Characterisation of the H+-ATPase in plasma membranes isolated from the green alga Chlamydomonas reinhardtii

Plasma membranes from the green alga Chlamydomonas reinhardtii were purified by differential centrifugation and two-phase partitioning in an aqueous polymer system. The isolated plasma membranes were virtually free from contaminating chloroplasts, mitochondria, endoplasmic reticulum and Golgi membranes as shown by marker enzyme and pigment analysis. The isolated plasma membranes exhibited vanadate sensitive ATPase activity, indicating the presence of a P-type ATPase. This was verified by using antibodies against P-type ATPase from Arabidopsis , which crossreacted with a protein of 109 kDa. The ATPase activity was inhibited to more than 90% by vanadate (K_i= 0.9 μ M ) but not affected by inhibitors specific for F- or V-type ATPases. demonstrating the purity of the plasma membranes. Mg-ATP was the substrate, and the rate of ATP-hydrolysis followed simple Michaelis-Menten kinetics giving a K_m= 0.46 m M . Free Mg²⁺ stimulated the activity, K_1/2= 0.68 m M . Maximal activity was obtained at pH 8. The ATPase activity was latent but stimulated 10 to 20-fold in the presence of detergents. This indicates that the isolated plasma membrane vesicles were tightly sealed and mostly right-side-out, making the ATPase inaccessible to the hydrophilic substrate ATP. In the presence of the Brij 58, the isolated plasma membranes performed ATP dependent H⁺-pumping as shown by the optical pH probe acridine orange. H⁺-pumping was dependent on the presence of valinomycin and K⁺ ions and completely abolished by vanadate. Addition of Brij 58 has been shown to produce 100% sealed inside-out vesicles of plant plasma membranes (Johansson et al. 1995, Plant J. 7: 165–173) and this was also the case for plasma membranes from the green alga Chlamydomonas reinhardtii.     

Effects of decreased atmospheric deposition on the sulfur budgets of two Dutch moorland pools

The chemical composition of surface waters of two Dutch moorland pools and of incident precipitation, was monitored from 1982 to 1990. For this period, sulfur and water budgets were calculated using a hydrochemical model developed for well-mixed non-stratifying lakes. Total atmospheric deposition of S decreased significantly after 1986 at both locations. A model describing the sulfur budget in terms of input, output and reduction/oxidation processes predicted a fast decrease of pool water SO₄ ²⁻ concentrations after a decrease of atmospheric input. However, SO₄ ²⁻ concentrations in the surface water was lowered only slightly or remained constant. Apparently a source within the lake caused the unexpectedly high SO₄ ²⁻ concentrations. The possible supply of SO₄ ²⁻ from the sediment through regulation by (K-)Al-SO₄ containing minerals or desorption of SO₄ ²⁻ from positively charged surfaces in the sediment was evaluated. Solubility calculations of pore water with respect to alunite, basaluminite and jurbanite indicated that SO₄ ²⁻ concentration was not regulated by these minerals. It is suggested here (1) that desorption of SO₄ ²⁻ from peaty sediments may account for the estimated SO₄ ²⁻ supply provided that the adsorption complex is periodically recharged by partial oxidation of the upper bottom sediments and (2) that because of exposure of a part of the pool bottom to the atmosphere during dry summers and subsequent oxidation of reduced S, the amount of SO₄ ²⁻ may be provided which complements the decreasing depositional SO₄ ²⁻ input. In future research these two mechanisms need to be investigated.     

