scholarly journals Streptolysin-O induces release of glycosylphosphatidylinositol-anchored alkaline phosphatase from ROS cells by vesiculation independently of phospholipase action

1995 ◽  
Vol 305 (2) ◽  
pp. 529-537 ◽  
Author(s):  
M Xie ◽  
M G Low
Keyword(s):  
Alkaline Phosphatase ◽  
Phospholipase D ◽  
Bovine Serum ◽  
Binding Agent ◽  
Extracellular Medium ◽  
Intact Cells ◽  
Streptolysin O ◽  
Binding Agents ◽  
Triton X ◽  
Cholesterol Binding

Streptolysin-O (SLO), a cholesterol-binding agent, was used for studies on the release of glycosylphosphatidylinositol (GPI)-anchored alkaline phosphatase (AP) from ROS cells. Treatment of cells with SLO resulted in a time- and concentration-dependent release of AP into the extracellular medium. This release was potentiated by Ca2+ and bovine serum, but not by GPI-specific phospholipase D (GPI-PLD) purified from bovine serum. The released AP distributed to the detergent phase after Triton X-114 phase separation. This result suggested that the released AP contained an intact GPI anchor, and thus both proteolysis and anchor degradation by anchor-specific hydrolases, including GPI-PLD, as the potential mechanisms for SLO-mediated AP release were ruled out. The released AP sedimented at 100,000 g. A substantial amount of lipids was detected in the 100,000 g pellet. Cholesterol and sphingomyelin were enriched in SLO-released material, compared with intact cells. These results were consistent with vesiculation as the mechanism for SLO induction of AP release. Two other cholesterol-binding agents, saponin and digitonin, were also able to release AP, possibly by a similar vesiculation mechanism, whereas others, including nystatin, filipin and beta-escin, failed to elicit any AP release. Eight GPI-anchored proteins were identified in ROS cells, and all were substantially enriched in the vesicles released by SLO. Taken together, these results do not provide any support for the hypothesis that the clustering of GPI-anchored proteins in the plasma membrane is responsible for their resistance to GPI-PLD cleavage.

scholarly journals Effects of cholesterol depletion by cyclodextrin on the sphingolipid microdomains of the plasma membrane

1998 ◽  
Vol 335 (2) ◽  
pp. 433-440 ◽  
Author(s):  
Subburaj ILANGUMARAN ◽  
Daniel C. HOESSLI
Keyword(s):  
Plasma Membrane ◽  
Surface Proteins ◽  
Cholesterol Depletion ◽  
Density Gradients ◽  
Streptolysin O ◽  
Binding Agents ◽  
Liquid Ordered ◽  
Detergent Resistant Membranes ◽  
Triton X ◽  
Cholesterol Binding

Sphingolipid microdomains are thought to result from the organization of plasma membrane sphingolipids and cholesterol into a liquid ordered phase, wherein the glycosylphosphatidylinositol (GPI)-anchored proteins are enriched. These domains, resistant to extraction by cold Triton X-100, can be isolated as buoyant membrane complexes (detergent-resistant membranes) in isopycnic density gradients. Here the effects of methyl-β-cyclodextrin (MBCD), a specific cholesterol-binding agent that neither binds nor inserts into the plasma membrane, were investigated on the sphingolipid microdomains of lymphocytes. MBCD released substantial quantities of GPI-anchored Thy-1 and glycosphingolipid GM1, and also other surface proteins including CD45, and intracellular Lck and Fyn kinases. From endothelial cells, MBCD released GPI-anchored CD59, and CD44, but only a negligible amount of caveolin. Most MBCD-released Thy-1 and CD59 were not sedimentable and thus differed from Thy-1 released by membrane-active cholesterol-binding agents such as saponin and streptolysin O, or Triton X-100. Unlike that released by Triton X-100, only part of the Thy-1 molecules released by MBCD was buoyant in density gradients and co-isolated with GM1. Finally, treatment of Triton X-100-isolated detergent-resistant membranes with MBCD extracted most of the cholesterol without affecting the buoyant properties of Thy-1 or GM1. We suggest that (1) MBCD preferentially extracts cholesterol from outside, rather than within the sphingolipid microdomains and (2) this partly solubilizes GPI-anchored and transmembrane proteins from the glycerophospholipid-rich membrane and releases sphingolipid microdomains in both vesicular and non-vesicular form.

