| 1. |
- Abedinpour, Parisa, et al.
(författare)
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Isolation of a caveolae-enriched fraction from rat lung by affinity partitioning and sucrose gradient centrifugation
- 2003
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Ingår i: Analytical Biochemistry. - 1096-0309. ; 313:1, s. 1-8
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Tidskriftsartikel (refereegranskat)abstract
- Caveolae were isolated from rat lungs by a combination of affinity partitioning and sucrose gradient centrifugation. After homogenization of the lungs directly in a polyethylene glycol–dextran two-phase system and conventional phase partitioning, the polyethylene glycol-rich top phase was affinity partitioned with fresh bottom phase containing dextran-linked wheat-germ agglutinin. The lectin selectively attracted plasma membranes to the bottom phase. The isolated plasma membrane fraction was treated with Triton X-100 or, alternatively, sonicated before centrifugation in a stepwise sucrose gradient. Caveolin-enriched material collected at the 5/24% sucrose boundary. This material also contained 5′-nucleotidase activity and actin. Electron microscopy showed the material to consist of a homogeneous population of 50- to 100-nm vesicles. This purification protocol should allow the facile purification of caveolae also from other tissues, facilitating structural and functional studies.
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| 2. |
- Barinaga-Rementeria Ramírez, Irene, et al.
(författare)
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Affinity partitioning for membrane purification exploiting the biotin-NeutrAvidin interaction - Model study of mixed liposomes and membranes
- 2002
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Ingår i: Journal of Chromatography A. - 0021-9673. ; 971:1-2, s. 117-127
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Tidskriftsartikel (refereegranskat)abstract
- Biotinylated negatively charged liposomes as well as membranes were affinity partitioned in an aqueous poly(ethylene glycol)-dextran two-phase system using NeutrAvidin conjugated to dextran as affinity ligand. Both liposomes and membranes redistributed from top to bottom phase upon addition of NeutrAvidin-dextran. The presence of 35-60 mM Li2SO4 was necessary both to force the components into the top phase without ligand and for ligand-dependent redistribution into the bottom phase. Attaching biotin via a hexanamidohexanoyl spacer and an increased density of biotin or NeutrAvidin enhanced the affinity separation. The separation conditions in these model experiments provide a basis for affinity partitioning of membranes using other affinity ligands.
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| 3. |
- Barinaga-Rementeria Ramírez, Irene, et al.
(författare)
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Affinity partitioning of biotinylated mixed liposomes: effect of charge on biotin-NeutrAvidin interaction
- 2000
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Ingår i: Journal of Chromatography. B. - 1387-2273. ; 743:1-2, s. 389-396
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Tidskriftsartikel (refereegranskat)abstract
- The partitioning behaviour of biotinylated mixed liposomes in aqueous poly(ethylene glycol)/dextran two-phase systems containing NeutrAvidin-dextran suggests that the biotin-NeutrAvidin affinity interaction is charge dependent. Biotinylated phosphatidylcholine liposomes with a low negative surface charge distributed in the NeutrAvidin-containing bottom phase at neutral pH, but the introduction of additional negative charges by including phosphatidylserine or the surfactant sodium dodecylsulfate in the liposomes caused them to distribute in the poly(ethylene glycol)-rich top phase instead. By gradually lowering the pH of the affinity two-phase system below the isoelectric point (6.3) of NeutrAvidin, negatively charged phosphatidylserine/phosphatidylcholine liposomes increasingly were attracted by NeutrAvidin to the bottom phase. It is suggested that acidic amino acids present at the rim of the biotin-binding pocket of NeutrAvidin may interact electrostatically with charged residues of the closely apposed liposome surface affecting the affinity interaction.
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| 4. |
- Barinaga-Rementeria Ramírez, Irene, et al.
