scholarly journals The formation of oligoglucans linked to lipid during synthesis of β-glucan by characterized membrane fractions isolated from peas

1975 ◽  
Vol 148 (1) ◽  
pp. 107-117 ◽  
Author(s):  
C T Brett ◽  
D H Northcote
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Chemical Properties ◽  
Membrane Vesicles ◽  
Sucrose Density Gradient ◽  
Phosphate Esters ◽  
Synthetic Activity ◽  
Smooth Membrane ◽  
Glucan Synthesis ◽  
Gradient Fractionation

Membrane fractions were obtained from peas roots by using a method that permitted the isolation of a fraction rich in relatively intact dictyosome stacks. No chemical fixatives were used. The method involved incubation of the roots with cellulase, followed by gentle homogenization and sucrose-density-gradient fractionation of the homogenate. The fractions were characterized by electron microscopy. All fractions were enzymically active in incorporating glucose from UDP-glucose into water-insoluble glycolipids containing both single glucose residues and glucose oligosaccharides. Some or all of the linkages of glucose to lipid were through phosphate esters. A substance containing glucose oligosaccharides attached to or very strongly adsorbed on to protein was also formed. The membrane fractions also incorporated glucose from UDP-glucose into alkali-soluble and alkali-insoluble β-glucans, which like the oligosaccharides contained β(1leads to 3) and β-(1leads to4) linkages. The distribution of the enzymic activities and the chemical properties of the lipid-linked and protein-linked oligosaccharides suggest that they may be intermediates in β-glucan synthesis. The synthetic activity is associated with smooth-membrane vesicles which may be derived from the plasma membrane.

The unique stability of the marsupial sperm acrosomal membranes examined by unprotected freeze-thawing and treatment with the detergent Triton X-100

1993 ◽  
Vol 5 (1) ◽  
pp. 1 ◽  
Author(s):  
Y Sistina ◽  
M Lin ◽  
KE Mate ◽  
ES Robinson ◽  
JC Rodger
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Membrane Vesicles ◽  
Tammar Wallaby ◽  
Significant Loss ◽  
Monodelphis Domestica ◽  
Brushtail Possum ◽  
Plasma Membrane Vesicles ◽  
High Concentrations ◽  
Unique Stability

In this study of the unique stability of the marsupial acrosome, experiments were carried out on the acrosomes of spermatozoa of the tammar wallaby (Macropus eugenii), common brushtail possum (Trichosurus vulpecula) and grey short-tailed opossum (Monodelphis domestica). Light microscopy showed that 4% of opossum and 15% of possum and wallaby spermatozoa lost their acrosomes after freeze-thawing. Electron microscopy revealed that freeze-thawing also induced changes in the acrosomal matrix of some acrosome intact spermatozoa. In both possum and wallaby, freeze-thawing increased the number of spermatozoa with vesiculation of the acrosomal matrix. Freeze-thawing disrupted the plasma membrane of spermatozoa but the acrosomal membranes remained intact. Immediately on addition of high concentrations of TX-100 (0.02% and 0.04%) there was significant loss of acrosomes and motility in possum and wallaby spermatozoa. Lower concentrations of TX-100 (< or = 0.01%) did not affect motility for up to 30 min in all three species, and there was no significant loss of acrosomes. Although loss of acrosomes did not occur under mild detergent treatment, 56% of wallaby and 70% of possum spermatozoa had altered acrosomes after 30 min in 0.01% TX-100. Electron microscopy revealed that acrosomes were undergoing a vesiculation process similar to that seen after freeze-thawing. Often the plasma membrane of detergent-treated spermatozoa was disrupted and had formed plasma membrane vesicles. However, the acrosomal membranes remained intact despite major changes to the acrosomal matrix. The study confirmed the remarkable stability of the marsupial acrosome and suggested that this is probably based in the acrosomal membranes.

scholarly journals Distribution of membranes, especially of plasma-membrane fragments, during zonal centrifugations of homogenates from glucose-repressed Saccharomyces Cerevisiae

