 under aerobic, oxygen limited and anaerobic conditions. Ethanol yields were highest under oxygen limited conditions with xylose and xylose/glucose. Anaerobic conditions were best for glucose fermentations.The effect of four metabolic inhibitors (azide, carbonyl cyanide m-chlorophenyl hydrazone (CCCP), oligomycin A and valinomycin-K⁺) were then studied under oxygen limited conditions. Only azide had a significant influence on ethanol production. At 2¢10^-4 M concentrations, ethanol yield increased up to two times and xylitol levels were repressed by 90% for xylose and glucose/xylose fermentations. 4.2×10^-3 M azide gave highest ethanol yields in glucose fermentations. At this concentration of azide, however, cell growth was inhibited, which seemed to prevent ethanol production in xylose fermentations. The effect of azide is discussed in terms of fine-tuning the respiratory activity necessary for metabolism.     

Effect of stereospecific hydroxylation of N6-(Δ 2-Isopentenyl)adenosine on cytokinin activity

The cytokinin activities of cis and trans ribosylzeatin isomers and that of N⁶-(²-isopentenyl)adenosine were compared in four bioassays. The trans isomer was found to be more active than the cis isomer in stimulation of cucumber cotyledon expansion (100x), retention of chlorophyll in detached leaf pieces (7x), induction and stimulation of chlorophyll synthesis in cucumber cotyledons (20x) and of betacyanin synthesis in Amaranthus caudatus seedlings grown in the dark (60x). The N⁶-(²-isopentenyl)adenosine adenosine was less active than the trans ribosylzeatin in all four bioassays and more active than the cis ribosylzeatin in induction and stimulation of betacyanin and chlorophyll synthesis. These results show that the hydroxylation of the trans methyl group in the N⁶ side chain of N⁶-(²-isopentenyl)adenosine increases the biological activity and that this activity is either decreased or not significantly changed when the cis methyl group is hydroxylated.Abbreviations i⁶Ado N⁶-(²-isopentenyl)adenosine or 6-(3-methyl-2-butenylamino)-9--D-ribofuranosylpurine - t-to⁶Ado trans-ribosylzeatin or 6-(4-hydroxy-3-methyl-trans-2-butenylamino)-9--D-ribofuranosylpurine - c-io⁶Ado cis-ribosylzeatin or 6-(4-hydroxy-3-methyl-cis-2-butenylamino)-9--D-ribofuranosylpurine - HPLC high pressure liquid chromatography     

The Heat Shock Gene, <Emphasis Type="Italic">htpG</Emphasis>, and Thermotolerance in the Cyanobacterium, <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803

The htpG null mutant was obtained by inserting a chloramphenicol resistance cassette (Cm ^r) in the htpG coding sequence. The htpG null mutant (htpG), hsp16.6, and the double mutant, htpG::hsp16.6 cells showed little growth disadvantage at 30°C and 37°C, but not at 40°C. This suggests that HtpG and HSP16.6 proteins do not have an essential role during growth at normal and mildly elevated temperatures. Cell growth, cell survival rate, and oxygen electrode measurements demonstrated that htpG, hsp16.6, and htpG::hsp16.6 cells were sensitive to heat stress. Decreased basal and acquired thermotolerance was observed when mutants were heat shocked, with htpG::hsp16.6 being the most sensitive. A comparison of mutants showed that hsp16.6 was more sensitive to heat shock than htpG. Received: 19 November 2002 / Accepted: 19 December 2002     

Negative-ion fast atom bombardment tandem mass spectrometry for characterization of sulfated unsaturated disaccharides from heparin and heparan sulfate