(författare)
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Purification of caveolae by affinity two-phase partitioning using biotinylated antibodies and NeutrAvidin-dextran
- 2004
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Ingår i: Analytical Biochemistry. - : Elsevier BV. - 1096-0309 .- 0003-2697. ; 331:1, s. 17-26
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Tidskriftsartikel (refereegranskat)abstract
- A new concept for affinity two-phase partitioning was tested. The partitioning was based on the interaction of target membranes with a primary antibody which, in turn, interacted with a biotinylated secondary antibody and NeutrAvidin-dextran in a poly(ethylene glycol)/dextran two-phase system. Caveolae selectively redistributed from the top phase to the NeutrAvidin-dextran-containing bottom phase by employing anti-caveolin as the primary antibody. This immunoaffinity approach was more selective than the established sucrose gradient centrifugation method and resulted in highly purified caveolae from Triton X-100-treated liver and lung plasma membranes. The same approach, employing other selective primary antibodies, should facilitate the purification also of other membrane fractions. (C) 2004 Elsevier Inc. All rights reserved.
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| 5. |
- Ekblad, Lars, et al.
(författare)
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Aqueous two-phase affinity partitioning of biotinylated liposomes using neutral avidin as affinity ligand
- 1998
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Ingår i: Journal of Chromatography A. - 0021-9673. ; 815:2, s. 189-195
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Tidskriftsartikel (refereegranskat)abstract
- Biotinylated small unilamellar liposomes were affinity partitioned in an aqueous poly(ethylene glycol)-dextran two-phase system using avidin coupled to dextran as affinity ligand. In the absence of affinity ligand more than 90% of the liposomes partitioned in the poly(ethylene glycol)-rich top phase, whereas in its presence more than 95% partitioned in the dextran-rich bottom phase. For this redistribution to occur 10 mM and above of lithium sulphate, or other appropriate salts, had to be added to the two-phase system. Without added salt the liposomes with complexed avidin-dextran instead partitioned in the top phase. An extended mixing time for the system was required for maximum redistribution. Less than two biotin residues per liposome, coupled via a C6 spacer arm, was required to redistribute the liposomes to the bottom phase.
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| 6. |
- Ekblad, Lars, et al.
(författare)
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Localization of phosphatidylinositol 4-kinase isoenzymes in rat liver plasma membrane domains
- 2001
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Ingår i: Biochimica et Biophysica Acta - Molecular and Cell Biology of Lipids. - 1388-1981. ; 1531:3, s. 209-221
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Tidskriftsartikel (refereegranskat)abstract
- The presence of different isoenzymes of phosphatidylinositol 4-kinase in isolated rat liver plasma membranes and their further distribution in plasma membrane domains was examined. Both wortmannin-sensitive and -insensitive PtdIns 4-kinase activities were detected in highly purified plasma membranes obtained by aqueous two-phase affinity partitioning. The wortmannin-sensitive enzyme was identified as the 230 kDa isoform by Western blotting, whereas the 92 kDa isoform was not detected in plasma membranes. The apparent molecular weights of these isoforms were 205 and 105 kDa on SDS polyacrylamide gel electrophoresis, but approximately 500 and 230 kDa respectively on gel filtration, suggesting that both enzymes either are dimers or composed of heterologous subunits. Approximately 25% of the total 230 kDa isoenzyme present in liver, and only ca 5% of the wortmannin-insensitive one, was associated with the plasma membrane fraction. Plasma membrane domains were isolated by a combination of sucrose and Nycodenz gradient centrifugations. The 230 kDa isoform was identified in the blood sinusoidal domain, but not in the bile canalicular one, and was also found in lateral plasma membranes. The wortmannin-insensitive isoenzyme was present only in this latter material. The functional implications of this distribution of PtdIns 4-kinase isoenzymes in plasma membrane regions are discussed.
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| 7. |
- Ekblad, Lars, et al.