1976 ◽  
Vol 154 (3) ◽  
pp. 751-763 ◽  
Author(s):  
T Nurminen ◽  
L Taskinen ◽  
H Suomalainen
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
High Speed ◽  
Membrane Vesicles ◽  
Appreciable Amount ◽  
Main Peak ◽  
Mitochondrial Density ◽  
Minor Peak ◽  
Smooth Membrane ◽  
A Minor

1. The distributions of several enzymes and other marker components were examined after zonal centrifugations of whole homogenates from glucose-repressed Saccharomyces cerevisiae on sucrose and iso-osmotic Ficoll, and the composition and morphology of the fractions were investigated. 2. After high-speed zonal centrifugation most of the protein, acid and alkaline phosphatases, alkaline pyrophosphatase, adenosine monophosphatase, β-fructofuranosidase, α-mannosidase, NADPH-cytochrome c oxidoreductase and an appreciable amount of phospholipid and sterol were non-sedimentable, i.e. were at densities below 1.09 (g/cm3). Most of the RNA was at p=1.06-1.08 in Ficoll and at p=1.09-1.11 in sucrose. 3. The bulk of the Mg2+-dependent adenosine triphosphatase (Mg-ATPase) was coincident with the main peak of phospholipid and sterol, at median density 1.10, which was also rich in smooth-membrane vesicles. In Ficoll, a minor peak of phospholipid and sterol at p-1.12-1.15 contained a smaller part of the oligomycin-insensitive Mg-ATPase and heavy membrane fragments. In sucrose, several minor peaks of Mg-ATPase were in the mitochondrial density range, and a peak of oligomycin-insensitive Mg-ATPase coincident with a minor peak of phospholipid and sterol at around p-1.25 contained heavy membrane fragments of high carbohydrate content, especially mannose. 4. Further purification of the oligomycin-insensitive Mg-ATPase containing membrane preparations was performed on Urografin gradients. 5. It is argued that the oligomycin-insensitive Mg-ATPase containing membranes are fragments of the plasma membrane, but have different densities because they contain different amounts of glycoprotein particles.

scholarly journals Phorbol ester-sensitive phospholipase D is mainly localized in the endoplasmic reticulum of BHK cells

1996 ◽  
Vol 320 (3) ◽  
pp. 885-890 ◽  
Author(s):  
Christina DECKER ◽  
Maria Jesus MIRO OBRADORS ◽  
Daniel J. SILLENCE ◽  
David ALLAN
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Phospholipase D ◽  
Phorbol Ester ◽  
Membrane Vesicles ◽  
Sucrose Density Gradient ◽  
Subcellular Fractionation ◽  
Plasma Membrane Vesicles ◽  
Intact Cells ◽  
Site Of Action

The localization of phorbol ester-sensitive phospholipase D (PLD) in baby hamster kidney cells has been investigated by determining the subcellular distribution of the phosphatidylbutanol produced when the cells are incubated with phorbol 12-myristate 13-acetate and n-butanol. Results derived by isolation of plasma membrane vesicles from intact cells or by subcellular fractionation on a sucrose density gradient suggest the PLD is specific for phosphatidylcholine and its primary site of action is not the plasma membrane but the endoplasmic reticulum.

scholarly journals ISOLATION OF PLASMA MEMBRANE FRAGMENTS FROM HELA CELLS

1969 ◽  
Vol 41 (2) ◽  
pp. 378-392 ◽  
Author(s):  
Charles W. Boone ◽  
Lincoln E. Ford ◽  
Howard E. Bond ◽  
Donald C. Stuart ◽  
Dianne Lorenz
Keyword(s):  
Plasma Membrane ◽  
Hela Cells ◽  
Plasma Membranes ◽  
Reductase Activity ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Sucrose Density Gradient ◽  
Plasma Membrane Vesicle ◽  
Whole Cells ◽  
Continuous Sucrose Gradient