Negative-ion fast atom bombardment tandem mass spectrometry has been used in the characterization of non-, mono-, di- and trisulfated disaccharides from heparin and heparan sulfate. The positional isomers of the sulfate group of monosulfated disaccharides were distinguished from each other by negative-ion fast atom bombardment tandem mass spectra, which provide an easy way of identifying the positional isomers. This fast atom bombardment collision induced dissociation mass spectrometry/mass spectrometry technique was also applied successfully to the characterization of di- and trisulfated disaccharides.Abbreviations FABMS fast atom bombardment mass spectrometry - CID collision induced dissociation - MIKE mass analysed ion kinetic energy - MS/MS mass spectrometry/mass spectrometry - HPLC high performance liquid chromatography - UA d-gluco-4-enepyranosyluronic acid - CS chondroitin sulfate - DS dermatan sulfate - HA hyaluronan - Hep heparin - HS heparan sulfate - UA(14) GlcNAc 2-acetamido-2-deoxy-4-O-(-d-gluco-4-enepyranosyluronic acid)-d-glucose - UA(14)GlcNAc6S 2-acetamido-2-deoxy-4-O-(-d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-glucose - UA2S(14)GlcNAc 2-acetamido-2-deoxy-4-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-d-glucose - UA2S(14)GlcNAc6S 2-acetamido-2-deoxy-4-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-glucose - UA(14)GlcN6S 2-amino-2-deoxy-4-O-(-d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-glucose - UA2S(14)GlcN 2-amino-2-deoxy-4-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-d-glucose - UA2S(14)GlcN6S 2-amino-2-deoxy-4-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-glucose - UA(14)GlcNS 2-deoxy-2-sulfamino-4-O-(-d-gluco-4-enepyranosyluronic acid)-d-glucose - UA(14)GlcNS6S 2-deoxy-2-sulfamino-4-O-(-d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-glucose - UA2S(14)GlcNS 2-deoxy-2-sulfamino-4-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-d-glucose - UA2S(14)GlcNS6S 2-deoxy-2-sulfamino-4-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-glucose - UA(13)GalNAc 2-acetamido-2-deoxy-3-O-(-d-Gluco-4-enepyranosyluronic acid)-d-galatose - UA(13)GalNAc4S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-4-O-sulfo-d-galactose - UA(13)GalNAc6S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-galactose - UA2S(13)GalNAc 2-acetamido-2-deoxy-3-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-d-galactose - UA2S(13)GalNAc4S 2-acetamido-2-deoxy-3-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-4-O-sulfo-d-galactose - UA2S(13)GalNAc6S 2-acetamido-2-deoxy-3-O-(2-O-sulfo--d-gluco-4-enepyranosyluronic acid)-6-O-sulfo-d-galactose - UA(13)GalNAcDiS 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-4,6-di-O-sulfo-d-galactose - UA(13)GlcNAc 2-acetamido-2-deoxy-3-O-(-d-gluco-4-enepyranosyluronic acid)-d-glucose.     

Regulation of intracellular H+ balance in Zymomonas mobilis 113 during the shift from anaerobic to aerobic conditions

Summary The energetics, enzyme activities and end-product synthesis of Zymomonas mobilis 113 in continuous culture were studied after the shift from an anaerobic to an aerobic environment. Aeration diminished ethanol yield and lactic acid concentration, but increased glucose consumption rate and production of acetic acid. After the shift to aerobic conditions reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H]-oxidase activity was stimulated. Washed cell suspensions consumed oxygen with glucose, lactate and ethanol as substrates. The aerobic Z. mobilis 113 regulated their intracellular redox balance by production and reoxidation of the end products, coupled with the formation of NAD(P)H. An increase in transmembrane pH gradient (pH) and a decrease in intracellular ATP concentration were observed after the shift to aerobic conditions. At low medium redox potential (Eh) values the H⁺ balance was regulated in an energy-independent way via end-product excretion. Under aerobic conditions this was supplemented by ATP-dependent H⁺ excretion by the membrane H⁺-ATPase.Abbreviations D dilution rate (h^-1) - S ₀ initial glucose concentration (g/l) - Y _x/s growth yield (g/mol) - Y _p/s product yield (g/g) - q _s specific rate of substrate utilization (g/g per hour) - q _p specific rate of ethanol formation (g/g per hour) - qo ₂ specific rate of CO₂ production (mmol/g per hour) - specific growth rate (h^-1) - X dry biomass concentration (g/l) - Eh redox potential of culture medium (mV) - pH transmembrane pH gradient (pH units) - pH_in intracellular pH - SASE sum of activities of specific enmymes of Entner-Doudoroff pathway     

