scholarly journals Fractionation of rat liver plasma-membrane regions by two-phase partitioning

1986 ◽  
Vol 235 (3) ◽  
pp. 685-691 ◽  
Author(s):  
P Gierow ◽  
M Sommarin ◽  
C Larsson ◽  
B Jergil
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Gradient Centrifugation ◽  
Fold Increase ◽  
Phase System ◽  
Two Phase ◽  
Phase Partitioning ◽  
Aqueous Polymer ◽  
Counter Current

Rat liver plasma membranes, enriched in blood-sinusoidal or bile-canalicular regions by differential and sucrose-gradient centrifugation, were further purified by partitioning in an aqueous polymer two-phase system. This method separates membranes according to differences in surface properties rather than size and density. A several-fold increase in the ratio of leucine aminopeptidase (a bile-canalicular marker) and 5′-nucleotidase to asialo-orosomucoid binding (a blood-sinusoidal marker) was obtained in one fraction, whereas another fraction gave a 2-3-fold increase in ratio of blood-sinusoidal to bile-canalicular markers. Furthermore, the markers for both regions of the plasma membrane, as well as markers for Golgi membranes and lysosomes, showed a heterogeneous behaviour on counter-current distribution.

scholarly journals Heterogeneity of smooth endoplasmic reticulum from rat liver studied by two-phase partitioning

1989 ◽  
Vol 262 (1) ◽  
pp. 55-61 ◽  
Author(s):  
P Gierow ◽  
B Jergil
Keyword(s):  
Rat Liver ◽  
Smooth Endoplasmic Reticulum ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Phase System ◽  
Marker Enzymes ◽  
Two Phase ◽  
Sucrose Density Gradient Centrifugation ◽  
Phase Partitioning ◽  
Microsomal Membranes

Smooth microsomal membranes, prepared from rat liver by sucrose-density-gradient centrifugation, were subfractionated by counter-current distribution in an aqueous two-phase system consisting of poly(ethylene glycol) and Dextran T500. A comparison of the distribution curves of marker enzymes, together with theoretically calculated curves, indicated the presence of at least five membrane subfractions, differing in the ratios of the marker enzymes. Glucose-6-phosphatase and arylesterase distributed in one manner, and NADPH-cytochrome c reductase and NADH-ferricyanide reductase in another. Evidence for further heterogeneities in the distribution of marker enzymes in smooth microsomes was obtained by analysing the membrane domain structure using a recently described method [Albertsson (1988) Q. Rev. Biophys. 21, 61-98]. Phenobarbital treatment did not influence the behaviour of the marker enzymes.

Purification of glucosyltransferase from cell-lysate of Streptococcus mutans by counter-current chromatography using aqueous polymer two-phase system

2004 ◽  
Vol 805 (1) ◽  
pp. 155-160 ◽  
Author(s):  
Akio Yanagida ◽  
Mitsuhiro Isozaki ◽  
Yoichi Shibusawa ◽  
Heisaburo Shindo ◽  
Yoichiro Ito
Keyword(s):  
Streptococcus Mutans ◽  
Phase System ◽  
Two Phase ◽  
Cell Lysate ◽  
Aqueous Polymer ◽  
Counter Current

scholarly journals Comparative studies on the soluble and plasma membrane associated nitrate reductase from Cucumis sativus L.

2014 ◽  
Vol 63 (3-4) ◽  
pp. 309-314 ◽  
Author(s):  
Grażyna Kłobus ◽  
Jolanta Marciniak ◽  
Józef Buczek
Keyword(s):  
Plasma Membrane ◽  
Nitrate Reductase ◽  
Plasma Membranes ◽  
Polyclonal Antiserum ◽  
Soluble Form ◽  
Phase System ◽  
Two Phase ◽  
Cucumis Sativus L ◽  
Cucumber Roots ◽  
Biochemical Comparison

The biochemical comparison between two forms of nitrate reductase from cucumber roots: the soluble enzyme and the plasma membrane-associated one was made. Soluble nitrate reductase was purified on the blue-Sepharose 4B. The nitrate reductase bound with plasma membranes was isolated from cucumber roots by partition of microsomes in the 6.5% dextran-PEG two phase system. The molecular weight of native enzyme estimated with HPLC was 240 kDa and 114 kDa for the soluble and membrane bounded enzyme, respectively. Temperature induced phase separation in Triton X-114 indicated a huge difference in hydrophobicity of the plasma membrane associated nitrate reductase and soluble form of enzyme. Small differences were observed in partial activities of plasma membrane nitrate reductase and soluble nitrate reductase. Also experiments with polyclonal antiserum raised against the native nitrate reductase showed some differences in the immunological properties of both forms of the nitrate reductase. The above results indicated that in cucumber roots two different forms of the nitrate reductase are present.

scholarly journals Nonidet P-40 extraction of lymphocyte plasma membrane. Characterization of the insoluble residue

1984 ◽  
Vol 219 (1) ◽  
pp. 301-308 ◽  
Author(s):  
A A Davies ◽  
N M Wigglesworth ◽  
D Allan ◽  
R J Owens ◽  
M J Crumpton
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fold Increase ◽  
Insoluble Fraction ◽  
Insoluble Residue ◽  
Protein Ratio