A method for isolating plasma membrane fragments from HeLa cells is described. The procedure starts with the preparation of cell membrane "ghosts," obtained by gentle rupture of hypotonically swollen cells, evacuation of most of the cell contents by repeated washing, and isolation of the ghosts on a discontinuous sucrose density gradient. The ghosts are then treated by minimal sonication (5 sec) at pH 8.6, which causes the ghost membranes to pinch off into small vesicles but leaves any remaining larger intracellular particulates intact and separable by differential centrifugation. The ghost membrane vesicles are then subjected to isopycnic centrifugation on a 20–50% w/w continuous sucrose gradient in tris-magnesium buffer, pH 8.6. A band of morphologically homogeneous smooth vesicles, derived principally from plasma membrane, is recovered at 30–33% (peak density = 1.137). The plasma membrane fraction contained a Na-K-activated ATPase activity of 1.5 µmole Pi/hr per mg, 3% RNA, and 13.8% of the NADH-cytochrome c reductase activity of a heavier fraction from the same gradient which contained mitochondria and rough endoplasmic vesicles. The plasma membranes of viable HeLa cells were marked with 125I-labeled horse antibody and followed through the isolation procedure. The specific antibody binding of the plasma membrane vesicle fraction was increased 49-fold over that of the original whole cells.

Electrogenic ATP-dependent Cl- transport by plasma membrane vesicles from Aplysia intestine

1988 ◽  
Vol 254 (1) ◽  
pp. R127-R133 ◽  
Author(s):  
G. A. Gerencser
Keyword(s):  
Plasma Membrane ◽  
Transport Process ◽  
Transport Mechanism ◽  
Plasma Membranes ◽  
Membrane Vesicles ◽  
Sucrose Density Gradient ◽  
Differential Centrifugation ◽  
Plasma Membrane Vesicles ◽  
Cl Transport ◽  
Vesicular Membrane

A Cl--stimulated adenosinetriphosphatase (ATPase) activity and an ATP-dependent Cl- transport process were found in Aplysia enterocyte plasma membranes. In an attempt to further elucidate this transport process plasma membrane vesicles from Aplysia enterocytes were prepared utilizing differential centrifugation and sucrose density gradient techniques. Electrogenicity of the ATP-dependent Cl- transport was confirmed in three ways. First, an inwardly directed valinomycin-induced K+ diffusion potential, making the vesicle interior electrically positive, enhanced ATP-driven Cl- uptake compared with vesicles lacking the ionophore. Second, ATP plus Cl- increased intravesicular negativity measured by lipophilic triphenylmethylphosphonium distribution across the vesicular membrane. Third, both vanadate and thiocyanate inhibited the ATP plus Cl--dependent intravesicular negativity. These results are consistent with the hypothesis that the active electrogenic Cl- transport mechanism in Aplysia intestine could be a Cl--stimulated ATPase found in the enterocyte plasma membrane.

scholarly journals Plasma Membrane Vesicles from Mouse 15091A Tumor Cells: Agent of Platelet Aggregating Activity

1977 ◽  
Author(s):  
G.J. Gasic ◽  
J.L. Catafalmo ◽  
G.P. Gasic ◽  
S.J. Shattil ◽  
G.J. Stewart
Keyword(s):  
Plasma Membrane ◽  
Tumor Cells ◽  
Cell Transformation ◽  
Gradient Centrifugation ◽  
Amorphous Material ◽  
Membrane Vesicles ◽  
Sucrose Density Gradient ◽  
Plasma Membrane Vesicles ◽  
Sucrose Density Gradient Centrifugation ◽  
Artificial Plasma

