scholarly journals Identification of Membrane Proteins in the Hyperthermophilic ArchaeonPyrococcus FuriosusUsing Proteomics and Prediction Programs

2001 ◽  
Vol 2 (5) ◽  
pp. 275-288 ◽  
Author(s):  
James F. Holden ◽  
Farris L. Poole II ◽  
Sandra L. Tollaksen ◽  
Carol S. Giometti ◽  
Hanjo Lim ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Membrane Fraction ◽  
Pyrococcus Furiosus ◽  
Cell Fractionation ◽  
Cytoplasmic Fraction ◽  
Atpase Subunit ◽  
Consensus Prediction ◽  
Associated Proteins ◽  
2D Gel ◽  
Membrane Components

Cell-free extracts from the hyperthermophilic archaeonPyrococcus furiosuswere separated into membrane and cytoplasmic fractions and each was analyzed by 2D-gel electrophoresis. A total of 66 proteins were identified, 32 in the membrane fraction and 34 in the cytoplasmic fraction. Six prediction programs were used to predict the subcellular locations of these proteins. Three were based on signal-peptides (SignalP, TargetP, and SOSUISignal) and three on transmembrane-spanning α-helices (TSEG, SOSUI, and PRED-TMR2). A consensus of the six programs predicted that 23 of the 32 proteins (72%) from the membrane fraction should be in the membrane and that all of the proteins from the cytoplasmic fraction should be in the cytoplasm. Two membrane-associated proteins predicted to be cytoplasmic by the programs are also predicted to consist primarily of transmembrane-spanningβ-sheets using porin protein models, suggesting that they are, in fact, membrane components. An ATPase subunit homolog found in the membrane fraction, although predicted to be cytoplasmic, is most likely complexed with other ATPase subunits in the membrane fraction. An additional three proteins predicted to be cytoplasmic but found in the membrane fraction, may be cytoplasmic contaminants. These include a chaperone homolog that may have attached to denatured membrane proteins during cell fractionation. Omitting these three proteins would boost the membrane-protein predictability of the models to near 80%. A consensus prediction using all six programs for all 2242 ORFs in theP. furiosusgenome estimates that 24% of the ORF products are found in the membrane. However, this is likely to be a minimum value due to the programs’ inability to recognize certain membrane-related proteins, such as subunits associated with membrane complexes and porin-type proteins.

Identification of nuclei associated proteins by 2D-gel electrophoresis and mass spectrometry

2001 ◽  
Vol 109 (1) ◽  
pp. 3-11 ◽  
Author(s):  
Jonas Bergquist ◽  
Johan Gobom ◽  
Anders Blomberg ◽  
Peter Roepstorff ◽  
Rolf Ekman
Keyword(s):  
Mass Spectrometry ◽  
Gel Electrophoresis ◽  
2D Gel Electrophoresis ◽  
Associated Proteins ◽  
2D Gel

scholarly journals Structural membrane proteins and loosely associated proteins of the sarcoplasmic reticulum

1974 ◽  
Vol 139 (3) ◽  
pp. 509-513 ◽  
Author(s):  
A. Margreth ◽  
U. Carraro ◽  
G. Salviati
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Membrane Proteins ◽  
Osmotic Shock ◽  
Protein Composition ◽  
Polyacrylamide Gels ◽  
Associated Proteins

The protein composition of sarcoplasmic-reticulum vesicles, either unpurified or after fractionation on sucrose gradients, and with or without previous osmotic shock and sonication, was investigated by electrophoresis in acid polyacrylamide gels. The pattern of release of loosely bound proteins is discussed with respect to their localization in the interior of the vesicles.

scholarly journals The High-Molecular-Weight Cytochrome c Cyc2 of Acidithiobacillus ferrooxidans Is an Outer Membrane Protein

2002 ◽  
Vol 184 (1) ◽  
pp. 313-317 ◽  
Author(s):  
Andrés Yarzábal ◽  
Gaël Brasseur ◽  
Jeanine Ratouchniak ◽  
Karen Lund ◽  
Danielle Lemesle-Meunier ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Outer Membrane ◽  
High Molecular Weight ◽  
Acidithiobacillus Ferrooxidans ◽  
Ferrous Iron ◽  
Membrane Fraction ◽  
Cytoplasmic Membrane ◽  
Respiratory Pathway ◽  
Membrane Components ◽  
Membrane Level

ABSTRACT A high-molecular-weight c-type cytochrome, Cyc2, and a putative 22-kDa c-type cytochrome were detected in the membrane fraction released during spheroplast formation from Acidithiobacillus ferrooxidans. This fraction was enriched in outer membrane components and devoid of cytoplasmic membrane markers. The genetics, as well as the subcellular localization of Cyc2 at the outer membrane level, therefore make it a prime candidate for the initial electron acceptor in the respiratory pathway between ferrous iron and oxygen.

