scholarly journals PREPARATION OF PLASMA MEMBRANE FROM ISOLATED NEURONS

1972 ◽  
Vol 53 (3) ◽  
pp. 654-661 ◽  
Author(s):  
Fritz A. Henn ◽  
Hans-Arne Hansson ◽  
Anders Hamberger
Keyword(s):  
Plasma Membrane ◽  
Density Gradient ◽  
Electron Micrographs ◽  
Membrane Fraction ◽  
Neuronal Cell ◽  
Sucrose Density Gradient ◽  
Isolated Neurons ◽  
Fold Enrichment ◽  
Neuronal Cell Bodies ◽  
Neuronal Plasma Membrane

A bulk fraction enriched with respect to neuronal cell bodies was used as starting material for the isolation of neuronal plasma membrane The cells were gently homogenized in isotonic sucrose and a crude membrane containing fraction sedimented at 3000 g. Subsequently, the membrane fraction was purified on a discontinuous sucrose density gradient between 35% and 25 5% sucrose (w/w). Enzymatic analyses showed a 4–5-fold enrichment in plasma membrane markers, and a 10–15% contamination of mitochondrial and microsomal material. Electron micrographs of the membrane fraction confirmed the enzymatic data Fragmented membranes were found, mainly in vesicular form No ribosomes, but a few mitochondria and some multilamellar membranes were seen

GANGLIOSIDES IN DEVELOPING RAT BRAIN: ISOLATION AND COMPOSITION OF SUBCELLULAR MEMBRANES ENRICHED IN GANGLIOSIDES

1967 ◽  
Vol 45 (5) ◽  
pp. 671-688 ◽  
Author(s):  
M. W. Spence ◽  
L. S. Wolfe
Keyword(s):  
Rat Brain ◽  
Density Gradient ◽  
Membrane Fraction ◽  
Dry Weight ◽  
Sucrose Density Gradient ◽  
Particulate Material ◽  
Neonatal Rat ◽  
Mitochondrial Fractions ◽  
Developing Rat ◽  
Neonatal Rat Brain

Neurones in the central nervous system of vertebrates contain considerable amounts of a complex group of acidic glycosphingolipids, the gangliosides. Determination of gangliosides, cholesterol, phospholipid phosphorus, neutral glycolipid hexose, and protein in developing rat brain showed that the deposition of gangliosides is predominantly a premyelin event. A light membrane fraction enriched in gangliosides relative to protein was obtained from neonatal rat-brain crude mitochondrial fractions by sucrose density-gradient centrifugations. Adult rat-brain fractions of similar density were not enriched in gangliosides because of the presence of myelin. If the initial homogenization and the separation of the crude mitochondrial fraction were carried out in 0.32 M sucrose at pH 9.2, the ganglioside enrichment of the light membrane fraction from both adult and neonatal rat brain was doubled. By further separation of the light membrane fraction on a second density gradient, particulate material was obtained from neonatal rat brain which consisted almost entirely of vesicular membrane elements. Based on dry weight, it contained gangliosides 7–9%, phospholipids 36–40%, cholesterol 5–7%, neutral glycolipids 1–3%, protein 28–29%, and cations (Na+, K+, Ca2+, Mg2+) 8%. This membrane fraction is likely derived from the neuronal plasma membrane or the endoplasmic reticulum.

scholarly journals SURFACE STRUCTURE OF ISOLATED NEURONS

1970 ◽  
Vol 47 (2) ◽  
pp. 319-331 ◽  
Author(s):  
Anders Hamberger ◽  
Hans-Arne Hansson ◽  
Johan Sjöstrand
Keyword(s):  
Electron Microscopy ◽  
Hypoglossal Nerve ◽  
Light And Electron Microscopy ◽  
Neuronal Cell ◽  
Spherical Particles ◽  
Hypoglossal Nucleus ◽  
Isolated Neurons ◽  
Tissue Sections ◽  
Neuronal Cell Bodies ◽  
Transmission Electron