A model of O·2 - generation in the complex III of the electron transport chain

Oxidation of semiquinone by O₂ in the Q cycle is known to be one of the sources of superoxide anion (O·₂ ^-) in aerobic cells. In this paper, such a phenomenon was analyzed using the chemical kinetics model of electron transfer from succinate to cytochrome c, including coenzyme Q, the complex III non-heme iron protein FeS_III and cytochromes b₁, b_h and c₁. Electron transfers from QH₂ to FeS_III and cytochrome b₁ were assumed to occur according to direct transfer mechanism (dynamic channelling) involving the formation of FeS^red _III -Q·^- and Q·^--cytochrome b₁ complexes. For oxidation/reduction reactions involving cytochromes b_h and b₁, the dependence of the equilibrium and elementary rate constants on the membrane potential () was taken into consideration. The rate of O·₂ ^- generation was found to increase dramatically with increase in above the values found in State 3. On the other hand, the rate of cytochrome c reduction decreased sharply at the same values of the membrane potential. This explains experimental data that the O·2^- generation at State 4 appears to be very much faster than at State 3. A mild uncoupling in State 4 can markedly decrease the superoxide generation due to a decrease in below the above mentioned critical level. pH appears to be equally effective as in stimulation of superoxide production which depends, in fact, upon the ^- _H + level.     

Energy conservation in the extreme thermophile Thermus thermophilus HB8

Whole cells of the extreme thermophile Thermus thermophilus HB8 contained a membrane-bound respiratory chain (comprised of nicotinamide nucleotide transhydrogenase, NADH dehydrogenase, menaquinone, and cytochromes b, c, aa₃, o), which exhibited a maximumH⁺/O quotient of approximately 8 g-ion H⁺·g-atom O^-1 for the oxidation of endogenous substrates. Whole cell respiration at 70° at the expense of endogenous substrates or ascorbate-TMPD generated a transmembrane protonmotive force (p) of up to 197 mV and an intracellular phosphorylation poteintial (Gp), measured under similar conditions, of approximately 43.9 kJ·mol^-1.The measured Gp/p ratio thus indicated anH⁺/ATP quotient of approximately 2.3 g-ion H⁺·mole ATP^-1. Glucose-limited continuous cultures of T. thermophilus at 60°, 70° and 78.5° exhibited extremely low moler growth yields (Y _O2 ^max 27.6 g cells·mol O ₂ ^-1 ; Y _glucose ^max 64.4 g cells ·mol glucose^-1) compared with mesophilic bacteria of similar respiratory chain composition and proton translocation efficiency. These low yields are probably at least partly explained by the extremely high permeability of the cytoplasmic membrane to H⁺, which thus causes the cells to respire rapidly in order to maintain the protonmotive force at a level commensurate with cell growth.Abbreviations TPMP⁺ triphenylmethylphosphonium cation - FCCP carbonylcyanide p-trifluoromethoxy phenythydrazone - TMPD N,N,N,N-tetramethyl-p-phenylene diamine     

Quantitation of the energetic state in mitochondria and submitochondrial vesicles with 8-anilino-1-naphthalene sulfonic acid

Some of the apparently anomalous findings made with the fluorescent probe 8-anilino-1-naphthalene sulfonic acid (ANS) have been reinvestigated using rat liver mitochondria. The results have been found compatible with current views on energy conservation.The direction of fluorescence and proton flux changes under different conditions have been delineated. The relation of these results to consideration of membrane polarity and organization is discussed.The reliability of ANS fluorescence changes in determining the level of energization of mitochondria and submitochondrial preparations is discussed.Abbreviations used ANS 8-anilino-1-naphthalene sulfonic acid - F _E and H⁺ _E O₂ dependent change in fluorescence and H⁺ in mitochondria and SMP - SMP submitochondrial preparation