(författare)
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Purification of rabbit lacrimal gland plasma membranes by aqueous two-phase affinity partitioning
- 2000
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Ingår i: Journal of Chromatography. B. - 1387-2273. ; 743:1-2, s. 397-401
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Tidskriftsartikel (refereegranskat)abstract
- We describe the purification of lacrimal gland plasma membranes by affinity partitioning using a two-phase system containing polyethylene glycol and dextran in which wheat germ agglutinin conjugated to dextran is used as affinity ligand. When partitioning a microsomal fraction, the plasma membrane marker 5'-nucleotidase was obtained in the affinity ligand-containing bottom phase, whereas the endoplasmic reticulum marker NADH-ferricyanide reductase remained in the top phase. The affinity partitioning behaviour of components involved in exocytosis and cellular signalling was also examined.
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| 8. |
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| 9. |
- Everberg, Henrik, et al.
(författare)
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Isolation of Escherichia coli inner membranes by metal affinity two-phase partitioning
- 2006
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Ingår i: Journal of Chromatography A. - : Elsevier BV. - 0021-9673. ; 1118:2, s. 244-252
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Tidskriftsartikel (refereegranskat)abstract
- As reduction of sample complexity is a central issue in membrane proteomic research, the need for new pre-fractionation methods is significant. Here we present a method for fast and efficient enrichment of Escherichia coli inner membranes expressing a His-tagged integral membrane L-fucose-proton symporter (FucP). An enriched inner membrane fraction was obtained from a crude membrane mixture using affinity two-phase partitioning in combination with nickel-nitrilotri acetic acid (Ni-NTA) immobilized on agarose beads. Due to interaction between the beads and FucP, inner membranes were selectively partitioned to the bottom phase of a polymer/polymer aqueous two-phase system consisting of poly(ethylene glycol) (PEG) and dextran. The partitioning of membranes was monitored by assaying the activity of an inner membrane marker protein and measuring the total protein content in both phases. The enrichment of inner membrane proteins in the dextran phase was also investigated by proteomic methodology, including sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), trypsin digestion and liquid chromatography in combination with tandem mass spectrometry (LC-MS/MS). Using a high level of significance (99.95%) in the subsequent database search, 36 proteins assigned to the inner membrane were identified in the bottom phase, compared to 29 when using the standard sucrose gradient centrifugation method for inner membrane isolation. Furthermore, metal affinity two-phase partitioning was up to 10 times faster than sucrose gradient centrifugation. The separation conditions in these model experiments provide a basis for the selective isolation of E. coli membranes expressing His-tagged proteins and can therefore facilitate research on such membrane proteomes. (c) 2006 Elsevier B.V. All rights reserved.
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| 10. |
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| 11. |
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| 12. |
- Jergil, Bengt, et al.
(författare)
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Purification of plasma membranes by affinity partitioning
- 2000
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Ingår i: Aqueous Two-Phase Systems : Methods and Protocols - Methods and Protocols. - New Jersey : Humana Press. - 1940-6061. - 9780896035416 - 9781592590285 ; 11, s. 193-200
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Bokkapitel (övrigt vetenskapligt/konstnärligt)
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| 13. |
- Jergil, Bengt, et al.
(författare)
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Purifications of plasma membranes by affinity partioning.
- 2000
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Ingår i: Aqueous Two-Phase Systems: Methods and protocols (Methods in Biotechnology ; 11). - 0896035417 ; 11, s. 193-200
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Bokkapitel (övrigt vetenskapligt/konstnärligt)abstract
- This chapter describes the purification of rat liver plasma membranes by affinity partitioning in an aqueous polymer two-phase system using the lectin wheat germ agglutinin (WGA) as affinity ligand. Affinity partitioning is advantageous for conventional membrane fractionation techniques in being highly selective, allowing the rapid and high-yield purification of membranes. In addition, the aqueous polymer environment is gentle to membrane structure and function, which is of importance when studying labile structures and components. A two-phase system will form when aqueous solutions of two structurally different polymers are mixed at sufficiently high concentrations (1). A commonly used polymer pair is polyethylene glycol 3350 (PEG) and Dextran T500. These polymers will form a two-phase system at concentrations above 5.4% (w/w) of each, the top phase being enriched in PEG and the bottom phase in Dextran. A conventional two-phase system like this may be used for the fractionation of biological material, including membranes. The distribution of material between the phases is modulated by altering polymer concentration and salt contents of the system (2), but the selectivity is often insufficient for the ready separation of membranes.