Purified preparations of lymphocyte plasma membrane were extracted exhaustively with Nonidet P-40 in Dulbecco's phosphate-buffered saline medium. The insoluble fraction, as defined by sedimentation at 10(6) g-min, contained about 10% of the membrane protein as well as cholesterol and phospholipid. The lipid/protein ratio, cholesterol/phospholipid ratio and sphingomyelin content were increased in the residue. Density-gradient centrifugation suggested that the lipid and protein form a common entity. As judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the Nonidet P-40-insoluble fractions of the plasma membranes of human B lymphoblastoid cells and pig mesenteric lymph-node lymphocytes possessed similar qualitative polypeptide compositions but differed quantitatively. Both residues comprised major polypeptides of Mr 28 000, 33 000, 45 000 and 68 000, together with a prominent band of Mr 120 000 in the human and of Mr 200 000 in the pig. The polypeptides of Mr 28 000, 33 000, 68 000 and 120 000 were probably located exclusively in the Nonidet P-40-insoluble residue, which also possessed a 4-fold increase in 5′-nucleotidase specific activity. The results indicate that a reproducible fraction of lymphocyte plasma membrane is insoluble in non-ionic detergents and that this fraction possesses a unique polypeptide composition. By analogy with similar studies with erythrocyte ghosts, it appears likely that the polypeptides are located on the plasma membrane's cytoplasmic face.

PLASMA MEMBRANE ATPASE ISOLATED FROM GREEN BELL PEPPER FRUIT BY TWO-PHASE PARTITIONING OR SUCROSE DENSITY GRADIENT

1994 ◽  
Vol 42 (1) ◽  
pp. 15-27
Author(s):  
R. Ronen ◽  
Z. Lipsker ◽  
L. Sonego ◽  
Susan Lurie
Keyword(s):  
Plasma Membrane ◽  
Density Gradient ◽  
Sucrose Density Gradient ◽  
Bell Pepper ◽  
Phase System ◽  
Plasma Membrane Atpase ◽  
Two Phase ◽  
Phase Partitioning ◽  
Pepper Fruit ◽  
Membrane Atpase

Plasma membrane was isolated from mature green bell pepper fruit by two-phase partitioning or by sucrose density gradient. The yield of plasma membrane was higher from the sucrose density gradient, but the two-phase system was less contaminated by other membranes, particularly those from chloroplasts and mitochondria. In the two-phase partitioned membranes, ATPase activity was stimulated by Triton X-100 by 100% and in sucrose density gradient membranes by 40%. Plasma membranes from two-phase partitioning exhibited simultaneous proton pumping and ATP hydrolysis, while the sucrose density purified membranes did not. Immunoblotting with ATPase antibody showed enrichment of plasma membrane ATPase in both the U3 phase of the two-phase system, and the 34% sucrose fraction of the sucrose gradient. However, the two-phase partitioned membranes were superior to those prepared by sucrose density for investigating functions of the ATPase.

scholarly journals Alkaline ribonuclease and phosphodiesterase activity in rat liver plasma membranes

1973 ◽  
Vol 132 (3) ◽  
pp. 449-458 ◽  
Author(s):  
Terence D. Prospero ◽  
Malcolm L. E. Burge ◽  
Kenneth A. Norris ◽  
Richard H. Hinton ◽  
Eric Reid
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Gradient Centrifugation ◽  
Ribonuclease Activity ◽  
Nuclear Fraction ◽  
Phosphodiesterase Activity ◽  
Ph Optimum ◽  
Liver Plasma Membranes ◽  
Rat Liver Plasma Membranes

The ribonuclease and phosphodiesterase activities of rat liver plasma membranes, purified from the crude nuclear fraction by centrifugation in an A-XII zonal rotor and flotation, were examined and compared. The plasma membrane is responsible for between 65 and 90% of the phosphodiesterase activity of the cell and between 25 and 30% of the particulate ribonuclease activity measured at pH8.7 in the presence of 7.5mm-MgCl2. Both enzymes were most active between pH8.5 and 8.9. Close to the pH optimum, both enzymes were more active in Tris buffer than in Bicine or glycine buffer. Both plasma-membrane phosphodiesterase and ribonuclease were strongly activated by Mg2+, there being at least a 12-fold difference between the activity in the presence of Mg2+ and of EDTA. There is, however, a difference in the response of the enzymes to Mg2+ and EDTA in that the phosphodiesterase is fully activated by 1.0mm-MgCl2 and fully inhibited by 1.0mm-EDTA, whereas the ribonuclease requires 7.5mm-MgCl2 for full activation and 5mm-EDTA for full inhibition. Density-gradient centrifugation has indicated that on solubilization in Triton X-100 most of the ribonuclease activity is released into a small fragment of the same size as that containing the phosphodiesterase activity. The relationship between the two activities is discussed in view of these results.

scholarly journals Plasma Membranes from Oats Prepared by Partition in an Aqueous Polymer Two-Phase System

1982 ◽  
Vol 70 (5) ◽  
pp. 1429-1435 ◽  
Author(s):  
Susanne Widell ◽  
Tomas Lundborg ◽  
Christer Larsson
Keyword(s):  
Plasma Membranes ◽  
Phase System ◽  
Two Phase ◽  
Aqueous Polymer

Subfractional Analysis of Cyanobacterial Membranes and Isolation of Plasma Membranes by Aqueous Polymer Two-Phase Partitioning

1994 ◽  
Vol 218 (1) ◽  
pp. 103-111 ◽  
Author(s):  
B. Norling ◽  
V. Mirzakhanian ◽  
F. Nilsson ◽  
D.J. Morre ◽  
B. Andersson
Keyword(s):  
Plasma Membranes ◽  
Two Phase ◽  
Phase Partitioning ◽  
Aqueous Polymer
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