Permeabilization of MDCK cells with cholesterol binding agents: dependence on substratum and confluency

1994 ◽  
Vol 267 (1) ◽  
pp. C166-C176 ◽  
Author(s):  
A. Esparis-Ogando ◽  
C. Zurzolo ◽  
E. Rodriguez-Boulan
Keyword(s):  
Mdck Cells ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Culture Conditions ◽  
Ovary Cells ◽  
Tight Junction Formation ◽  
Streptolysin O ◽  
Binding Agents ◽  
Darby Canine Kidney ◽  
Cholesterol Binding

We studied systematically the susceptibility of Madin-Darby canine kidney (MDCK) cells to permeabilization by two cholesterol binding agents, digitonin and streptolysin-O (SLO), under different culture conditions. Monolayers grown on polycarbonate filter chambers (Transwells) required twice the concentration of digitonin effective on monolayers grown on glass or plastic (80 vs. 40 micrograms/ml) to allow antibody penetration or the release of 90% of the cytosolic protein lactate dehydrogenase (LDH). Neither the apical nor the basolateral surface showed preferential susceptibility to digitonin. Confluent MDCK cells, cultured either on filters or on impermeable substrates, showed poor antibody permeability after addition of commercial SLOs, even when used at concentrations 100 times higher (20 U/ml) than those effective on nonepithelial Chinese hamster ovary cells. Surprisingly, culture conditions that prevent tight junction formation and the acquisition of a polarized phenotype (< 10 microM Ca2+) increased dramatically the susceptibility to permeabilization by SLO. On restoration of normal Ca2+ levels, susceptibility to SLO quickly decreased. Thus conditions that lead to the full establishment of polarity result in decreased sensitivity to disruption by digitonin and SLO.

scholarly journals Factors affecting the ability of glycosylphosphatidylinositol-specific phospholipase D to degrade the membrane anchors of cell surface proteins

1991 ◽  
Vol 279 (2) ◽  
pp. 483-493 ◽  
Author(s):  
M G Low ◽  
K S Huang
Keyword(s):  
Alkaline Phosphatase ◽  
Cell Surface ◽  
Phospholipase D ◽  
Bovine Serum ◽  
Cell Membranes ◽  
Surface Proteins ◽  
Phospholipid Bilayer ◽  
Placental Alkaline Phosphatase ◽  
Gpi Anchor ◽  
Cell Surface Proteins

Mammalian serum and plasma contain high levels of glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD). Previous studies with crude serum or partially purified GPI-PLD have shown that this enzyme is capable of degrading the GPI anchor of several purified detergent-solubilized cell surface proteins yet is unable to act on GPI-anchored proteins located in intact cells. Treatment of intact ROS17/2.8, WISH or HeLa cells (or membrane fractions prepared from them) with GPI-PLD purified from bovine serum by immunoaffinity chromatography gave no detectable release of alkaline phosphatase into the medium. However, when membranes were treated with GPI-PLD in the presence of 0.1% Nonidet P-40 substantial GPI anchor degradation (as measured by Triton X-114 phase separation) was observed. The mechanism of this stimulatory effect of detergent was further investigated using [3H]myristate-labelled variant surface glycoprotein and human placental alkaline phosphatase reconstituted into phospholipid vesicles. As with the cell membranes the reconstituted substrates exhibited marked resistance to the action of purified GPI-PLD which could be overcome by the inclusion of Nonidet P-40. Similar results were obtained when crude bovine serum was used as the source of GPI-PLD. These data indicate that the resistance of cell membranes to the action of GPI-PLD is not entirely due to the action of serum or membrane-associated inhibitory factors. A more likely explanation is that, in common with many other eukaryotic phospholipases, the action of GPI-PLD is restricted by the physical state of the phospholipid bilayer in which the substrates are embedded. These data may account for the ability of endothelial and blood cells to retain GPI-anchored proteins on their surfaces in spite of the high levels of GPI-PLD present in plasma.

Intestinal alkaline phosphatase is secreted bidirectionally from villous enterocytes

1989 ◽  
Vol 257 (1) ◽  
pp. G14-G23 ◽  
Author(s):  
N. L. Sussman ◽  
R. Eliakim ◽  
D. Rubin ◽  
D. H. Perlmutter ◽  
K. DeSchryver-Kecskemeti ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Phospholipase D ◽  
Sodium Dodecyl ◽  
Serum Analysis ◽  
Intestinal Cell ◽  
Intestinal Alkaline Phosphatase ◽  
Isoelectric Focussing ◽  
Intestinal Cell Line ◽  
Extracellular Release ◽  
Triton X

A fraction of intestinal alkaline phosphatase (IAP) is secreted into blood. To study this process, enzyme secretion was examined in a fetal (IRD-98) and a differentiated (Caco-2) intestinal cell line. Tissue-unspecific alkaline phosphatase (AP) activity in the IRD-98 cells increased 20-fold after addition of 1.5 mM sodium butyrate and 40 mM NaCl, but no AP activity was secreted into the medium. In contrast, newly synthesized IAP in Caco-2 cells was secreted into the medium. AP secretion increased with time and was inhibited by monensin. Medium AP was still partially bound to membranes as assessed by Triton X-114 phase separation and could be released by the addition of serum. Analysis by sodium dodecyl sulfate polyacrylamide gels and by isoelectric focussing showed that secreted AP gave a pattern similar to that of the AP released from membranes by phospholipase D treatment. When Caco-2 cells were grown on filters, AP activity was found in both basolateral (75%) and luminal (25%) media. These data demonstrate that the secretion of a particulate AP with extracellular release from the membrane can account for the appearance of the intestinal isozyme in both the serum and the lumen.

