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A small anaerobically induced tomato transcription factor was isolated from a subtractive library. This factor, designated ABZ1 (anaerobic basic leucine zipper), is anaerobically induced in fruits, leaves and roots and encodes a nuclear localized protein. ABZ1 shares close structural and sequence homology with the S-family of small basic leucine zipper (bZIP) transcription factors that are implicated in stress response. Nuclear localization of ABZ1 is mediated by the basic region and occurs under normoxic conditions. ABZ1 binds to G-box-like target sites as a dimer. Binding can be abolished by heterodimerization with a truncated protein retaining the leucine zipper but lacking the DNA binding domain. The protein binds in a sequence specific manner to the CaMV 35S promoter which is down regulated when ABZ1 is coexpressed. This correlates with the anaerobic down regulation of the 35S promoter in tomato and tobacco. These results may suggest that small bZIP proteins are involved in the negative regulation of gene expression under anaerobic conditions. [ABSTRACT FROM AUTHOR]

Chlamydomonas reinhardtii , a unicellular green alga, contains a hydrogenase enzyme, which is induced by anaerobic adaptation of the cells. Using the suppression subtractive hybridization (SSH) approach, the differential expression of genes under anaerobiosis was analyzed. A␣PCR fragment with similarity to the genes of bacterial Fe-hydrogenases was isolated and used to screen an anaerobic cDNA expression library of C. reinhardtii . The cDNA sequence of hydA contains a 1494-bp ORF encoding a protein with an apparent molecular mass of 53.1 kDa. The transcription of the hydrogenase gene is very rapidly induced during anaerobic adaptation of the cells. The deduced amino-acid sequence corresponds to␣two␣polypeptide sequences determined by sequence analysis of the isolated native protein. The Fe-hydrogenase contains a short transit peptide of 56 amino acids, which routes the hydrogenase to the chloroplast stroma. The isolated protein belongs to a new class of Fe-hydrogenases. All four cysteine residues and 12 other␣amino acids, which are strictly conserved in the active site (H-cluster) of Fe-hydrogenases, have been identified. The N-terminus of the C. reinhardtii protein is markedly truncated compared to other nonalgal Fe-hydrogenases. Further conserved cysteines that coordinate additional Fe–S-cluster in other Fe-hydrogenases are missing. Ferredoxin PetF, the natural electron donor, links the hydrogenase from C. reinhardtii to the photosynthetic electron transport chain. The hydrogenase enables the survival of the green algae under anaerobic conditions by transferring the electrons from reducing equivalents to the enzyme. [ABSTRACT FROM AUTHOR]

Purification of the ferripyoverdine reductase from Pseudomonas aeruginosa, strain PAO1, lead to the isolation of a soluble protein of Mr 27000–28000, as determined by HPLC sieving filtration and by denaturating gel electrophoresis. In the presence of NADH as the reductant, ferripyoverdine as the iron substrate, ferrozine as an iron(II)-trapping agent and FMN, this protein displayed an iron-reductase activity which resulted in the formation of ferrozine-iron(II) complex, providing that the enzymic assay was run under strict anaerobiosis. FMN was absolutely required for the activity to occur, but the lack of a visible spectrum and the lack of fluorescence for the protein in solution suggested that ferripyoverdine reductase is not a flavin-containing protein and that covalently bound FMN is not a prerequisite for the enzymatic reaction. A search of ferripyoverdine reductase by immunological detection amongst the different cellular compartments of P. aeruginosa lead to the conclusion that the soluble enzyme, which represented more than 95% of the total cellular enzyme, is not located in the periplasm but specifically in the cytoplasm. A strongly immunoreacting material, corresponding to a protein with identical Mr as the ferripyoverdine reductase of P. aeruginosa PAO1, was detected in all the eighteen fluorescent pseudomonad strains belonging to the P. aeruginosa, P. fluorescens, P. putida and P. chlororaphis species, as well as in P. stutzeri, a non-fluorescent species, suggesting that the enzyme acting as a ferripyoverdine reductase in P. aeruginosa PAO1 is ubiquitous among the Pseudomonas. [ABSTRACT FROM AUTHOR]

