Photoaffinity spin-labeling of the Ca2+ ATPase in sarcoplasmic reticulum : Evidence for oligomeric structure

A spin labeled fatty acid (16-doxylstearic acid) was linked to a photochemical reacting group (azido derivative). When the molecule is introduced, at a low concentration, into rabbit sarcoplasmic reticulum membranes, the spectrum before illumination is identical to the spectrum obtained with the corresponding spin labeled fatty acid. After illumination, a large immobilized components is seen. It corresponds to about 70% of the ESR signal of the effectively bound label, at room temperature. The fraction of immobilized component varies with temperature, from 100% at 0°C to 50% at 35°C. Addition of a small amount of detergent (dodecyl octaethylene glycol monoether), under non solubilizing conditions, decreases the fraction of signal due to a strongly immobilized probe. A possible interpretation is that the immobilized signal reflects protein bound spin labels trapped in Ca²⁺ ATPase oligomers, which are partially dissociated by detergent addition or temperature increase.     

Membrane solubilization by detergent: use of brominated phospholipids to evaluate the detergent-induced changes in Ca2+-ATPase/lipid interaction

The solubilization and delipidation of sarcoplasmic reticulum Ca2+-ATPase by different nonionic detergents were measured from changes in turbidity and recovery of intrinsic fluorescence of reconstituted ATPase in which tryptophan residues had been quenched by replacement of endogenous phospholipids with brominated phospholipids. It was found that incorporation of C12E8 or dodecyl maltoside (DM) at low concentrations in the membrane, resulting in membrane "perturbation" without solubilization, displaced a few of the phospholipids in contact with the protein; perturbation was evidenced by a parallel drop in ATPase activity. As a result of further detergent addition leading to solubilization, the tendency toward delipidation of the immediate environment of the protein was stopped, and recovery of enzyme activity was observed, suggesting reorganization of phospholipid and detergent molecules in the solubilized ternary complex, as compared to the perturbed membrane. After further additions of C12E8 or DM to the already solubilized membrane, the protein again experienced progressive delipidation which was only completed at a detergent concentration about 100-fold higher than that necessary for solubilization. Delipidation was correlated with a decrease in enzyme activity toward a level similar to that observed during perturbation. On the other hand, Tween 80, Tween 20, and Lubrol WX failed to solubilize SR membranes and to induce further ATPase delipidation when added after preliminary SR solubilization by C12E8 or dodecyl maltoside. For Tween 80, this can be related to an inability to solubilize pure lipid membrane; in contrast, Tween 20 and Lubrol WX were able to solubilize liposomes but not efficiently to solubilize SR membranes. In all three cases, insertion of the detergent in SR membranes is, however, demonstrated by perturbation of enzyme activity. Correlation between detergent structure and ability to solubilize and delipidate the ATPase suggests that one parameter impeding ATPase solubilization might be the presence of a bulky detergent polar headgroup, which could not fit close to the protein surface. We also conclude that in the active protein/detergent/lipid ternary complexes, solubilized by C12E8 or dodecyl maltoside, most phospholipids remain closely associated with the ATPase hydrophobic surface as in the membranous form. Binding of only a few detergent molecules on this hydrophobic surface may be sufficient for inhibition of ATPase activity observed at high ATP concentration, both during perturbation and in the completely delipidated, solubilized protein.(ABSTRACT TRUNCATED AT 400 WORDS)     

Mode of interaction of polyoxyethyleneglycol detergents with membrane proteins

Binding of dodecyloctaethyleneglycol monoether (C12E3) and purified Triton X-100 to various integral membrane proteins was studied by chromatographic procedures. Binding capacity decreased in the following order: bovine rhodopsin greater than photochemical reaction center greater than sarcoplasmic reticulum Ca2+-ATPase. The detergents were bound in different amounts to the proteins and less than corresponding to the aggregation number of the pure micelles. Appreciable binding of C12E8 to Ca2+-ATPase was observed far below the critical micelle concentration, consistent with interaction of the membrane protein with non-micellar detergent. Model calculations indicate that the detergents cannot combine with the membrane proteins, forming an oblate ring similar to that of pure detergent micelles, such as has been previously proposed for e.g. cytochrome b5 [Robinson and Tanford (1975) Biochemistry, 14, 365-378]. Other arrangements (prolate and monolayer rings), in which all detergent molecules are in contact with the protein, are considered as alternatives for covering the hydrophobic surface of the membrane protein with a continuous layer of detergent.     

Diatom assemblages in superficial sediments from the Gulf of Riga,eastern Baltic Sea

Twenty one superficial sediment samples from areas of high and moderate eutrophication in the Gulf of Riga were studied with respect to siliceous microfossils, mainly diatoms.The results seem to imply that the number of taxa and the abundance of the frustules are affected by runoff from rivers and the degree of eutrophication in different parts of the Gulf. Areas with high eutrophication, e.g. the river estuaries, have diatom assemblages of varying composition, while in areas with moderate eutrophication the composition is almost constant and the influx of freshwater diatoms and littoral periphyton small. The high abundance and low diversity of brackish-marine and brackish, planktonic diatoms seem to be a result of an influx of nutrients and pollutants in the water and bottom sediments. The sea ice diatoms occurring in the Baltic waters and also in the Gulf of Riga tend to be resistant to eutrophication or are even favoured by it.     

The distribution of coastal fish eDNA sequences in the Anthropocene

Aim

Coastal fishes have a fundamental role in marine ecosystem functioning and contributions to people, but face increasing threats due to climate change, habitat degradation and overexploitation. The extent to which human pressures are impacting coastal fish biodiversity in comparison with geographic and environmental factors at large spatial scale is still under scrutiny. Here, we took advantage of environmental DNA (eDNA) metabarcoding to investigate the relationship between fish biodiversity, including taxonomic and genetic components, and environmental but also socio-economic factors.