scholarly journals The Fusion of Lipid and DNA Nanotechnology

Genes ◽  
2019 ◽  
Vol 10 (12) ◽  
pp. 1001 ◽  
Author(s):  
Es Darley ◽  
Jasleen Kaur Daljit Singh ◽  
Natalie A. Surace ◽  
Shelley F. J. Wickham ◽  
Matthew A. B. Baker
Keyword(s):  
Membrane Proteins ◽  
Lipid Membranes ◽  
Dna Nanotechnology ◽  
Bilayer Membrane ◽  
Diverse Range ◽  
Impermeable Barrier ◽  
Self Assembling ◽  
Associated Proteins ◽  
Fundamental Biological Process

Lipid membranes form the boundary of many biological compartments, including organelles and cells. Consisting of two leaflets of amphipathic molecules, the bilayer membrane forms an impermeable barrier to ions and small molecules. Controlled transport of molecules across lipid membranes is a fundamental biological process that is facilitated by a diverse range of membrane proteins, including ion-channels and pores. However, biological membranes and their associated proteins are challenging to experimentally characterize. These challenges have motivated recent advances in nanotechnology towards building and manipulating synthetic lipid systems. Liposomes—aqueous droplets enclosed by a bilayer membrane—can be synthesised in vitro and used as a synthetic model for the cell membrane. In DNA nanotechnology, DNA is used as programmable building material for self-assembling biocompatible nanostructures. DNA nanostructures can be functionalised with hydrophobic chemical modifications, which bind to or bridge lipid membranes. Here, we review approaches that combine techniques from lipid and DNA nanotechnology to engineer the topography, permeability, and surface interactions of membranes, and to direct the fusion and formation of liposomes. These approaches have been used to study the properties of membrane proteins, to build biosensors, and as a pathway towards assembling synthetic multicellular systems.

scholarly journals Large-scale quantitative LC-MS/MS analysis of detergent-resistant membrane proteins from rat renal collecting duct

2008 ◽  
Vol 295 (3) ◽  
pp. C661-C678 ◽  
Author(s):  
Ming-Jiun Yu ◽  
Trairak Pisitkun ◽  
Guanghui Wang ◽  
Juan F. Aranda ◽  
Patricia A. Gonzales ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Large Scale ◽  
Collecting Duct ◽  
Rat Kidney ◽  
Apical Region ◽  
Annexin 2 ◽  
Associated Proteins ◽  
Medullary Collecting Duct ◽  
Renal Collecting Duct

In the renal collecting duct, vasopressin controls transport of water and solutes via regulation of membrane transporters such as aquaporin-2 (AQP2) and the epithelial urea transporter UT-A. To discover proteins potentially involved in vasopressin action in rat kidney collecting ducts, we enriched membrane “raft” proteins by harvesting detergent-resistant membranes (DRMs) of the inner medullary collecting duct (IMCD) cells. Proteins were identified and quantified with LC-MS/MS. A total of 814 proteins were identified in the DRM fractions. Of these, 186, including several characteristic raft proteins, were enriched in the DRMs. Immunoblotting confirmed DRM enrichment of representative proteins. Immunofluorescence confocal microscopy of rat IMCDs with antibodies to DRM proteins demonstrated heterogeneity of raft subdomains: MAL2 (apical region), RalA (predominant basolateral labeling), caveolin-2 (punctate labeling distributed throughout the cells), and flotillin-1 (discrete labeling of large intracellular structures). The DRM proteome included GPI-anchored, doubly acylated, singly acylated, cholesterol-binding, and integral membrane proteins (IMPs). The IMPs were, on average, much smaller and more hydrophobic than IMPs identified in non-DRM-enriched IMCD. The content of serine 256-phosphorylated AQP2 was greater in DRM than in non-DRM fractions. Vasopressin did not change the DRM-to-non-DRM ratio of most proteins, whether quantified by tandem mass spectrometry (LC-MS/MS, n = 22) or immunoblotting ( n = 6). However, Rab7 and annexin-2 showed small increases in the DRM fraction in response to vasopressin. In accord with the long-term goal of creating a systems-level analysis of transport regulation, this study has identified a large number of membrane-associated proteins expressed in the IMCD that have potential roles in vasopressin action.