1. During aerobic cation uptake in liver mitochondria, the hydrophobic pH indicator bromothymol blue undergoes a multiphase response: phase 1 (rapid acidification), phase 2 (slow alkalinization), phase 3 (rapid alkalinization) and phase 4 (reacidification). 2. Titrations with ruthenium red and malonate indicate that the various phases depend on the relative rates of cation uptake and proton translocation: at high rates of cation uptake, phase 1 disappears and phases 2 and 3 are transformed in a monotonic process of alkalinization. 3. The comparison of the bromothymol blue response with the arsenazo III, 2',7'-bis(carboxyethyl)5(6)carboxyfluorescein (BCECF) and safranine responses indicates that: (a) phase 2 (slow alkalinization) corresponds to a slow rise of matrix pH and a parallel decline of membrane potential; (b) phase 3 (rapid alkalinization) corresponds to termination of proton translocation and initiation of the processes of cation elf lux and proton reuptake. All the above processes reach completion during phase 4. 4. Although bromothymol blue always behaves as a membrane-bound indicator, the extent to which it reflects the matrix or the cytosolic pH is a function of file membrane-potential-determined asymmetric distribution: in parallel with the lowering of the membrane potential, the dye chromophore is shifted from the cytosolic to the matrix side membrane layer. 5. A model is discussed which describes the behaviour of bromothymol blue as pH indicator recording the changes in membrane layers facing either the matrix or the cytosolic side. The complex response of the dye during cation uptake is due to two independent processes, one of pH change and another of dye intramembrane shift. Computer simulations of the dye response, based on the conversion of a kinetic model into an electrical network and closely reproducing the experimental observations, are reported. [ABSTRACT FROM AUTHOR]

1. The kinetics of acidification and realkalinization of the matrix after addition of nigericin to respiring and non-respiring mitochondria, recorded by intramitochondrial pH indicators such as neutral red and 2',7'bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF), is complementary to that recorded by extramitochondrial pH indicators. The extent of acidification decreases with the logarithm of thc KCl concentration and is inhibited by P1 and ammonium ions.2. Proton translocation during respiration has been compared with proton extraction from matrix bulk water. During oxygen pulses to EOTA-untreated rnitochondria, BCECF records an extraction of protons from matrix bulk water of about 2-3 nmol H+/mg, reduced to 1-2 nmol H+/mg in EGTA-treated mitochondria. Since the amount of proton translocation required to achieve steady state is of the order of 6-7 nmol H+/mg, it appears that 75-90% of the protons are not extracted from matrix bulk water. Only a slight response is recorded by neutral red.3. The effect of permeant cations and of uncouplers on the distribution of proton extraction between membrane and matrix bulk water has been studied in presteady state. During Sr2+ uptake, proton extrusion into cytosolic bulk water, as well as proton extraction from matrix bulk water, corresponds almost to 100% of the protons translocated by the redox proton pumps. In the absence of Sr2+, parallel to the disappearance of the proton extrusion in cytosolic bulk water, the proton extraction from matrix bulk water diminishes to about 20% of the proton translocation.4. The mechanism by which divalent cation uptake and protonophoric uncouplers affect thc distribution of proton extraction between matrix bulk water and membrane domains and the nature of the membrane domains are discussed. [ABSTRACT FROM AUTHOR]

