Fourier transform infrared spectroscopic study of ion binding and intramolecular interactions in the polar head of digalactosyldiacylglycerol

Lipid bilayers composed of digalactosyldiacyl-glycerol (DGDG), that is, Galp1-6Galp1-3DAG, a non-ionic lipid of the thylakoid membrane of chloroplasts, aggregate in aqueous media containing mono- and divalent cations in amounts above a threshold concentration (C_t) of about 1.0, 4.7 and 10.0 mM for Ca²⁺, Mg²⁺ and Na⁺, respectively. In this work, we found that above C_t the DGDG membranes do not undergo fusion and that the aggregation can be reversed, or disrupted. This means that the perturbation induced by the salts results from adsorption, or complexation of the ions in the polar head of DGDG. To investigate this question, we used Fourier transform infrared (FTIR) spectroscopy to identify the molecular sites in DGDG which are modified by interaction, or adduct formation with CaCl₂, MgCl₂ and NaCl. We also determined whether the ions affect the intramolecular hydrogen bonding between the sn₂ ester C = O and the carbon-6 of the -anomer of galactose (Gal). The major conclusions are: (i) the salts do not affect, at least directly, the, ester carbonyl region of DGDG, (ii) the most probable sites of binding, or adsorption, for the ions are the ring oxygen, and (iii) the ring hydroxyls are the sites of either ion complexation or intra- and intermolecular H-bonding in interacting DGDG membranes. Within this framework, the complexation of the ions with Gal might induce total or partial dehydration of the galactolipid headgroup and thus provides the means to overcome the repulsive hydration forces that hinder aggregation of the DGDG membranes.Abbreviations DGDG digalactosyldiacylglycerol - EDTA ethylenediaminetetracetic acid - FTIR Fourier transform infrared - Gal galactose - GIDG D-glucosyldiacylglycerol - Glyc glycerol - LHCII chloroplast light harvesting complex II - MGDG monogalactosyldiacylglycerol - PC phosphatidylcholine - PG phosphatidylglycerol - PS phosphatidylserine - SQDG sulfoquinovosyl-diacylglycerol Correspondence to: M. Fragata     

TheEscherichia coli mannitol permease as a model for transport via the bacterial phosphotransferase system

The bacterial phosphoenolpyruvate-dependent carbohydrate phosphotransferase system (PTS) consists of several proteins whose primary functions are to transport and phosphorylate their substrates. The complexity of the PTS undoubtedly reflects its additional roles in chemotaxis to PTS substrates and in regulation of other metabolic processes in the cell. The PTS permeases (Enzymes II) are the membrane-associated proteins of the PTS that sequentially recognize, transport, and phosphorylate their specific substrates in separate steps, and theEscherichia coli mannitol permease is one of the best studied of these proteins. It consists of two cytoplasmic domains (EIIA and EIIB) involved in mannitol phosphorylation and an integral membrane domain (EIIC) which is sufficient to bind mannitol, but which transports mannitol at a rate that is dependent on phosphorylation of the EIIA and EIIB domains. Recent results show that several residues in a hydrophilic, 85-residue segment of the EIIC domain are important for the binding, transport, and phosphorylation of mannitol. This segment may be at least partially exposed to the cytoplasm of the cell. A model is proposed in which this region of the EIIC domain is crucial in coupling phosphorylation of the EIIB domain to transport through the EIIC domain of the mannitol permease.     

Photosynthetic water oxidation: The protein framework

Approximately 20 protein subunits are associated with the PS II complex, not counting subunits of peripheral light-harvesting antenna complexes. However, it is not yet established which proteins specifically are involved in the water-oxidation process. Much evidence supports the concept that the D1/D2 reaction center heterodimer not only plays a central role in the primary photochemistry of Photosystem II, but also is involved in electron donation to P680 and in ligation of the manganese cluster. This evidence includes (a) the primary donor to P680 has been shown to be a redox-active tyrosyl residue (Tyr161) in the D1 protein, and (b) site-directed mutagenesis and computer-assisted modeling of the reaction center heterodimer have suggested several sites with a possible function in manganese ligation. These include Asp170, Gln165 and Gln189 of the D1 protein and Glu69 of the D2 protein as well as the C-terminal portion of the mature D1 protein. Also, hydrophilic loops of the chlorophyll-binding protein CP43 that are exposed at the inner thylakoid surface could be essential for the water-splitting process.In photosynthetic eukaryotes, three lumenal extrinsic proteins, PS II-O (33 kDa), PS II-P (23 kDa) and PS II-Q (16 kDa), influence the properties of the manganese cluster without being involved in the actual catalysis of water oxidation. The extrinsic proteins together may have multiple binding sites to the integral portion of PS II, which could be provided by the D1/D2 heterodimer and CP47. A major role for the PS II-O protein is to stabilize the manganese cluster. Most experimental evidence favors a connection of the PS II-P protein with binding of the Cl^- and Ca²⁺ ions required for the water oxidation, while the PS II-Q protein seems to be associated only with the Cl^- requirement. The two latter proteins are not present in PS II of prokaryotic organisms, where their functions may be replaced by a 10–12 kDa subunit and a newly discovered low-potential cytochrome c-550.Abbreviations PS II Photosystem II - PCC Pasteur Culture Collection     

