Purification of peroxisomes and mitochondria from spinach leaf by percoll gradient centrifugation

A procedure was developed to purify simultaneously peroxisomes and mitochondria from spinach (Spinacia oleracea L.) leaf under isoosmotic and low viscosity conditions. This method involved differential centrifugation and density gradient centrifugation on four layers of Percoll. Chlorophyll-free preparations of highly intact and active organelles were obtained and cross-contamination was negligible. Both organelles were stable for several hours, even if they remained in Percoll. Purified mitochondria were able to carry out the oxidation of different substrates with excellent respiratory control and ADP:O ratios. The method described in the present work was also suitable to purify mitochondria and peroxisomes from potato (Solanum tuberosum L.) tubers.     

Pyruvate,orthophosphate dikinase from Acetobacter aceti

A pyruvate, orthophosphate dikinase (EC 2.7.9.1) has been isolated from Acetobacter aceti grown on pyruvate as the only source of carbon and energy. The enzyme was purified 65-fold, and its molecular weight was determined to be about 330,000 by gel filtration.The optimum pH was 8.0 in the forward direction [phosphoenolpyruvate (PEP) formation] and 7.1 for the backward reaction (pyruvate production). In both directions Mg²⁺ was required (forward K _m 1.70 mM; reverse K _m 0.87 mM) and no other divalent cation was able to replace it. The K _m values for pyruvate, ATP, and P_i were 27 M, 0.20 mM, and 0.83 mM, respectively, in the forward direction. The K _m values for PEP, AMP, and PP_i were 0.13 mM, 6 M, and 62 M, respectively, for the reverse reaction. The substrate-product pairs pyruvate-PEP, ATP-AMP, P_i-PP_i were competitive inhibitors to each other in both directions. These product inhibition studies suggest for the enzyme from A. aceti nonclassical three-site Tri (Uni Uni) Ping-Pong kinetics.Abbreviations PEP phosphoenolpyruvate - OAA oxaloacetate - MW molecular weight - SDS sodium dodecyl sulphate - TEMG buffer 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 5 mM MgCl₂, 1 mM glutathione     

Intracellular localization, isolation and characterization of two distinct varieties of superoxide dismutase from Neurospora crassa

1. Neurospora crassa was found to contain two distinct superoxide dismutases. 2. Most of the activity is associated with the cytosolic fraction and was shown to be the Cu/Zn-containing form of the protein. 3. Mitochondria isolated from Neurospora crassa showed two distinct superoxide dismutases: a cyanide-sensitive Cu/Zn-containing protein and a cyanide-insensitive form which probably contains manganese. 4. Localization experiments, using selective marker enzymes and digitonin fractionation, indicated that the cyanide-sensitive form is localized in the intermembrane space, whereas the cyanide-insensitive form is confined to the mitochondrial matrix space. 5. The cytosolic Cu/Zn-containing superoxide dismutase was isolated in high yields and extensively characterized by using e.p.r. spectroscopy, isoelectric focusing and analytical ultracentrifugation. 6. E.p.r. spectroscopy was used to monitor changes in the copper environment of the native protein after the addition of a number of potential inhibitors and after high-pH treatment. 7. Both of the cyanide-sensitive Cu/Zn-containing enzymes (cytosolic and mitochondrial) appeared to have identical properties which in turn were different from the cyanide-insensitive enzyme. 8. It is probable that the cyanide-insensitive enzyme was not previously detected, owing to its low amount (less than 10% of the total activity), greater lability than the cyanide-sensitive enzyme and the necessity of obtaining a mitochondrial-enriched fraction before its isolation.     

Feasibility of Labile Zn Phytoextraction Using Enhanced Tobacco and Sunflower: Results of Five- and One-Year Field-Scale Experiments in Switzerland

Phytoextraction with somaclonal variants of tobacco and sunflower mutant lines (non-GMs) with enhanced metal uptake and tolerance can be a sustainable alternative to conventional destructive decontamination methods, especially for stripping bioavailable zinc excess in topsoil. The overall results of a 5-year time series experiment at field scale in north-eastern Switzerland confirm that the labile Zn pool in soil can be lowered by 45–70%, whereas subplots without phytoextraction treatment maintained labile Zn concentrations. In 2011, the phytoextraction experiment site was enlarged by a factor of 3, and the labile 0.1 M NaNO₃ extractable Zn concentration in the soil was reduced up to 58% one period after harvest. A Mass Balance Analysis confirmed soil Zn decontamination in line with plant Zn uptake. The plants partially take Zn from the non-labile pool of the total. The sustainability of Zn phytoextraction in subplots that no longer exceed the Swiss trigger value is now assessed over time. In contrary to the phytoextraction of total soil Zn which needs a long cleaning up time, the bioavailable Zn stripping is feasible within a few years period.     

Evidence for a lack of phospholipid transfer protein in the stroma of spinach chloroplasts

The possible activity of phospholipid transfer protein in stroma extracts from spinach leaf has been investigated. Stroma, prepared from purified intact chloroplasts, was dialyzed and passed through various chromatography columns. None of the protein fractions eluted was able to stimulate the transfer of phosphatidylglycerol (PG) or phosphatidylcholine (PC) from liposomes to mitochondria, suggesting the lack of phospholipid transfer protein in the stroma from mature spinach chloroplasts.     

Activation of the heat shock response in plants by chlorophenols: transgenic Physcomitrella patens as a sensitive biosensor for organic pollutants

The ability to detect early molecular responses to various chemicals is central to the understanding of biological impact of pollutants in a context of varying environmental cues. To monitor stress responses in a model plant, we used transgenic moss Physcomitrella patens expressing the beta-glucuronidase reporter (GUS) under the control of the stress-inducible promoter hsp17.3B. Following exposure to pollutants from the dye and paper industry, GUS activity was measured by monitoring a fluorescent product. Chlorophenols, heavy metals and sulphonated anthraquinones were found to specifically activate the hsp17.3B promoter (within hours) in correlation with long-term toxicity effects (within days). At mildly elevated physiological temperatures, the chemical activation of this promoter was strongly amplified, which considerably increased the sensitivity of the bioassay. Together with the activation of hsp17.3B promoter, chlorophenols induced endogenous chaperones that transiently protected a recombinant thermolabile luciferase (LUC) from severe heat denaturation. This sensitive bioassay provides an early warning molecular sensor to industrial pollutants under varying environments, in anticipation to long-term toxic effects in plants. Because of the strong cross-talk between abiotic and chemical stresses that we find, this P. patens line is more likely to serve as a direct toxicity bioassay for pollutants combined with environmental cues, than as an indicator of absolute toxicity thresholds for various pollutants. It is also a powerful tool to study the role of heat shock proteins (HSPs) in plants exposed to combined chemical and environmental stresses.