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| 14. |
- Karlsson, Margareta, 1942-
(författare)
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Caveolae in insulin signalling in human and rat adipocytes
- 2003
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The pancreatic hormone insulin is a key hormone in maintenance of metabolic homeostasis but it also exerts control on gene expression and cell growth. This thesis presents results on fhe role of caveolae in insulin signalling in human and rat adipocytes. Caveolae are invaginations of the plasma membrane, characterised by the structural protein caveolin. Caveolae and caveolin have been implicated in a variety of functions, like uptake of molecular cargo into the cell, cholesterol transport and signal transduction. After isolation of caveolae and using electron microscopy on cell membranes, the insulin receptor was demonstrated to be localised in caveolae of human adipocytes. We also used biochemical and morphological methods to show that the glucose transporter GLUT4 was translocated to caveolae in response to insulin in rat adipocytes, indicating fhat the caveola is the locale for glucose uptake in adipocytes.Adipocytes fhat were depleted of cholesterol using ß-cyclodextrin lacked caveolae invaginations. In cells fhus depleted of cholesterol and caveolae, fhe insulin receptor itself was not affected, but insulin signalling to metabolic control was inhibited. In rat adipocytes, insulin signalling to mitogenic control was not affected. In human fat cells, however, insulin's mitogenic signalling was dependent on caveolae/cholesterol. In contrast to other cells studied, including rat adipocytes, where the insulin receptor substrate (IRS-1) is mainly cytosolic, in human adipocytes IRS-1 was found in the plasma membrane and in caveolae. These results show the importance of choosing the relevant system to work with, since there are clear species differences.We performed an analysis of the lipid composition of purified caveolae from rat adipocytes. As expected, cholesterol constitutes a major part of caveolae, but there is also an enrichment of sphingomyelin and the gangliosides GM1, GM3, GD3 and GD1a, while there is less protein, compared to the surrounding plasma membrane.Taken together, caveolae appear as hnbs for insulin signalling. Caveolae seem necessary for fhe maintenance of metabolic signalling, like glucose uptake, and defects in caveolae may thus be the cause of insulin resistance.
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| 15. |
- Loog, Mart
(författare)
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Studies on the Differential Specificity of Protein Kinases and Its Applications
- 2001
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Protein kinases are enzymes that catalyse the phosphoryl transfer from the g-phosphate of ATP to acceptor amino acids in proteins. The specificity of selected model protein kinases was studied at three different levels using a) novel bi-substrate-analogue inhibitors, b) synthetic peptide substrates and c) mutated protein substrate analogues. A new class of protein kinase bi-substrate-analogue inhibitors was designed on the basis of adenosine-5’-carboxylic acid derivatives, where a short arginine containing peptide was attached to the 5'-carbon atom of the adenosine sugar moiety via a linker chain. These compounds showed high inhibitory potential against two basophilic protein kinases, the protein kinase A (PKA) and protein kinase C (PKC), with IC50 values in the nanomolar range, but no inhibitory activity towards the acidophilic kinases CK1 and CK2. The inhibitors were efficiently applied for affinity purification of PKA using MgATP as well as L-arginine as eluting agents. Ca2+-dependent protein kinase (CDPK-1) was purified from maize seedlings and its substrate specificity was studied using a set of synthetic peptides. These were derived from the phosphorylatable sequence RVLSRLHS(15)VRER of maize sucrose synthase 2 (SuSy2), and a consensus sequence motif A/LXRXXSXRZR (where X denotes a position with no strict amino acid requirements and Z a position strictly not tolerating arginine) was defined from a study using arrays of systematically varied peptides attached to cellulose membrane (SPOTsTM membranes). The SuSy2 derived peptides were also found to be efficient substrates for mammalian PKC, but showed low reactivity in the case of PKA. On the basis of this peptide motif, a positionally oriented peptide library approach based on ESI-MS detection of phosphopeptides in initial velocity conditions was designed for quantitative kinetic characterization of protein kinase specificity profiles. On the basis of the obtained data an optimal peptide substrate for PKC, FRRRRSFRRR, was designed. The specificity of protein kinase A was studied using site-directed mutagenesis in the phosphorylation site of L-type pyruvate kinase (L-PK), and comparison of the obtained data with the data from previous studies on structurally altered peptide substrates revealed that amino acid alterations in short peptide substrates cause stronger effects on the phosphorylation rate than the corresponding alterations in the protein substrate L-PK.