In situ hybridization and immunocytochemical localization of SERCA2 encoded Ca2+ pump in rabbit heart and stomach

Heart tissue contains large amounts of the protein encoded by the Ca²⁺ pump gene SERCA2. The SERCA2 RNA can be spliced alternatively to produce mRNA encoding the proteins SERCA2a and SERCA2b which differ in their C-terminal sequences. In this study we report the tissue distribution of SERCA2a and SERCA2b isoforms byin situ hybridization to rabbit heart and stomach. The expression of SERCA2 mRNA was high in myocardial cells, being the highest in the atrial region. In contrast, there was more SERCA2 protein in Western blots in ventricles than in atria. Myocardial cells expressed predominantly the mRNA for the isoform SERCA2a. Whereas the stomach smooth muscle and the neuronal plexus expressed SERCA2 at levels much lower than myocardial cells, the expression was very high in the stomach mucosa. Mucosa contained mainly the mRNA for SERCA2b. From immunocytochemistry it was concluded that the anti-heart SR Ca²⁺ pump antibody IID8 reacted much better with heart and surface mucosal cells in the stomach than with the stomach smooth muscle, and that IID8 reactivity was intracellular. In contrast PM4A2B, an antibody against the plasma membrane Ca²⁺ pump, reacted well with heart and stomach smooth muscle, plexus and mucosa, and its localization appeared to be in the plasma membrane. Thus, stomach smooth muscle expressed SERCA2b mRNA and protein at low levels, mucosa expressed SERCA2b mRNA and protein at high levels, atria and ventricle expressed SERCA2a mRNA and protein at high levels, mRNA being more in atria, but protein being more in ventricles.Deceased August 14, 1992     

甲基毒死蜱对红细胞膜乙酰胆碱酯酶的抑制作用及其与膜脂相互关系

以人红细胞膜为材料,研究了甲基毒死蜱与膜上乙酰胆碱酯酶(AChE)的相互作用及其与膜脂的关系。结果显示,甲基毒死蜱对人红细胞膜AChE有明显的抑制作用,与膜温育30min,其半数抑制浓度约为0.10 mmol/L。动力学分析表明,其抑制作用为非竞争性。0.2％Triton X-100并不改变AChE对甲基毒死蜱的敏感性,亦即AChE上甲基毒死蜱的作用部位与其所处的脂质微环境无关。     

An endogenous factor which interacts with synaptosomal membrane Na+, K+-ATPase activation by K+

In previous papers, the isolation of brain soluble fractions able to modify neuronal Na⁺, K⁺-ATPase activity has been described. One of those fractions-peak I-stimulates membrane Na⁺, K⁺-ATPase while another-peak II-inhibits this enzyme activity, and has other ouabain-like properties. In the present study, synaptosomal membrane Na⁺, K⁺-ATPase was analyzed under several experimental conditions, using ATP orp-nitrophenylphosphate (p-NPP) as substrate, in the absence and presence of cerebral cortex peak II. Peak II inhibited K⁺-p-NPPase activity in a concentration dependent manner. Double reciprocal plots indicated that peak II uncompetitively inhibits K⁺-p-NPPase activity regarding substrate, Mg²⁺ and K⁺ concentration. Peak II failed to block the known K⁺-p-NPPase stimulation caused by ATP plus Na⁺. At various K⁺ concentrations, percentage K⁺-p-NPPase inhibition by peak II was similar regardless of the ATP plus Na⁺ presence, indicating lack of correlation with enzyme phosphorylation. Na⁺, K⁺-ATPase activity was decreased by peak II depending on K⁺ concentration. It is postulated that the inhibitory factor(s) present in peak II interfere(s) with enzyme activation by K⁺.     