Freehand, isolated neuronal perikarya from the hypoglossal nucleus of the rabbit have been examined with light-and electron-microscopy (transmission and scanning). The surface of the cell bodies was largely covered with spherical particles which were 0.5–2 µ in diameter. Transmission electron microscopy proved that the spherical particles were synaptic nerve terminals. Crush of the hypoglossal nerve which leads to chromatolysis and swelling of the neuronal cell bodies results in a conspicuous reduction in the number of terminals attached to the surface of hypoglossal neurons. This effect was observed both for isolated neurons and in tissue sections. The effect is considered in relation to earlier reported variations in the adherence of neuropil to isolated neuronal perikarya. The functional importance of nerve ending detachment in connection with nerve injury is discussed.

scholarly journals Subcellular localization of NAD(P)H oxidase(s) in human neutrophilic polymorphonuclear leucocytes

1978 ◽  
Vol 176 (1) ◽  
pp. 175-178 ◽  
Author(s):  
D B Iverson ◽  
P Wang-Iverson ◽  
J K Spitznagel ◽  
L R DeChatelet
Keyword(s):  
Cell Membrane ◽  
Nadph Oxidase ◽  
Subcellular Localization ◽  
Density Gradient ◽  
Membrane Fraction ◽  
Sucrose Density Gradient ◽  
Human Neutrophils ◽  
Polymorphonuclear Leucocytes

NADH and NADPH oxidase activities in a homogenate of human neutrophils co-sediment in a linear sucrose density gradient under either velocity or isopycnic conditions of centrifugation. The position of these activities in the gradient does not correspond to any known subcellular granule or to the cell-membrane fraction. These data suggest that the oxidase activities may reside in a unique granule that has previously not been recognized.

scholarly journals Drosophila ßHeavy-Spectrin is required in polarized ensheathing glia that form a diffusion-barrier around the neuropil

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Nicole Pogodalla ◽  
Holger Kranenburg ◽  
Simone Rey ◽  
Silke Rodrigues ◽  
Albert Cardona ◽  
...  
Keyword(s):  
Nervous System ◽  
Plasma Membrane ◽  
Diffusion Barrier ◽  
Neuronal Cell ◽  
Apical Plasma Membrane ◽  
Neuronal Cell Bodies ◽  
The Central Nervous System ◽  
Basolateral Plasma Membrane ◽  
Nervous System Function ◽  
Spectrin Cytoskeleton

AbstractIn the central nervous system (CNS), functional tasks are often allocated to distinct compartments. This is also evident in the Drosophila CNS where synapses and dendrites are clustered in distinct neuropil regions. The neuropil is separated from neuronal cell bodies by ensheathing glia, which as we show using dye injection experiments, contribute to the formation of an internal diffusion barrier. We find that ensheathing glia are polarized with a basolateral plasma membrane rich in phosphatidylinositol-(3,4,5)-triphosphate (PIP3) and the Na+/K+-ATPase Nervana2 (Nrv2) that abuts an extracellular matrix formed at neuropil-cortex interface. The apical plasma membrane is facing the neuropil and is rich in phosphatidylinositol-(4,5)-bisphosphate (PIP2) that is supported by a sub-membranous ßHeavy-Spectrin cytoskeleton. ßHeavy-spectrin mutant larvae affect ensheathing glial cell polarity with delocalized PIP2 and Nrv2 and exhibit an abnormal locomotion which is similarly shown by ensheathing glia ablated larvae. Thus, polarized glia compartmentalizes the brain and is essential for proper nervous system function.

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 Organization of the phosphoinositide cycle. Assessment of inositol transferase activity in purified plasma membranes

1993 ◽  
Vol 290 (1) ◽  
pp. 179-183 ◽  
Author(s):  
O M Santiago ◽  
L I Rosenberg ◽  
M E Monaco
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Density Gradient ◽  
Plasma Membranes ◽  
Sucrose Density Gradient ◽  
Tumour Cells ◽  
Assay System ◽  
Mammary Tumour ◽  
Transferase Activity ◽  
Phosphoinositide Cycle