The Uptake of Adenine by Isolated Human Platelet Membranes

1974 ◽  
Vol 32 (02/03) ◽  
pp. 457-464
Author(s):  
Paul C. French ◽  
Jan J. Sixma ◽  
Holm Holmsen
Keyword(s):  
Human Platelet ◽  
Facilitated Diffusion ◽  
Adenine Phosphoribosyltransferase ◽  
Ph Optimum ◽  
Intact Cells ◽  
Platelet Membranes ◽  
Group Translocation ◽  
Triton X

SummaryAdenine uptake into isolated platelet membranes had about the same Km (151 ± 21 • 9 nM) as uptake into intact cells (159 ± 21 nM) and was also competitively inhibited by papaverine and hypoxanthine. No uptake occurred at 0° and accumulated adenine was converted to AMP. AMP was not firmly bound to protein as judged by chromatography of triton X-100 solubilized membranes on Sephadex G25. The pH optimum for adenine uptake was at pH 5-5. Exogenous 5-phosphoribosyl-l-pyrophos- phate strongly stimulated uptake. These data may be explained by uptake of adenine by facilitated diffusion followed by conversion to AMP by adenine phosphoribosyltransferase but group translocation cannot be entirely excluded.

scholarly journals Epithelial structure revealed by chemical dissection and unembedded electron microscopy.

1984 ◽  
Vol 99 (1) ◽  
pp. 203s-208s ◽  
Author(s):  
E G Fey ◽  
D G Capco ◽  
G Krochmalnic ◽  
S Penman
Keyword(s):  
Nuclear Matrix ◽  
Intermediate Filament ◽  
Protein Composition ◽  
Intact Cells ◽  
Thin Sections ◽  
Transmission Electron ◽  
Kidney Epithelial Cell ◽  
Epithelial Structure ◽  
Triton X ◽  
Whole Mounts

Cytoskeletal structures obtained after extraction of Madin-Darby canine kidney epithelial cell monolayers with Triton X-100 were examined in transmission electron micrographs of cell whole mounts and unembedded thick sections. The cytoskeleton, an ordered structure consisting of a peripheral plasma lamina, a complex network of filaments, and chromatin-containing nuclei, was revealed after extraction of intact cells with a nearly physiological buffer containing Triton X-100. The cytoskeleton was further fractionated by extraction with (NH4)2SO4, which left a structure enriched in intermediate filaments and desmosomes around the nuclei. A further digestion with nuclease and elution with (NH4)2SO4 removed the chromatin. The stable structure that remained after this procedure retained much of the epithelial morphology and contained essentially all of the cytokeratin filaments and desmosomes and the chromatin-depleted nuclear matrices. This structural network may serve as a scaffold for epithelial organization. The cytoskeleton and the underlying nuclear matrix intermediate filament scaffold, when examined in both conventional embedded thin sections and in unembedded whole mounts and thick sections, showed the retention of many of the detailed morphological aspects of the intact cells, which suggests a structural continuum linking the nuclear matrix, the intermediate filament network, and the intercellular desmosomal junctions. Most importantly, the protein composition of each of the four fractions obtained by this sequential procedure was essentially unique. Thus, the proteins constituting the soluble fraction, the cytoskeleton, the chromatin fraction, and the underlying nuclear matrix-intermediate filament scaffold are biochemically distinct.

scholarly journals The roles of phospholipase D and a GTP-binding protein in guanosine 5′-[γ-thio]triphosphate-stimulated hydrolysis of phosphatidylcholine in rat liver plasma membranes

1990 ◽  
Vol 272 (3) ◽  
pp. 749-753 ◽  
Author(s):  
K M Hurst ◽  
B P Hughes ◽  
G J Barritt
Keyword(s):  
Rat Liver ◽  
Phospholipase D ◽  
Plasma Membranes ◽  
Regulatory Protein ◽  
Gtp Binding ◽  
Liver Plasma Membranes ◽  
Low Concentrations ◽  
Rat Liver Plasma Membranes ◽  
Triton X ◽  
Hydrolysis Of