Sulfate reduction and S-oxidation in a moorland pool sediment

In an oligotrophic moorland pool in The Netherlands, S cycling near the sediment/water boundary was investigated by measuring (1) SO₄ ^2– reduction rates in the sediment, (2) depletion of SO₄ ^2– in the overlying water column and (3) release of³⁵S from the sediment into the water column. Two locations differing in sediment type (highly organic and sandy) were compared, with respect to reduction rates and depletion of SO₄ ^2– in the overlying water.Sulfate reduction rates in sediments of an oligotrophic moorland pool were estimated by diagenetic modelling and whole core³⁵SO₄ ^2– injection. Rates of SO₄ ^2– consumption in the overlying water were estimated by changes in SO₄ ^2– concentration over time in in situ enclosures. Reduction rates ranged from 0.27–11.2 mmol m^–2 d^–1. Rates of SO₄ ^2– uptake from the enclosed water column varied from –0.5, –0.3 mmol m^–2 d^–1 (November) to 0.43–1.81 mmol m^–2 d^–1 (July, August and April). Maximum rates of oxidation to SO₄ ^2– in July 1990 estimated by combination of SO₄ ^2– reduction rates and rates of in situ SO₄ ^2– uptake in the enclosed water column were 10.3 and 10.5 mmol m^–2 d^–1 at an organic rich and at a sandy site respectively.Experiments with³⁵S^2– and³⁵SO₄ ^2– tracer suggested (1) a rapid formation of organically bound S from dissimilatory reduced SO₄ ^2– and (2) the presence of mainly non SO₄ ^2–-S derived from reduced S transported from the sediment into the overlying water. A³⁵S^2– tracer experiment showed that about 7% of³⁵S^2– injected at 1 cm depth in a sediment core was recovered in the overlying water column.Sulfate reduction rates in sediments with higher volumetric mass fraction of organic matter did not significantly differ from those in sediments with a lower mass fraction of organic matter.Corresponding author     

Reconstitution of mitochondrial oligomycin and dicyclohexylcarbodiimide-sensitive ATPase

1. Oligomycin and dicyclohexylcarbodiimide-sensitive ATPase was isolated from beef-heart mitochondria and treated with 3.5 M NaBr in order to remove F1. The residue, called F0, was found to consist of seven components. Five of these are stained by Coomassie blue after dodecylsulfate-polyacrylamide-gel electrophoresis. Two of them correspond to the oligomycin-sensitivity-conferring protein and coupling factor F6, with apparent molecular weights of 21,000 and 9,400, respectively. Three additional polypeptides of molecular weights 23,000, 10,500 and 8,600 were not identified with known proteins. Two components not stained with Coomassie blue were detected by autoradiography of the gels of F0 preincubated with [14C]dicyclohexylcarbodiimide. These two components probably represent monomeric and oligomeric forms of the dicyclohexylcarbodiimide-binding protein. 2. F0 induced an oligomycin and dicyclohexylcarbodiimide-sensitive enhancement of K+ + valinomycin-driven proton translocation across the membrane of artificial phospholipid vesicles. 3. The interaction of F0 with purified, soluble beef heart F1 was investigated. F0 was capable of binding F1 and conferring oligomycin and dicyclohexylcarbodiimide sensitivity and cold stability on its ATPase activity. Furthermore F0 was found to diminish the specific activity of F1-ATPase. A comparison of these effects at varying F0/F1 ratios shows that F0 binds F1 in both an oligomycin-sensitive and an oligomycin-insensitive manner, and that both types of binding involve a conferral of cold stability and a decrease in specific activity. High F0/F1 ratios favoured in oligomycin-sensitive type of binding, indicating that F1 binds preferentially to oligomycin-sensitivity-conferring sites. Treatment of ATPase complex with trypsin resulted in an F0 with a decreased proportion of oligomycin-sensitivity-conferring binding sites and a diminished ability to lower the specific activity an cold lability of F1. 4. Reconstitution of F0 treated with trypsin and F1, oligomycin-sensitivity-conferring protein and F6 showed that at a constant amount of F1 bound, both oligomycin-sensitivity-conferring protein and F6 increased the oligomycin sensitivity of ATPase activity. It was therefore concluded that both of these coupling factors are involved in the conferral of oligomycin sensitivity. 5. The effect of the order of addition of F1, oligomycin-sensitivity-conferring protein and F6 to F0 on the reconstitution of oligomycin-sensitive ATPase activity, and of F1 and oligomycin-sensitivity-conferring protein to submitochondrial particles on the reconstitution of respiratory control, was investigated. The highest values of oligomycin sensitivity and respiratory control were obtained when F1 was added as the first component, indicating that F1 plays a directing role in the organisation of the components.     