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| 16. |
- Michelsen, P, et al.
(författare)
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Quantification of polyphosphoinositides using selected ion monitoring electrospray mass spectrometry
- 1995
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Ingår i: Rapid Communications in Mass Spectrometry. - : Wiley. - 1097-0231 .- 0951-4198. ; 9:12, s. 1109-1114
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Tidskriftsartikel (refereegranskat)abstract
- Polyphosphoinositides (PIP) and (PIP2) show prominent negative singly and doubly charged deprotonated molecules in electrospray mass spectrometry. These ions can be used for quantification of PIP and PIP2 in the low picomole range, without prior chromatographic separation, using selected ion monitoring and consecutive measurements of the signals from the deprotonated singly charged molecules. The dose response curves for both compounds are linear. In a complex matrix consisting of polar lipids (Folch extract) PIP and PIP2 monitored at m/z 965.4 and 1045.5 (stearoyl and arachidonoyl) were determined in the low picomole range, at a flow rate of 100 mu L/min, Collision-induced decomposition of PIP and PIP2 using a mixture of xenon and argon at 25 eV afforded identical high mass ions formed by loss of a molecule of water from PIP and a phosphate group and a molecule of water from PIP2. The results indicate that polyphosphoinositides, and biologically relevant changes in their concentrations, can be quantified directly in cells and cellular membranes by selected-ion monitoring with electrospray mass spectrometry.
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| 17. |
- Olsson, H, et al.
(författare)
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Phosphatidylcholine enhances the activity of rat liver type II phosphatidylinositol-kinase
- 1993
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Ingår i: FEBS Letters. - 1873-3468. ; 327:3, s. 6-332
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Tidskriftsartikel (refereegranskat)abstract
- A PtdIns 4-kinase was purified extensively from rat liver exocytotic vesicles. The enzyme had a low Km for ATP, was inhibited by adenosine, and had an apparent molecular mass of 54 kDa, indicating it to be a type II PtdIns-kinase. The activity of the purified enzyme was enhanced several-fold by PtdCho, and to some extent by other phospholipids with basic polar head groups, and was inhibited by PtdSer. Kinetic analyses, presenting the substrate in mixed micelles of Triton X-100, PtdIns and PtdCho, showed that the effect of PtdCho was both to increase Vmax and to decrease the apparent Km for micellar PtdIns.
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| 18. |
- Olsson, Henric, et al.
(författare)
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Presence of a novel form of phosphatidylinositol 4-kinase in rat liver
- 1995
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Ingår i: FEBS Letters. - 1873-3468. ; 361:2-3, s. 282-286
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Tidskriftsartikel (refereegranskat)abstract
- Rat liver microsomes contain two distinct forms of PtdIns 4-kinase which were resolved by heparin-Sepharose chromatography. One enzyme was identified as the type II PtdIns kinase previously isolated from exocytotic vesicles. The other enzyme, however, was a novel PtdIns 4-kinase isoform with properties differing from any other PtdIns kinase so far characterized. Both kinases were recognized by a monoclonal antibody specific for type II PtdIns 4-kinase, but the novel enzyme was considerably less sensitive to inhibition by adenosine and Ca2+ than type II enzymes, and in addition was specifically inhibited by submillimolar concentrations of dithioerythritol. The presence of a novel PtdIns 4-kinase isoform in rat liver raises the question of whether this enzyme is unique for this organ or whether it has a more widespread distribution but so far has avoided detection.
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| 19. |
- Persson, A, et al.
