Micrococcus lysodeikticus membrane ATPase. effect of trypsin on stimulation of a purified form of the enzyme and identification of its natural inhibitor

A soluble purified form of Micrococcus lysodeikticus ATPase (form B_AT, from strain B, active, trypsin-stimulated) was stimulated 100% by trypsin and this stimulation was inhibited by preincubation of the protease with phenyl methyl sulphonylfluoride. This form of the enzyme was also stimulated 125–150% by filtration on Sephadex G-200. Analysis by sodium dodecyl sulphate-gel electrophoresis showed that stimulation of this form of M. lysodeikticus ATPase was always accompanied by the disappearance of a subunit of mol. wt. 25 000 (ε subunit). It suggests that this subunit is the natural inhibitor of M. lysodeikticus ATPase. In the case of ATPase stimulation by trypsin, a partial and limited degradation of the α subunit was also observed. The interaction between the ε subunit and the rest of the ATPase complex was reversibly affected by pH, suggesting its non-covalent nature.     

Confomational and molecular responses to pH variation of the purified membrane adenosine triphospate of Micrococcus lysodeikticus

A preparation of ATPase from the membranes of Micrococcus lysodeikticus, solubilized and more than 95 %. pure, showed two main bands in analytical polyacrylamide gel electrophoresis. They did not correspond to isoenzymes because one band could be converted into the other by exposure to a mildly alkaline pH value. The conversion was paralleled by changes in molecular weight, circular dichroism and catalytic properties. Denaturation by pH at 25 °C was followed by means of circular dichroism, ultracentrifugation and polyacrylamide gel electrophoresis. A large conformational transition took place in the acid range with midpoints at about pH = 3.6 (I = 10^?4 M), 4.3 (I = 0.03 M) and 5.3 (I = 0.1 M). The transition was irreversible. Strong aggregation of the protein occurred in this range of pH. The final product was largely random coil, but even at pH 1.5 dissociation into individual subunits was not complete. However, partial dissociation took place at pH 5 (I = 0.028 M). At this pH value the enzyme was inactive, but 20–30 % of the activity could be recovered when the pH was returned to 7.5.In the alkaline region the midpoint of the transition occurred near pH = 11 (I = 0.028 M). The pK of most of the tyrosine residues of the protein was about 10.9. The unfolding was irreversible and the protein was soon converted into peptide species with molecular weights lower than those determined for the subunits by gel clectrophoresis in the presence of sodium dodecyl sulphate. Conventional proteolysis did not account for the transformation.     

Interaction of ethidium bromide with the transport system for monovalent cations in yeast

Summary Ethidium was found to be taken up by yeast cells in a process that, at certain concentrations has the main following characteristics: a) a substrate is required; b) it presents cooperative kinetics, withn, according to the Hill equation 3; c) ethidium can be concentrated more than 100-fold; d) the uptake is inhibited by Ca²⁺; e) the uptake of the dye is inhibited by monovalent cations with a selectivity pattern similar to that observed in their transport by yeast; f) ethidium inhibits the uptake of K⁺, and, at concentrations up to about 250 m produces a competitive inhibition on the uptake of Rb⁺; and g) ethidium produces the same effects as K⁺ on respiration and the extrusion of H⁺. It is concluded that ethidium is taken up by yeast cells in a selective way by the same transport system normally employed for monovalent cation uptake.     

Photoreceptor Pigment for Blue Light in Neurospora crassa

Irradiating the mycelium of Neurospora crassa with moderate intensities of blue light causes a reversible photoreduction of a b-type cytochrome. The action spectrum for the photoreduction of cytochrome b is very similar to the absorption spectrum of flavin pigments. Prolonged irradiation of the mycelium with strong blue light irreversibly bleaches flavin-like pigments and as these pigments are bleached the photoresponse of cytochrome b is lost. We conclude from these and other data that a flavin is the photoreceptor pigment for the photoreduction of cytochrome b. The close similarity between the action spectrum for the photoreduction of cytochrome b and action spectra for a number of physiological photoresponses suggests that this photoreceptor pigment controls a wide variety of photobiological processes in a wide diversity of organisms.     

