Isolation of Synapse Sub-Domains by Subcellular Fractionation Using Sucrose Density Gradient Centrifugation: Purification of the Synaptosome, Synaptic Plasma Membrane, Postsynaptic Density, Synaptic Membrane Raft, and Postsynaptic Density Lattice

2019 ◽  
pp. 21-42
Author(s):  
Tatsuo Suzuki ◽  
Yoshinori Shirai ◽  
Weidong Li
Keyword(s):  
Plasma Membrane ◽  
Density Gradient ◽  
Gradient Centrifugation ◽  
Postsynaptic Density ◽  
Sucrose Density Gradient ◽  
Subcellular Fractionation ◽  
Synaptic Membrane ◽  
Synaptic Plasma Membrane ◽  
Membrane Raft ◽  
Sucrose Density Gradient Centrifugation

The Organelle Pathology and Demonstration of Mitochondrial Superoxide Dismutase Deficiency in Two Patients with Dubin–Johnson–Sprinz Syndrome

1978 ◽  
Vol 54 (5) ◽  
pp. 549-553
Author(s):  
T. J. Peters ◽  
Carol A. Seymour
Keyword(s):  
Superoxide Dismutase ◽  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Subcellular Fractionation ◽  
Sucrose Density Gradient Centrifugation ◽  
Mitochondrial Superoxide ◽  
Liver Biopsies ◽  
Mitochondrial Superoxide Dismutase

1. Liver biopsies from two patients with the Dubin—Johnson—Sprinz syndrome were subjected to analytical subcellular fractionation by sucrose-density-gradient centrifugation and enzymic micro-assays. 2. A selective deficiency of mitochondrial superoxide dismutase has been demonstrated in these patients. 3. The significance of this enzyme deficiency is discussed in relation to the organelle pathology of the syndrome.

scholarly journals Solubilization of pig lymphocyte plasma membrane and fractionation of some of the components

1971 ◽  
Vol 123 (5) ◽  
pp. 967-975 ◽  
Author(s):  
D. Allan ◽  
M. J. Crumpton
Keyword(s):  
Plasma Membrane ◽  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Biological Activities ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sucrose Density Gradient ◽  
Similar Distribution ◽  
Sucrose Density Gradient Centrifugation

The degree of solubilization of pig lymphocyte plasma membrane by sodium deoxycholate was determined at a variety of temperatures and detergent concentrations. Approx. 95% of the membrane protein was soluble in 2% deoxycholate at 23°C. Some of the biological activities of the membrane survived this treatment. The leucine β-naphthylamidase activity was more readily soluble than the 5′-nucleotidase and these enzymes could be separated by extraction with 0.5% deoxycholate at 0°C. Membrane solubilized in 2% deoxycholate at 23°C was fractionated by sucrose-density-gradient centrifugation in 1% deoxycholate. The phospholipid was separated from the protein, which formed a fairly symmetrical peak that sedimented slightly slower than ovalbumin; the leucine naphthylamidase and 5′-nucleotidase activities were resolved from each other and from the main protein peak. Similar separations were achieved by elution from Sephadex G-200 and Sepharose 6B in 1% deoxycholate. The main proteins, however, appeared to possess much higher molecular weights than those indicated by sucrose-density-gradient centrifugation. This disparity suggests that many of the membrane proteins have a rod-like shape, especially since the results of experiments with [14C]deoxycholate revealed that the proteins did not bind significant amounts of deoxycholate. In contrast, 5′-nucleotidase and leucine naphthylamidase appeared to be globular. Polyacrylamide-gel electrophoresis of membrane solubilized in sodium dodecyl sulphate gave a similar distribution of protein to that achieved by sucrose-density-gradient centrifugation. Trace amounts only of polypeptides of molecular weight less than 10000 were detected.

The subcellular localization of transglutaminase in normal liver and in glucagon-treated and partial hepatectomized rats

1983 ◽  
Vol 3 (12) ◽  
pp. 1085-1090 ◽  
Author(s):  
Sally E. Bruce ◽  
Timothy J. Peters
Keyword(s):  
Plasma Membrane ◽  
Subcellular Distribution ◽  
Specific Activity ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Normal Liver ◽  
Sucrose Density Gradient ◽  
Subcellular Fractionation ◽  
Sucrose Density Gradient Centrifugation ◽  
Significant Difference

Rat liver was homogenized in isotonic sucrose and subjected to analytical subcellular fractionation by sucrose density-gradient centrifugation. Transglutaminase, when assayed with putrescine and dimethylcasein as substrates, showed three distinct localizations, cytosol (73%), plasma membrane (20%), and nuclei (7%). The distribution was unaffected by homogenization in the presence of potassium chloride, indicating that the particulate activity was not due to adsorbed cytosolic enzyme. The specific activity and subcellular distribution of transglutaminase in rats which had received intra-peritoneal glucagon, stimulating endocytosis; or which had been subjected to sub-total hepatectomy 2, 16, or 32 h previously, showed no significant difference from control animals.

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.

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