We reported that cells from most tumors display platelet aggregating activity (PAA) in heparinized plasma and that this activity contributes to metastasis.Recently, we demonstrated that PAA can be used as a marker of cell transformation in virally infected rat cells. The material responsible for PAA is shed into culture medium. Characterization revealed a material which is particulate and sedimentable at 50,000x g for 60 min.; it contains proteins and lipids with a free cholesterol to phospholipid ratio of 0.556.Delipida-tion as well as complete solubilization abolished PAA.SDS-ME PAGE, 7.5% slab gels, revealed 20 bands.EM studies of 50,000x g pellets shed by 15091A cells indicated they contained numerous vesicles, some solid bodies, numerous free or vesicle associated small particles, and some amorphous material. Discontinuous sucrose density gradient centrifugation of the 50,000x g pellet yielded at the 1.07-1.17 g/cm3 interface a predominantly vesicular fraction which was the most active interfacial material. The vesicles, visible with phase contrast microscopy, resemble those produced by artificial plasma membrane vesiculation in various cell systems, including normal cells. Since PAA is only shown by transformed cells, vesicles from these must be different or much more numerous. Spontaneous vesiculation by tumor cells may be potentially important in understanding cell transformation and tumor metastases.

scholarly journals TheSchizosaccharomyces pombe spo3+Gene Is Required for Assembly of the Forespore Membrane and Genetically Interacts withpsy1+-encoding Syntaxin-like Protein

2001 ◽  
Vol 12 (12) ◽  
pp. 3955-3972 ◽  
Author(s):  
Taro Nakamura ◽  
Michiko Nakamura-Kubo ◽  
Aiko Hirata ◽  
Chikashi Shimoda
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Vegetative Growth ◽  
Fluorescent Protein ◽  
Membrane Vesicles ◽  
Null Mutant ◽  
Wild Type ◽  
Normal Morphology ◽  
Novel Proteins ◽  
Green Fluorescent

Formation of the forespore membrane, which becomes the plasma membrane of spores, is an intriguing step in the sporulation of the fission yeast Schizosaccharomyces pombe. Here we report two novel proteins that localize to the forespore membrane.spo3+encodes a potential membrane protein, which was expressed only during sporulation. Green fluorescent protein (GFP) fusion revealed that Spo3 localized to the forespore membrane. The spo3 disruptant was viable and executed meiotic nuclear divisions as efficiently as the wild type but did not form spores. One of the spo3 alleles,spo3-KC51, was dose-dependently suppressed bypsy1+, which encodes a protein similar to mammalian syntaxin-1A, a component of the plasma membrane docking/fusion complex. psy1+was essential for vegetative growth, and its transcription was enhanced during sporulation. As expected, Psy1 localized to the plasma membrane during vegetative growth. Interestingly, Psy1 on the plasma membrane disappeared immediately after first meiotic division and relocalized to the forespore membrane as the second division initiated. In thespo3 null mutant, the forespore membrane was initiated but failed to develop a normal morphology. Electron microscopy revealed that membrane vesicles were accumulated in the cytoplasm of immaturespo3Δ asci. These results suggest that Spo3 is a key component of the forespore membrane and is essential for its assembly acting in collaboration with the syntaxin-like protein.

Structural Study of H,K-ATPase by Electron Microscopy and Image Reconstruction

1990 ◽  
Vol 48 (1) ◽  
pp. 94-95
Author(s):  
Manijeh Mohraz ◽  
Sonal Sathe ◽  
P.R. Smith
Keyword(s):  
Electron Microscopy ◽  
Amino Acids ◽  
Plasma Membrane ◽  
Image Reconstruction ◽  
Sequence Homology ◽  
Membrane Vesicles ◽  
Two Dimensional ◽  
Β Subunit ◽  
Transport Atpases ◽  
Protein Rich

H,K-ATPase is an integral protein of the plasma membrane of parietal cells. It is believed to constitute the pump responsible for secretion of acid into stomach. Its catalytic subunit (Mr 110,000) shows striking sequence homology to those of other transport ATPases. Recent studies suggest that there is also a glycoprotein (ca 300 amino acids) associated with the H,K pump, which is very homologous to the β subunit of the Na,K-ATPase.The enzyme is isolated in protein-rich membrane vesicles from hog stomachs. Formation of two-dimensional crystals is induced in suspensions of the enzyme by methods that had proved successful for crystallization of the Na,K-ATPase.