The Active and the Inactive Form of the hAT1 Receptor Have an Identical Ligand-Binding Environment: An MPA Study on a Constitutively Active Angiotensin II Receptor Mutant

Several models of activation mechanisms were proposed for G protein-coupled receptors (GPCRs), yet no direct methods exist for their elucidation. The availability of constitutively active mutants has given an opportunity to study active receptor conformations within acceptable limits using models such as the angiotensin II type 1 (AT₁)¹ receptor mutant N111G-hAT₁ which displays an important constitutive activity. Recently, by using methionine proximity assay, we showed for the hAT₁ receptor that TMD III, VI, and VII form the ligand-binding pocket of the C-terminal amino acid of an antagonistic AngII analogue. In the present contribution, we investigated whether the same residues would also constitute the ligand-binding contacts in constitutively activated mutant (CAM) receptors. For this purpose, the same Met mutagenesis strategy was carried out on the N111G double mutants. Analysis of 43 receptors mutants in the N111G-hAT₁ series, photolabeled and CNBr digested, showed that there were only subtle structural changes between the wt-receptor and its constitutively active form.     

Multifactorial diversity sustains microbial community stability

Maintenance of a high degree of biodiversity in homogeneous environments is poorly understood. A complex cheese starter culture with a long history of use was characterized as a model system to study simple microbial communities. Eight distinct genetic lineages were identified, encompassing two species: Lactococcus lactis and Leuconostoc mesenteroides. The genetic lineages were found to be collections of strains with variable plasmid content and phage sensitivities. Kill-the-winner hypothesis explaining the suppression of the fittest strains by density-dependent phage predation was operational at the strain level. This prevents the eradication of entire genetic lineages from the community during propagation regimes (back-slopping), stabilizing the genetic heterogeneity in the starter culture against environmental uncertainty.     

Quality by design approach for viral clearance by protein a chromatography

Protein A chromatography is widely used as a capture step in monoclonal antibody (mAb) purification processes. Antibodies and Fc fusion proteins can be efficiently purified from the majority of other complex components in harvested cell culture fluid (HCCF). Protein A chromatography is also capable of removing modest levels of viruses and is often validated for viral clearance. Historical data mining of Genentech and FDA/CDER databases systematically evaluated the removal of model viruses by Protein A chromatography. First, we found that for each model virus, removal by Protein A chromatography varies significantly across mAbs, while remains consistent within a specific mAb product, even across the acceptable ranges of the process parameters. In addition, our analysis revealed a correlation between retrovirus and parvovirus removal, with retrovirus data generally possessing a greater clearance factor. Finally, we describe a multivariate approach used to evaluate process parameter impacts on viral clearance, based on the levels of retrovirus‐like particles (RVLP) present among process characterization study samples. It was shown that RVLP removal by Protein A is robust, that is, parameter effects were not observed across the ranges tested. Robustness of RVLP removal by Protein A also correlates with that for other model viruses such as X‐MuLV, MMV, and SV40. The data supports that evaluating RVLP removal using process characterization study samples can establish multivariate acceptable ranges for virus removal by the protein A step for QbD. By measuring RVLP instead of a model retrovirus, it may alleviate some of the technical and economic challenges associated with performing large, design‐of‐experiment (DoE)—type virus spiking studies. This approach could also serve to provide useful insight when designing strategies to ensure viral safety in the manufacturing of a biopharmaceutical product. Biotechnol. Bioeng. 2014;111: 95–103. © 2013 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

On the 13C/12C isotopic signal of day and night respiration at the mesocosm level

While there is currently intense effort to examine the ¹³C signal of CO₂ evolved in the dark, less is known on the isotope composition of day‐respired CO₂. This lack of knowledge stems from technical difficulties to measure the pure respiratory isotopic signal: day respiration is mixed up with photorespiration, and there is no obvious way to separate photosynthetic fractionation (pure c_i/c_a effect) from respiratory effect (production of CO₂ with a different δ¹³C value from that of net‐fixed CO₂) at the ecosystem level. Here, we took advantage of new simple equations, and applied them to sunflower canopies grown under low and high [CO₂]. We show that whole mesocosm‐respired CO₂ is slightly ¹³C depleted in the light at the mesocosm level (by 0.2–0.8‰), while it is slightly ¹³C enriched in darkness (by 1.5–3.2‰). The turnover of the respiratory carbon pool after labelling appears similar in the light and in the dark, and accordingly, a hierarchical clustering analysis shows a close correlation between the ¹³C abundance in day‐ and night‐evolved CO₂. We conclude that the carbon source for respiration is similar in the dark and in the light, but the metabolic pathways associated with CO₂ production may change, thereby explaining the different ¹²C/¹³C respiratory fractionations in the light and in the dark.     

Genetic portrait of Lisboa immigrant population from Cabo Verde with mitochondrial DNA analysis

Journal of Genetics -