Membrane bound and soluble adenosine triphosphatase of Escherichia coli K 12. kinetic properties of the basal and trypsin-stimulated activities

Basal and trypsin-stimulated adenosine triphosphatase activities of Escherichia coli K 12 have been characterized at pH 7.5 in the membrane-bound state and in a soluble form of the enzyme. The saturation curve for Mg2+/ATP = 1/2 was hyperbolic with the membrane-bound enzyme and sigmoidal with the soluble enzyme. Trypsin did not modify the shape of the curves. The kinetic parameters were for the membrane-bound ATPase: apparent Km = 2.5 mM, Vmax (minus trypsin) = 1.6 mumol-min-1-mg protein-1, Vmax (plus trypsin) = 2.44 mumol-min-1-mg protein-1; for the soluble ATPase: [S0.5] = 1.2 mM, Vmax (-trypsin) = 4 mumol-min-1-mg protein-1; Vmax (+ trypsin) = 6.6 mumol-min-1-mg protein-1. Hill plot analysis showed a single slope for the membrane-bound ATPase (n = 0.92) but two slopes were obtained for the soluble enzyme (n = 0.98 and 1.87). It may suggest the existence of an initial positive cooperativity at low substrate concentrations followed by a lack of cooperativity at high ATP concentrations. Excess of free ATP and Mg2+ inhibited the ATPase but excess of Mg/ATP (1/2) did not. Saturation for ATP at constant Mg2+ concentration (4 mM) showed two sites (groups) with different Kms: at low ATP the values were 0.38 and 1.4 mM for the membrane-bound and soluble enzyme; at high ATP concentrations they were 17 and 20 mM, respectively. Mg2+ saturation at constant ATP (8 mM) revealed michealian kinetics for the membrane-bound ATPase and sigmoid one for the protein in soluble state. When the ATPase was assayed in presence of trypsin we obtained higher Km values for Mg2+. These results might suggest that trypsin stimulates E. coli ATPase by acting on some site(s) involved in Mg2+ binding. Adenosine diphosphate and inorganic phosphate (Pi) act as competitive inhibitors of Escherichia coli ATPase. The Ki values for Pi were 1.6 +/- 0.1 mM for the membrane-bound ATPase and 1.3 +/- 0.1 mM for the enzyme in soluble form, the Ki values for ADP being 1.7 mM and 0.75 mM for the membrane-bound and soluble ATPase, respectively. Hill plots of the activity of the soluble enzyme in presence of ADP showed that ADP decreased the interaction coefficient at ATP concentrations below its Km value. Trypsin did not modify the mechanism of inhibition or the inhibition constants. Dicyclohexylcarbodiimide (0.4 mM) inhibited the membrane-bound enzyme by 60-70% but concentrations 100 times higher did not affect the residual activity nor the soluble ATPase. This inhibition was independent of trypsin. Sodium azide (20 muM) inhibited both states of E. coli ATPase by 50%. Concentrations 25-fold higher were required for complete inhibition. Ouabain, atebrin and oligomycin did not affect the bacterial ATPase.     

Biodegradation of a crude oil by three microbial consortia of different origins and metabolic capabilities

Microbial consortia were obtained three by sequential enrichment using different oil products. Consortium F1AA was obtained on a heavily saturated fraction of a degraded crude oil; consortium TD, by enrichment on diesel and consortium AM, on a mixture of five polycyclic aromatic hydrocarbons [PAHs]. The three consortia were incubated with a crude oil in order to elucidate their metabolic capabilities and to investigate possible differences in the biodegradation of these complex hydrocarbon mixtures in relation to their origin. The efficiency of the three consortia in removing the saturated fraction was 60% (F1AA), 48% (TD) and 34% (AM), depending on the carbon sources used in the enrichment procedures. Consortia F1AA and TD removed 100% of n-alkanes and branched alkanes, whereas with consortium AM, 91% of branched alkanes remained. Efficiency on the polyaromatic fraction was 19% (AM), 11% (TD) and 7% (F1AA). The increase in aromaticity of the polyaromatic fraction during degradation of the crude oil by consortium F1AA suggested that this consortium metabolized the aromatic compounds primarily by oxidation of the alkylic chains. The 500-fold amplification of the inocula from the consortia by subculturing in rich media, necessary for use of the consortia in bioremediation experiments, showed no significant decrease in their degradation capability. Journal of Industrial Microbiology & Biotechnology (2002) 28, 252–260 DOI: 10.1038/sj/jim/7000236 Received 12 July 2001/ Accepted in revised form 11 November 2001     

Assessing the impact of extreme adverse weather on the biological traits of a European storm petrel colony