Evidence for the presence of a receptor for the cytolethal distending toxin (CLDT) of Campylobacter jejuni on CHO and HeLa cell membranes and development of a receptor-based enzyme-linked immunosorbent assay for detection of CLDT

Abstract Using ligand blotting, it was found that partially purified cytolethal distending toxin prepared from and enterotoxigenic strain of Campylobacter jejuni , bound to two peptides of molecular masses of approximately 59 kDa and 45 kDa and to a single peptide of 59 kDa in protein blots prepared from HeLa and CHO cell membranes, respectively. In contrast, labile toxin of Escherichia coli and cholera toxin bound to a single peptide of the same molecular mass (15 kDa) on protein blots prepared from both CHO and HeLa cell crude membranes resolved by gel electrophoresis. This banding pattern was identical using SDS-solubilized membrane, with or without heat treatment, but no band was obtained when reduced (treatment with 2-mercaptoethanol) samples were used for the gel electrophoresis. The differences between receptors of cytolethal distending toxin and cholera toxin/labile toxin were exploited to develop a receptor-based enzyme-linked immunosorbent assay for detection of cytolethal distending toxin which involved the consecutive addition of either solubilized CHO or HeLa membranes, antigen and antibody. This enzyme-linked immunosorbent assay consistently detected crude cytolethal distending toxin diluted up to 16-fold. The receptor-based enzyme-linked immunosorbent assay for detection of cytolethal distending toxin developed in this study is a suitable alternative assay which can be performed easily in laboratories with minimal facilities and, more importantly, the results are available within a few hours as compared to times of up to 5 days in the conventional tissue culture detection of cytolethal distending toxin.     

Integrated membrane systems for gas separation in biotechnology: potential and prospects

Integrated non-porous membrane systems were applied for microbial combustible gas separation processes. Methane/CO₂ mixtures of various concentrations from methane fermentation processes (biogas) were separated using a membrane-separation complex of permabsorber type into individual components of technical grade (more than 95% purity). In experiments with three-component mixtures, using a selective membrane valve with various liquid carriers, all the gases of interest (H₂, CH₄ and CO₂) were obtained at greater than 90% purity in one separation step. The perspectives for the further application of non-porous membrane separating devices for various gaseous mixtures from different microbial processes are discussed.V. Teplyakov and E. Sostina are with the A.V. Topchiev institute of Petrochemical Synthesis, Russian Academy of Sciences, Membrane Research Center, Moscow 117912, Russia. E. Sostina is also, and A. Netrusov is with the Microbiology Department, Moscow University, Moscow 119899, Russia. I. Beckman is with the Chemistry Department, Moscow University, Moscow 119899, Russia.     

Membrane fatty acid composition of tissues is related to body mass of mammals

Phospholipids were extracted from tissues (heart, skeletal muscle, kidney cortex, liver and brain) of mammals representing a 9,000-fold range in body mass (mouse, rat, rabbit, sheep and cattle) and their fatty acid composition was determined. In heart, skeletal muscle and kidney cortex, there were significant allometric decreases in the Unsaturation Index (UI; average number of double bonds per 100 fatty acid molecules) with increasing body mass. There were significant inverse allometric relationships between body mass and the proportion of docosahexaenoic acid (2263) in heart and skeletal muscle. In heart, skeletal muscle and kidney cortex, larger mammals also had shorter fatty acid chains in their phospholipids and a higher proportion of monounsaturates. In liver, smaller mammals had a higher UI than larger mammals (except the rabbit, which had the lowest UI and very low proportions of 3 fatty acids). The brain of all mammals maintained a high UI with similar levels of polyunsaturated fatty acids, especially 2263. Our results suggest that in heart, skeletal muscle and kidney cortex the activity of the elongases and desaturases are reduced in large mammals compared to small mammals. The allometric trends in membrane composition may be involved in modifying membrane permeability. It is proposed that the elevated degree of polyunsaturation in the membranes of several tissues from small mammals is related to their higher metabolic activity.This work was supported by an Australian Commonwealth Postgraduate Research Scholarship from the University of Wollongong to P. Couture and by a grant from the Australian Research Council to A.J. Hulbert. We wish to thank Voytek Mantaj for technical assistance.     

Cross-flow ultrafiltration of proteins through asymmetric polysulfonic membranes: I. Retention curves and pore size distributions

Flux and retention of 0.1%w/w aqueous solutions of several proteins [lysozyme, pepsin, bovine serum albumin (BSA), lipase, and gamma-globulin] with molecular weights of 14.6, 36, 67, 801 and 150 kDa are studied when they are tangentially filtered, with transmembrane pressure differences until 1 MPa and circulation velocities in the re-tentate loop from 0.04 to 1.98 m/s (laminar regime), through two asymmetric polysulfone commercial membranes (E-100 with a nominal pore size of 0.01 mum and E-500 with a nominal pore size of 0.04 mum). Results are analyzed with the film theory for the concentration-polarization phenomenon, obtaining the mass transfer coefficient along with the apparent and true retention coefficients for the cell used, as a function of the feed circulation velocity and the molecular weight of the solute. The standard retention curves lead to pore size distributions differing from the nominal ones. These differences can be attributed to the modifications of the membranes when they are in operational conditions, probably due to protein adsorption. (c) 1995 John Wiley & Sons, Inc.