Les systèmes membranaires de Scopulariopsis brevicaulis. I. Isolement et caractérisation

1987 ◽  
Vol 33 (1) ◽  
pp. 70-77 ◽  
Author(s):  
Jean-Louis Fonvieille ◽  
Jane-Marie Touze-Soulet ◽  
Robert Dargent
Keyword(s):  
Density Gradient ◽  
Phosphotungstic Acid ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Continuous Density ◽  
Scopulariopsis Brevicaulis ◽  
Enzymatic Markers ◽  
Nadh Cytochrome

Membranes isolated (except mitochondrial membranes) from mycelium of Scopulariopsis brevicaulis were separated by continuous density gradient centrifugation using Percoll at a concentration of 10%. This method was compared with discontinuous sucrose density gradient centrifugation. Isolated membrane systems were characterized by enzymatic markers (NADPH and NADH cytochrome c reductases, ATPase, AMPase, IDPase) and by cytochemical reactions using electron microscopy (Thiéry test, phosphotungstic acid – chromic acid). The best separation of membranes was obtained with continuous density gradient centrifugation using 10% Percoll.

Influence de la cytochalasine A sur la composition de fractions subcellulaires d'hyphes en croissance du Mucor mucedo L. I. Composition du plasmalemme

1988 ◽  
Vol 34 (11) ◽  
pp. 1256-1265 ◽  
Author(s):  
Abdou Ahmed Abd El Razak El Mougith ◽  
Jean-Louis Fonvieille ◽  
Robert Dargent ◽  
Jacques Rami ◽  
Jane-Marie Touzé-Soulet
Keyword(s):  
Protein Content ◽  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Point Of View ◽  
Continuous Density ◽  
Mucor Mucedo ◽  
Phospholipid Ratio ◽  
Enzymatic Markers

The plasma membrane of young hyphae of Mucor mucedo L. growing in presence or absence of cytochalasine A was isolated by continuous density gradient centrifugation using Percoll at 10% or on discontinuous sucrose density gradient. Isolated membranes were characterized by enzymatic markers and cytochemical reactions, using electron microscopy. Lipid composition and protein content were determined. From the enzymatic point of view, the cytochalasine A induced a decrease (60%) in ATPase activity and with regard to the chemical composition of the membrane, a decrease in sterol content and in the sterol – phospholipid ratio as well as a decrease in protein content and an increase in the proportion of cysteine relative to other amino acids.

The Combining Ability of Glycoproteins IIb, IIIa and Ca2+ in EDTA-Treated Nonaggregable Platelets

1983 ◽  
Vol 50 (04) ◽  
pp. 848-851 ◽  
Author(s):  
Marjorie B Zucker ◽  
David Varon ◽  
Nicholas C Masiello ◽  
Simon Karpatkin
Keyword(s):  
Density Gradient ◽  
Combining Ability ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Electrophoretic Pattern ◽  
Sucrose Density Gradient ◽  
Irreversible Loss ◽  
Sucrose Density Gradient Centrifugation ◽  
Crossed Immunoelectrophoresis ◽  
Triton X

SummaryPlatelets deprived of calcium and incubated at 37° C for 10 min lose their ability to bind fibrinogen or aggregate with ADP when adequate concentrations of calcium are restored. Since the calcium complex of glycoproteins (GP) IIb and IIIa is the presumed receptor for fibrinogen, it seemed appropriate to examine the behavior of these glycoproteins in incubated non-aggregable platelets. No differences were noted in the electrophoretic pattern of nonaggregable EDTA-treated and aggregable control CaEDTA-treated platelets when SDS gels of Triton X- 114 fractions were stained with silver. GP IIb and IIIa were extracted from either nonaggregable EDTA-treated platelets or aggregable control platelets with calcium-Tris-Triton buffer and subjected to sucrose density gradient centrifugation or crossed immunoelectrophoresis. With both types of platelets, these glycoproteins formed a complex in the presence of calcium. If the glycoproteins were extracted with EDTA-Tris-Triton buffer, or if Triton-solubilized platelet membranes were incubated with EGTA at 37° C for 30 min, GP IIb and IIIa were unable to form a complex in the presence of calcium. We conclude that inability of extracted GP IIb and IIIa to combine in the presence of calcium is not responsible for the irreversible loss of aggregability that occurs when whole platelets are incubated with EDTA at 37° C.