scholarly journals Isolation of a plasma-membrane fraction from gastric smooth muscle. Comparison of the calcium uptake with that in endoplasmic reticulum

1983 ◽  
Vol 210 (2) ◽  
pp. 315-322 ◽  
Author(s):  
L Raeymaekers ◽  
F Wuytack ◽  
J Eggermont ◽  
G De Schutter ◽  
R Casteels
Keyword(s):  
Endoplasmic Reticulum ◽  
Smooth Muscle ◽  
Plasma Membrane ◽  
Membrane Fraction ◽  
Microsomal Fraction ◽  
Membrane Vesicles ◽  
Cholesterol Content ◽  
Sucrose Density Gradient ◽  
Plasma Membrane Vesicles ◽  
Plasma Membrane Fraction

1. A plasma-membrane fraction was isolated from the smooth muscle of the pig stomach by using differential and sucrose-density-gradient centrifugations. When the centrifugation was carried out after preloading the crude microsomal fraction with Ca2+ in the presence of oxalate, the contamination of the plasma-membrane fraction by endoplasmic reticulum was decreased and a fraction enriched in endoplasmic reticulum vesicles filled with calcium oxalate crystals was obtained. 2. The plasmalemmal and endoplasmic-reticulum membranes could be distinguished by differences in the activity of marker enzymes and in the cholesterol content and by their different permeability to oxalate and phosphate. Oxalate and phosphate stimulated the Ca2+ uptake in the endoplasmic reticulum much more than in the plasmalemmal vesicles. In the plasma-membrane vesicles 40 mM-phosphate was more effective for stimulating the Ca2+ uptake than was 5 mM-oxalate, but the reverse was seen in the endoplasmic reticulum. 3. The high cholesterol/phospholipid ratio of the crude microsomal fraction are of the majority of the vesicles present in the crude microsomal fraction are of plasmalemmal origin. 4. The Ca2+ pump of the plasmalemmal and endoplasmic-reticulum vesicles could be differentiated by their different sensitivities to calmodulin. However, the two Ca2+-transport ATPases did not differ by their sensitivity to vanadate nor by the energization of the Ca2+ transport by different nucleoside triphosphates.

p-Chloromercurobenzene sulfonate inhibition of active Cl- transport in plasma membrane vesicles from Aplysia gut

1990 ◽  
Vol 259 (6) ◽  
pp. R1111-R1116
Author(s):  
G. A. Gerencser
Keyword(s):  
Plasma Membrane ◽  
Transport Process ◽  
Transport Mechanism ◽  
Plasma Membranes ◽  
Membrane Vesicles ◽  
Sucrose Density Gradient ◽  
Absorptive Cell ◽  
Plasma Membrane Vesicles ◽  
Cl Transport ◽  
Cell Plasma

Both a Cl(+)-stimulated adenosinetriphosphatase (ATPase) activity and an ATP-dependent Cl- transport process were found in Aplysia foregut absorptive cell plasma membranes. In an attempt to further characterize this transport process, plasma membrane vesicles from Aplysia foregut absorptive cells were prepared utilizing differential centrifugation and sucrose density-gradient techniques. Sulfhydryl ligand participation in ATP-dependent Cl- transport was confirmed in three ways. First, 1,4-dithiothreitol partially restored a p-chloromercurobenzene sulfonate (PCMBS)-inhibited ATP-dependent Cl- transport. Second, 1,4-dithiothreitol restored intravesicular negativity inhibited by PCMBS. Third, 1,4-dithiothreitol had no effect on either ATP-dependent Cl- transport or ATP-dependent intravesicular negativity inhibited by N-ethylmaleimide. These results are consistent with the hypothesis that surface sulfhydryl groups participate in the functioning of the active electrogenic Cl- transport mechanism in Aplysia gut.

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