Two nitrate reductases, nitrate reductase A and nitrate reductase Z, exist in Escherichia coli. The nitrate reductase Z enzyme has been purified from the membrane fraction of a strain which is deleted for the operon encoding the nitrate reductase A enzyme and which harbours a multicopy plasmid carrying the nitrate reductase Z structural genes: it was purified 219 times with a yield of about 11%. It is an Mr-230000 complex containing 13 atoms iron and 12 atoms labile sulfur/molecule. The presence of a molybdopterin cofactor in the nitrate reductase Z complex was demonstrated by reconstitution experiments of the molybdenum-cofactor-deficient NADPH-dependent nitrate reductase activity from a Neurospora crassa nit-1 mutant and by fluorescence emission and excitation spectra of stable derivatives of molybdoterin extracted from the purified enzyme. Both nitrate reductases share common properties such as relative molecular mass, subunit composition and electron donors and acceptors. Nevertheless, they diverge by two properties: their electrophoretic migrations are very different (RF of 0.38 for nitrate reductase Z versus 0.23 for nitrate reductase A), as are their susceptibilities to trypsin. An immunological study performed with a serum raised against nitrate reductase Z confirmed the existence of common epitopes in both complexes but unambiguously demonstrated the presence of specific determinants in nitrate reductase Z. Furthermore, it revealed a peculiar aspect of the regulation of both nitrate reductases: the nitrate reductase A enzyme is repressed by oxygen, strongly inducible by nitrate and positively controlled by the fnr gene product; on the contrary, the nitrate reductase Z enzyme is produced aerobically, barely induced by nitrate and repressed by the fnr gene product in anaerobiosis. [ABSTRACT FROM AUTHOR]

Both from irreversible thermodynamics and from mass-action kinetics it can be derived that upon anaerobiosis in an oxygen-pulse experiment the protonmotive force across a mitochondrial membrane undergoes a sudden drop. Under representative conditions the protonmotive force after the drop (the residual protonmotive force) is shown to be less than 3 kJ. mol[SUP-1] as opposed to steady-state values for the protonmotive force of 19 kJ. mol[SUP-] It is concluded that corrections for proton leakage in pulse experiments by back extrapolation underestimate proton leakage. Consequently the observed H[SUP+]/O stoichiometries must underestimate the true H[SUP+]/O ratios. [ABSTRACT FROM AUTHOR]

Arthrobacter pyridinolis possesses alternative transport systems for D-fructose: a respiration-coupled transport system whereby D-fructose transport occurs with concomitant oxidation of L-malate, and a phosphoenolpyruvate:D-fructose phosphotransferase system. Studies of D-fructose uptake by whole cells in the presence and absence of cyanide demonstrate that respiration-coupled transport is used almost exclusively during the first half of logarithmic growth, after which it accounts for only 15-20% of D-fructose uptake. Phosphotransferase levels are low during log phase, peak during late log, and then slowly decline. In a mutant of A. pyridinolis which requires δ-aminolevulinic acid for growth, the growth rate, cell cytochrome content, and activity of the respiration-coupled transport system increased with increasing concentrations of δ-aminolevulinic acid up to 50 µg/ml. By contrast, phosphotransferase activity was highest in cells grown on limiting δ-aminolevulinic acid. L-Malate, which stimulates respiration-coupled transport, repressed the phosphotransferase system. The respiratory activity and the ability to release CO2 from internalized D-fructose was consistently low in D-fructose-grown cells. A cyanide-resistant cytochrome, tentatively identified as cytochrome d, appeared in the late exponential phase of growth. Isocitrate lyase activity, required for aerobic growth of this organism, declined markedly during the late exponential phase. Thus the phosphotransferase system is maximally induced, in this obligate aerobe, under conditions of relative anaerobiosis during which metabolism is primarily fermentative. [ABSTRACT FROM AUTHOR]

The mitochondrial ADP/ATP carrier in Saccharomyces cerevisiae is encoded by three independent genes, AAC1, AAC2, and AAC3.In this work, we analysed the 5' upstream; region of AAC1 by sequencing and by mapping the transcription initiation site of the gene. By monitoring the level of AAC1 mRNA and the β-galactosidase activity of AAC1-lacZ fusion constructs, we showed that expression of AAC1 is subjected to regulation by oxygen . In contrast to the other two AAC genes, the effect of oxygen on AAC1 is not mediated by heme and heme-dependent transcription factors. The AAC1 expression was reduced eightfold in annerobically grown cells compared to expression in cells grown aerobically, but it was not affected by the nature of carbon source used for growth. The data presented show that AAC1 expression while constitutive under all aerobic conditions tested is repressed during anaerobiosis in a heme-independent manner. [ABSTRACT FROM AUTHOR]