Location

Tropical, temperate and polar coastal areas.

Time period

Present day.

Major taxa studied

Marine fishes.

Methods

We analysed fish eDNA in 263 stations (samples) in 68 sites distributed across polar, temperate and tropical regions. We modelled the effect of environmental, geographic and socio-economic factors on α- and β-diversity. We then computed the partial effect of each factor on several fish biodiversity components using taxonomic molecular units (MOTU) and genetic sequences. We also investigated the relationship between fish genetic α- and β-diversity measured from our barcodes, and phylogenetic but also functional diversity.

Results

We show that fish eDNA MOTU and sequence α- and β-diversity have the strongest correlation with environmental factors on coastal ecosystems worldwide. However, our models also reveal a negative correlation between biodiversity and human dependence on marine ecosystems. In areas with high dependence, diversity of all fish, cryptobenthic fish and large fish MOTUs declined steeply. Finally, we show that a sequence diversity index, accounting for genetic distance between pairs of MOTUs, within and between communities, is a reliable proxy of phylogenetic and functional diversity.

Main conclusions

Together, our results demonstrate that short eDNA sequences can be used to assess climate and direct human impacts on marine biodiversity at large scale in the Anthropocene and can further be extended to investigate biodiversity in its phylogenetic and functional dimensions.     

Ligand-specific dose-response of heterologously expressed olfactory receptors.

Primary olfactory neuronal cultures exposed to odorant stimulation have previously exhibited concentration-related effects in terms of intracellular cAMP levels and adenylate cyclase activity [Ronnett, G.V., Parfitt, D.J., Hester, L.D. & Snyder, S.H. (1991) PNAS88, 2366-2369]. Maximal stimulation occurred for intermediate concentrations, whereas AC activity declined for both low and high odorant concentrations. We suspected that this behavior might be ascribed to the intrinsic response of the first molecular species concerned by odorant detection, i.e. the olfactory receptor itself. In order to check this hypothesis, we developed an heterologous expression system in mammalian cells to characterize the functional response of receptors to odorants. Two mammalian olfactory receptors were used to initiate the study, the rat I7 olfactory receptor and the human OR17-40 olfactory receptor. The cellular response of transfected cells to an odorant stimulation was tested by a spectrofluorimetric intracellular calcium assay, and proved in all cases to be dose-dependent for the known ligands of these receptors, with an optimal response for intermediate concentrations. Further experiments were carried out with the rat I7 olfactory receptor, for which the sensitivity to an odorant, indicated by the concentration yielding the optimal calcium response, depended on the carbon chain length of the aldehydic odorant. The response is thus both ligand-specific and dose-dependent. We thus demonstrate that a differential dose-response originates from the olfactory receptor itself, which is thus capable of efficient discrimination between closely related agonists.     

Properties of detergent-solubilized and membranous (Ca2+ + Mg2+)-activated ATPase from sarcoplasmic reticulum as studied by sulfhydryl reactivity and ESR spectroscopy. Effect of protein-protein interactions

(1) Sulfhydryl reactivity and electron spin resonance spectra of nitroxide maleimide spin labels, covalently attached to sarcoplasmic reticulum ATPase, were examined on both detergent-solubilized and membranous material. Monomeric and oligomeric ATPases were prepared by the use of dodecyloctaethylene glycol monoether as a solubilizing detergent. (2) Immediately after solubilization, the reaction curve of nonomeric ATPase with 5,5'-dithiobis(2-nitrobenzoate) was characterized by positive cooperativity (S-shaped as a function of time). In contrast, the SH reactivity of both oligomeric and membranous ATPases obeyed usual first-order kinetics and could be analyzed in terms of three classes of reactive site. All enzymatically active ATPase preparations responded to addition of ADP with a decrease in SH reactivity. During enzymatic inactivation of monomeric ATPase, the SH-modification rate was dramatically enhanced with loss of cooperative features. Ca2+ removal from the high-affinity sites stimulated SH reactivity before inactivation had taken place. (3) ESR spectroscopy indicated less motional constraints on monomeric than on oligomeric and membranous ATPases. Arrhenius plots of ESR spectral parameters suggest a conformational transition in both membranous and solubilized ATPases at about 22 degrees C. The transition was also present in EGTA-, but not in heat-inactivated ATPase. Although SH reactivity of monomeric ATPase was dramatically enhanced by EGTA inactivation, the results of ESR, circular dichroism and analytical ultracentrifugation experiments indicate limited conformational changes induced by EGTA treatment. (4) The data indicate marked differences in the properties of monomeric ATPase on the one hand and oligomeric and membranous enzymes on the other hand. They are consistent with previous functional evidence for the presence of ATPase in an associated state in the membrane (M?ller, J.V., Lind, K.E. and Andersen, J.P. (1980) J. Biol. Chem. 255, 1912-1920).     

Comparative immunochemical study of ribosomal proteins from various mammalian cells by two-dimensional immunoelectrophoresis

Summary Proteins isolated from ribosomal subunits of various mammalian cells were analysed comparatively by two different methods: a two-dimensional polyacrylamide gel electrophoresis system and a recently described two-dimensional immunoelectrophoresis technique. For this purpose, antisera were raised in rabbits against the total mixture of ribosomal proteins from murine cells. These sera were characterized by ring-test, double immunodiffusion and two-dimensional immunoelectrophoresis. They were shown to contain antibodies to a large number of ribosomal proteins. Immunoelectrophoretic analysis of 60S and 40S subunit proteins from rabbit, lamb, canine and human cells using anti-murine sera revealed a striking conservation of their antigenic properties. These results corroborated those obtained by two-dimensional polyacrylamide gel electrophoresis.