Different sites of adenosine formation in the heart

1981 ◽  
Vol 240 (6) ◽  
pp. H963-H970 ◽  
Author(s):  
W. Schutz ◽  
J. Schrader ◽  
E. Gerlach
Keyword(s):  
Guinea Pig ◽  
Membrane Fraction ◽  
Isolated Heart ◽  
Adenosine Kinase ◽  
Nucleoside Transport ◽  
Myocardial Tissue ◽  
Cell Fractionation ◽  
Adenosylhomocysteine Hydrolase ◽  
Alpha Beta ◽  
Uptake And Release

In an attempt to further define the site of myocardial adenosine formation, isolated guinea pig hearts were perfused with potent inhibitors of 5'-nucleotidase [alpha, beta-methylene adenosine 5'-diphosphate (AOPCP)] and of nucleoside transport [4-nitrobenzyl thioinosine (NBMPR)]. AOPCP (50 microM) inhibited the activity of cardiac ecto-5'-nucleotidase by 85% but did not influence the release of adenosine, inosine, and hypoxanthine formed at an accelerated rate by the heart during hypoxic perfusion (30% O2). In contrast, NBMPR (5 microM) diminished the hypoxia-induced release of adenosine and its degradatives and greatly potentiated the increase of myocardial tissue levels of respective purine compounds. Studies carried out with 5'-deoxyadenosine, an adenosine derivative that is not metabolized, indicate NBMPR to inhibit both uptake and release of adenosine in the isolated heart and in human erythrocytes. Cell fractionation studies on guinea pig ventricular muscle revealed that 5'-nucleotidase, though mainly associated with the membrane fraction, is also found in the cardiac cytosol (200,000-g supernatant), exhibiting a different substrate specificity. Furthermore, S-adenosylhomocysteine hydrolase as well as adenosine kinase and adenosine deaminase proved to be exclusively present in the cytosolic fraction. Our findings suggest that in the hypoxic heart a) ecto-5'-nucleotidase most likely is not involved in the formation of adenosine, b) release of adenosine from the heart requires adenosine to be transported across the sarcolemma membrane, and c) adenosine is predominantly formed intracellularly, a process involving cytosolic 5'-nucleotidase and/or S-adenosylhomocysteine hydrolase.

scholarly journals Physiological Responses of the Hyperthermophilic Archaeon “Pyrococcus abyssi” to DNA Damage Caused by Ionizing Radiation

2003 ◽  
Vol 185 (13) ◽  
pp. 3958-3961 ◽  
Author(s):  
Edmond Jolivet ◽  
Fujihiko Matsunaga ◽  
Yoshizumi Ishino ◽  
Patrick Forterre ◽  
Daniel Prieur ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Ionizing Radiation ◽  
Dna Synthesis ◽  
Gamma Ray ◽  
Pyrococcus Furiosus ◽  
Repair System ◽  
Cell Fractionation ◽  
Chromosomal Dna ◽  
Pyrococcus Abyssi

ABSTRACT The mechanisms by which hyperthermophilic Archaea, such as “Pyrococcus abyssi” and Pyrococcus furiosus, survive high doses of ionizing gamma irradiation are not thoroughly elucidated. Following gamma-ray irradiation at 2,500 Gy, the restoration of “P. abyssi” chromosomes took place within chromosome fragmentation. DNA synthesis in irradiated “P. abyssi” cells during the DNA repair phase was inhibited in comparison to nonirradiated control cultures, suggesting that DNA damage causes a replication block in this organism. We also found evidence for transient export of damaged DNA out of irradiated “P. abyssi” cells prior to a restart of chromosomal DNA synthesis. Our cell fractionation assays further suggest that “P. abyssi” contains a highly efficient DNA repair system which is continuously ready to repair the DNA damage caused by high temperature and/or ionizing radiation.

A study of Erythrocyte Membrane Proteins and Urinary Polypeptides in Conga Drumming Haemoglobinuria

1985 ◽  
Vol 69 (1) ◽  
pp. 105-108 ◽  
Author(s):  
Richard D. Levy ◽  
Ali A. Khaleeli ◽  
Beatrice L. Griffiths ◽  
Yvonne H. Edwards
Keyword(s):  
Membrane Proteins ◽  
Carbonic Anhydrase ◽  
Erythrocyte Membrane ◽  
Acute Phase ◽  
Protein Staining ◽  
Membrane Components ◽  
Erythrocyte Membrane Proteins ◽  
Erythrocyte Carbonic Anhydrase

1. The erythrocyte membrane proteins and glycoproteins and urinary polypeptides have been examined in a patient exhibiting intermittent pigmenturia associated with conga drumming. 2. Significant excretion of haemoglobin, albumin and probably erythrocyte carbonic anhydrase but not myoglobin occurred during the acute phase of the conga drumming-induced pigmenturia. This usually ceased within 24-48 h. 3. We found no evidence of aberrant erythrocyte membrane components on electrophoresis with either protein staining or a range of 125I-labelled lectins used for detection.

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