Climate change affects the climatic disturbance patterns and regimes and is altering the frequency and intensity of subtropical cyclones. These events can affect population dynamics of seabirds (e.g., survival, reproduction). In this work we tested the effect of adverse weather on a colony of European storm petrels (Hydrobates pelagicus) located in a small islet (Aketx) in northern Spain. Over a long-term monitoring period (1993–2014) we ringed 3728 petrels. From 2003 onwards we also monitored breeding success, the percentage of immature individuals and moult scores. We used Cormack-Jolly-Seber models and Underhill and Zucchini models to analyze the effects of climatic conditions on a number of biological traits (survival, breeding parameters, moulting patterns). Our analyses revealed a constant value of adult survival over the 26-year monitoring period. Recapture probability, however, tended to be positively influenced by NAO conditions in spring, and negatively influenced by NAO conditions in winter (although this would only affect to a fraction of first-captured birds). Moreover, the impact of adverse weather, especially in 2011 and 2014, resulted in an increasing proportion of yearlings in the breeding population, a lower breeding success and a delayed onset of moult. These effects were similar to those observed during the Prestige oil spill catastrophe.     

Relationships between leaf morphological traits, nutrient concentrations and isotopic signatures for Mediterranean woody plant species and communities

Background and aims

Soil factors are driving forces that influence spatial distribution and functional traits of plant species. We test whether two anchor morphological traits—leaf mass per area (LMA) and leaf dry matter content (LDMC)—are significantly related to a broad range of leaf nutrient concentrations in Mediterranean woody plant species. We also explore the main environmental filters (light availability, soil moisture and soil nutrients) that determine the patterns of these functional traits in a forest stand.

Methods

Four morphological and 19 chemical leaf traits (macronutrients and trace elements and δ¹³C and δ¹⁵N signatures) were analysed in 17 woody plant species. Community-weighted leaf traits were calculated for 57 plots within the forest. Links between LMA, LDMC and other leaf traits were analysed at the species and the community level using standardised major axis (SMA) regressions

Results

LMA and LDMC were significantly related to many leaf nutrient concentrations, but only when using abundance-weighted values at community level. Among-traits links were much weaker for the cross-species analysis. Nitrogen isotopic signatures were useful to understand different resource-use strategies. Community-weighted LMA and LDMC were negatively related to light availability, contrary to what was expected.

Conclusion

Community leaf traits have parallel shifts along the environmental factors that determine the community assembly, even though they are weakly related across individual taxa. Light availability is the main environmental factor determining this convergence of the community leaf traits.     

The type of competition modulates the ecophysiological response of grassland species to elevated CO2 and drought

The effects of elevated CO₂ and drought on ecophysiological parameters in grassland species have been examined, but few studies have investigated the effect of competition on those parameters under climate change conditions. The objective of this study was to determine the effect of elevated CO₂ and drought on the response of plant water relations, gas exchange, chlorophyll a fluorescence and aboveground biomass in four grassland species, as well as to assess whether the type of competition modulates that response. Elevated CO₂ in well‐watered conditions increased aboveground biomass by augmenting CO₂ assimilation. Drought reduced biomass by reducing CO₂ assimilation rate via stomatal limitation and, when drought was more severe, also non‐stomatal limitation. When plants were grown under the combined conditions of elevated CO₂ and drought, drought limitation observed under ambient CO₂ was reduced, permitting higher CO₂ assimilation and consequently reducing the observed decrease in aboveground biomass. The response to climate change was species‐specific and dependent on the type of competition. Thus, the response to elevated CO₂ in well‐watered grasses was higher in monoculture than in mixture, while it was higher in mixture compared to monoculture for forbs. On the other hand, forbs were more affected than grasses by drought in monoculture, while in mixture the negative effect of drought was higher in grasses than in forbs, due to a lower capacity to acquire water and mineral nutrients. These differences in species‐level growth responses to CO₂ and drought may lead to changes in the composition and biodiversity of the grassland plant community in future climate conditions.     

Sensitive fluorescence in situ hybridization signal detection in maize using directly labeled probes produced by high concentration DNA polymerase nick translation

The signal produced by fluorescence in situ hybridization (FISH) often is inconsistent among cells and sensitivity is low. Small DNA targets on the chromatin are difficult to detect. We report here an improved nick translation procedure for Texas red and Alexa Fluor 488 direct labeling of FISH probes. Brighter probes can be obtained by adding excess DNA polymerase I. Using such probes, a 30 kb yeast transgene, and the rp1, rp3 and zein multigene clusters were clearly detected.