(författare)
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The purification of membranes by affinity partitioning
- 1995
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Ingår i: FASEB Journal. - 1530-6860. ; 9:13, s. 1304-1310
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Tidskriftsartikel (refereegranskat)abstract
- We describe affinity partitioning as a preparative method for membranes and membraneous structures such as organelles, cells, and viruses. Biospecific affinity partitioning is carried out in aqueous polymer two- phase systems, commonly with polyethylene glycol and dextran as phase polymers, in an environment compatible with membrane structures. Ideally, two-phase conditions are chosen to partition the bulk of membrane material into one phase, while the affinity ligand, conjugated to the second phase polymer, will selectively pull the membranes to be isolated into this phase. Suitable ligands include lectins, antibodies, and receptor-specific agents. Because the method has so far been used successfully in rather few instances, all using high ligand receptor densities in target membranes, the discussion focuses on factors to be considered when developing affinity partitioning conditions using new ligands.
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| 20. |
- Santesson, Sabina, et al.
(författare)
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Affinity Two-Phase Partitioning in Acoustically Levitated Drops
- 2004
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 1520-6882 .- 0003-2700. ; 76:2, s. 303-308
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Tidskriftsartikel (refereegranskat)abstract
- Miniaturized (<1 L) biospecific affinity two-phase partitioning in an acoustically levitated drop is described. Miniaturization commonly gives unfavorable surface/volume ratios, but in the levitation approach adsorption problems are minimized since the only surrounding wall is the liquid/air interface of the drop. Biotinylated liposomes were partitioned in aqueous poly(ethylene glycol)/dextran two-phase drops with NeutrAvidin-dextran as the affinity ligand. A two-phase drop was trapped and manipulated in a node of a standing ultrasonic wave. Alternatively, a two-phase system was formed by levitation and evaporation of a polymer one-phase drop. Phase mixing was achieved by adjusting the ultrasonic field and phase separation by readjusting the field. NeutrAvidin-dextran brought about the redistribution of biotinylated liposomes from the poly(ethylene glycol)-rich phase into the dextran-rich phase. Thus, an entire affinity two-phase separation procedure, including mixing of the phases and incubation to allow affinity interactions to develop under constant volume, followed by phase separation under controlled evaporation, can be performed in a single levitated drop. This miniaturized technique would allow the separation of biologically active membranes or organelles from individual cells for analysis.
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| 21. |
- Westergren, Tomas, et al.
(författare)
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Phosphatidylinositol 4-Kinase Associated with Spinach Plasma Membranes. Isolation and Characterization of Two Distinct Forms
- 1999
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Ingår i: Plant Physiology. - 1532-2548. ; 121:2, s. 507-516
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Tidskriftsartikel (refereegranskat)abstract
- Highly purified plasma membranes from spinach (Spinacia oleracea L.) leaves contained phosphatidylinositol (PtdIns) kinase activity that was firmly associated with the membrane. The enzyme was solubilized by detergent treatment (2% [w/v] Triton X-100) and purified by heparin-Sepharose and Q-Sepharose chromatography. Two enzymically active fractions, QI and QII, both exhibiting PtdIns 4-kinase activity, were resolved and purified 100- to 300-fold over the plasma membrane. QI and QII shared similar high apparent Km values for ATP (approximately 0.45 mM) and PtdIns (approximately 0.2 mM) and were insensitive to inhibition by adenosine. While Mg2+ was the preferred divalent cation, Mn2+ could partly substitute in the reaction catalyzed by the QII enzyme but not in that catalyzed by QI. Mn2+ acted synergistically with suboptimal Mg2+ concentrations to activate not only the QII enzyme, but also to some extent QI. Both enzymes were inhibited by millimolar concentrations of Ca2+ and micromolar concentrations of wortmannin. The apparent molecular mass for QI was 120 kD, which was determined by SDS-PAGE and western blotting using an antibody against a peptide unique for lipid kinases and the binding of 3H-wortmannin, and for QII 65 kD as determined by immunodetection and renaturation of PtdIns kinase activity in the 65-kD region of polyacrylamide gels.
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