scholarly journals Purification of Torpedo californica post-synaptic membranes and fractionation of their constituent proteins

1980 ◽  
Vol 185 (3) ◽  
pp. 667-677 ◽  
Author(s):  
J Elliott ◽  
S G Blanchard ◽  
W Wu ◽  
J Miller ◽  
C D Strader ◽  
...  
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Selective Extraction ◽  
Synaptic Membranes ◽  
Detergent Solution ◽  
Major Step ◽  
Sucrose Density Gradient Centrifugation ◽  
Torpedo Californica

A rapid methof for preparation of membrane fractions highly enriched in nicotinic acetylcholine receptor from Torpedo californica electroplax is described. The major step in this purification involves sucrose-density-gradient centrifugation in a reorienting rotor. Further purification of these membranes can be achieved by selective extraction of proteins by use of alkaline pH or by treatment with solutions of lithium di-idosalicylate. The alkali-treated membranes retain functional characteristics of the untreated membranes and in addition contain essentially only the four polypeptides (mol.wts. 40000, 50000, 60000 and 65000) characteristic of the receptor purified by affinity chromatography. Dissolution of the purified membranes or of the alkali-treated purified membranes in sodium cholate solution followed by sucrose-density-gradient centrifugation in the same detergent solution yields solubilized receptor preparations comparable with the most highly purified protein obtained by affinity-chromatographic procedures.

scholarly journals Molecular size of the 5-HT3 receptor solubilized from NCB 20 cells

1990 ◽  
Vol 269 (3) ◽  
pp. 623-628 ◽  
Author(s):  
R M McKernan ◽  
C S Biggs ◽  
N Gillard ◽  
K Quirk ◽  
C I Ragan
Keyword(s):  
Physical Properties ◽  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Receptor Site ◽  
Molecular Size ◽  
Sucrose Density Gradient ◽  
Partial Specific Volume ◽  
Sucrose Density Gradient Centrifugation ◽  
Ics 205 930

The 5-HT3 hydroxytryptamine receptor from NCB 20 cells was solubilized and the molecular and hydrodynamic properties of the receptor were investigated. The receptor was identified by binding of the radioligand 3-NN′-[3H]dimethyl-8-azabicyclo[3.2.1]octanyl indol-3-yl carboxylate ester [(3H]Q ICS 205-930) to NCB 20 membranes (Bmax = 1.19 +/- 0.31 pmol/mg of protein; Kd = 0.43 +/- 0.076 nM) and was optimally solubilized with 0.5% deoxycholate. [3H]Q ICS 205-930 labelled one population of sites in solution (Bmax = 1.11 +/- 0.4 pmol/mg of protein; Kd = 0.48 +/- 0.06 nM; n = 4). The characteristics of [3H]Q ICS 205-930 binding were essentially unchanged by solubilization, and competition for [3H]Q ICS 205-930 binding by a series of 5-HT3 agonists and antagonists was consistent with binding to a 5-HT3 receptor site and was similar to that observed for 5-HT3 receptors solubilized from rat brain [McKernan, Quirk, Jackson & Ragan (1990) J. Neurochem. 54, 924-930]. Some physical properties of the solubilized receptor were investigated. The molecular size (Stokes radius) of the [3H]Q ICS 205-930-binding site was measured by gel-exclusion chromatography in a buffer containing 0.2% Lubrol and 0.5 M-NaCl and was determined as 4.81 +/- 0.15 nm (mean +/- S.E.M.; n = 6). Sucrose-density-gradient centrifugation was also performed under the same detergent and salt conditions to determine the partial specific volume (v) of the detergent-receptor site complex. This was found to be 0.794 ml.g-1. Sucrose-density-gradient centrifugation was carried out in both 1H2O and 2H2O to allow correction for detergent binding to the receptor. The Mr of the 5-HT3 receptor under these conditions was calculated as 249,000 +/- 18,000 (n = 3). The size and physical properties of the 5-HT3 receptor are similar to those observed for members of the family of ligand-gated ion channels.

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