Experiments were carried out to determine whether or not CDP-diacylglycerol:myo-inositol 3-phosphatidyltransferase (IT) activity (EC 2.7.8.11) could be detected in purified plasma-membrane fractions from WRK-1 rat mammary tumour cells. These cells have previously been shown to have a very active phosphoinositide cycle. Sucrose-density-gradient-purified plasma membranes contained no IT activity that could not be accounted for by endoplasmic-reticulum contamination. However, we also determined that the relative amount of IT activity in endoplasmic reticulum and plasma-membrane fractions could be altered by changing the concentration of detergent in the assay system.

scholarly journals Drosophila beta-Heavy-Spectrin is required in polarized ensheathing glia that form a diffusion-barrier around the neuropil

2021 ◽  
Author(s):  
Nicole Pogodalla ◽  
Holger Kranenburg ◽  
Simone Rey ◽  
Silke Rodrigues ◽  
Albert Cardona ◽  
...  
Keyword(s):  
Nervous System ◽  
Plasma Membrane ◽  
Diffusion Barrier ◽  
Neuronal Cell ◽  
Apical Plasma Membrane ◽  
Neuronal Cell Bodies ◽  
Insect Cns ◽  
The Central Nervous System ◽  
Nervous System Function ◽  
Spectrin Cytoskeleton

In the central nervous system (CNS), functional tasks are often allocated to distinct compartments. This is also evident in the insect CNS where synapses and dendrites are clustered in distinct neuropil regions. The neuropil is separated from neuronal cell bodies by ensheathing glia, which as we show using dye injection experiments forms an internal diffusion barrier. We find that ensheathing glial cells are polarized with a basolateral plasma membrane rich in phosphatidylinositol-(3,4,5)-triphosphate (PIP3) and the Na+/K+-ATPase Nervana2 (Nrv2) that abuts an extracellular matrix formed at neuropil-cortex interface. The apical plasma membrane is facing the neuropil and is rich in phosphatidylinositol-(4,5)-bisphosphate (PIP2) that is supported by a sub-membranous beta-Heavy-Spectrin cytoskeleton. beta-Heavy-spectrin mutant larvae affect ensheathing glial cell polarity with delocalized PIP2 and Nrv2 and exhibit an abnormal locomotion which is similarly shown by ensheathing glia ablated larvae. Thus, polarized glia compartmentalizes the brain and is essential for proper nervous system function.

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.

Cl- -HCO3- -stimulated ATPase in intestinal mucosa of Aplysia

1985 ◽  
Vol 248 (2) ◽  
pp. R241-R248 ◽  
Author(s):  
G. A. Gerencser ◽  
S. H. Lee
Keyword(s):  
Plasma Membrane ◽  
Atpase Activity ◽  
Intestinal Mucosa ◽  
Membrane Fraction ◽  
Transport Mechanism ◽  
Plasma Membranes ◽  
Sucrose Density Gradient ◽  
Disulfonic Acid ◽  
Differential Centrifugation ◽  
Optimum Ph

The serosa negative transepithelial potential difference across Aplysia intestine is generated by a Na+-independent, active electrogenic Cl- absorptive mechanism. In an attempt to clarify the Cl- absorptive mechanism an anion-stimulated ATPase was prepared from plasma membranes from Aplysia enterocytes utilizing differential centrifugation and sucrose density gradient techniques. ATPase activity, which could be activated by either Cl- or HCO3-, was found in the plasma membrane fraction. Maximal anion-ATPase activity was achieved with either 25 mM Cl- or 25 mM HCO3-. The apparent Km for Cl- activation of the ATPase was 10.3 mM, whereas apparent Km for HCO3- was 9.7 mM. ATP was the most effective nucleotide substrate for both HCO3- and Cl- -ATPase activities, whereas optimum pH for both activities was 7.8. These enzyme activities were inhibited more than 30% by thioacyanate (10 mM). Acetazolamide and vanadate were also found to strongly inhibit both Cl- and HCO3- -ATPase activities, whereas 10 microM 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid, 1 mM furosemide, or 1 mM ouabain had little or no effect. These results are consistent with the hypothesis that the active Cl- transport mechanism in Aplysia intestine could be a Cl- -HCO3- -stimulated ATPase found in the enterocyte plasma membrane.

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