1. Guanosine 5′-[gamma-thio]triphosphate (GTP[S]) stimulated by 50% the rate of release of [3H]choline and [3H]phosphorylcholine in rat liver plasma membranes labelled with [3H]choline. About 70% of the radioactivity released in the presence of GTP[S] was [3H]choline and 30% was [3H]phosphorylcholine. 2. The hydrolysis of phosphorylcholine to choline and the conversion of choline to phosphorylcholine did not contribute to the formation of [3H]choline and [3H]phosphorylcholine respectively. 3. The release of [3H]choline from membranes was inhibited by low concentrations of SDS or Triton X-100. Considerably higher concentrations of the detergents were required to inhibit the release of [3H]phosphorylcholine. 4. Guanosine 5′-[beta gamma-imido]triphosphate and guanosine 5′-[alpha beta-methylene]triphosphate, but not adenosine 5′-[gamma-thio]-triphosphate, stimulated [3H]choline release to the same extent as did GTP[S]. The GTP[S]-stimulated [3H]choline release was inhibited by guanosine 5′-[beta-thio]diphosphate, GDP and GTP but not by GMP. 5. It is concluded that, in rat liver plasma membranes, (a) GTP[S]-stimulated hydrolysis of phosphatidylcholine is catalysed predominantly by phospholipase D with some contribution from phospholipase C, and (b) the stimulation of phosphatidylcholine hydrolysis by GTP[s] occurs via a GTP-binding regulatory protein.

Utilization of Old Coconut (Cocos nucifera) Husk Waste as Potential Source of Natural Dye and its Dyeing Properties on Cotton Cloth

2021 ◽  
Vol 882 ◽  
pp. 280-286
Author(s):  
Aris Sugih Arto Kholil ◽  
Husniyyah Ulfah Adani ◽  
Annisa’ Mufsihah ◽  
Achmad Chafidz
Keyword(s):  
Cocos Nucifera ◽  
Natural Dye ◽  
Natural Dyes ◽  
Cotton Cloth ◽  
Color Fastness ◽  
Fastness Properties ◽  
Binding Agent ◽  
Binding Agents ◽  
Colored Cotton ◽  
Brown Color

Coconut husk is considered waste and its fibers can be used as a source of natural dyes for textiles. The objective of this research is to obtain natural dyes from old coconut (Cocos nucifera) husk waste fibers through the extraction process. Brown color was produced in the liquid-liquid extraction method. The brown color natural dyes were then used to dye cotton cloth. Three different binding agents were used during the fixation process, i.e. tunjung, naphtol salt, and alum. The coloring results of the natural dye on the cotton cloth were as follow: with alum binding agent the resulting color was light brown (cream), whereas tunjung binding agent produced a greenish brown color, and using napthol salt binding agent produced yellow color. The colored cotton cloths were tested for their color fastness properties against rubbing, soap washing, and sunlight exposure. The measurement scale used was grey scales, which was used to evaluate the color change (color fading) and color staining (color transfer) during color fastness testing. In general, based on the results, the colored cotton cloth using alum as binding agent showed better color fastness properties against rubbing, soap washing, and sunlight exposure compared to the ones using tunjung and naphtol salt binding agents.

scholarly journals A permeabilized cell system identifies the endoplasmic reticulum as a site of protein degradation.

1991 ◽  
Vol 115 (5) ◽  
pp. 1225-1236 ◽  
Author(s):  
F J Stafford ◽  
J S Bonifacino
Keyword(s):  
Protein Degradation ◽  
Secretory Pathway ◽  
Fibroblast Cell ◽  
Cell System ◽  
Current Evidence ◽  
Permeabilized Cell ◽  
Intact Cells ◽  
Permeabilized Cells ◽  
Streptolysin O ◽  
A Site

Analysis of the fate of a variety of newly synthesized proteins in the secretory pathway has provided evidence for the existence of a novel protein degradation system distinct from that of the lysosome. Although current evidence suggests that proteins degraded by this system are localized to a pre-Golgi compartment before degradation, the site of proteolysis has not been determined. A permeabilized cell system was developed to examine whether degradation by this pathway required transport out of the ER, and to define the biochemical characteristics of this process. Studies were performed on fibroblast cell lines expressing proteins known to be sensitive substrates for this degradative process, such as the chimeric integral membrane proteins, Tac-TCR alpha and Tac-TCR beta. By immunofluorescence microscopy, these proteins were found to be localized to the ER. Treatment with cycloheximide resulted in the progressive disappearance of intracellular staining without change in the ER localization of the chimeric proteins. Cells permeabilized with the pore-forming toxin streptolysin O were able to degrade these newly synthesized proteins. The protein degradation seen in permeabilized cells was representative of that seen in intact cells, as judged by the similar speed of degradation, substrate selectivity, temperature dependence, and involvement of free sulfhydryl groups. Degradation of these proteins in permeabilized cells took place in the absence of transport between the ER and the Golgi system. Moreover, degradation occurred in the absence of added ATP or cytosol, and in the presence of apyrase, GTP gamma S, or EDTA; i.e., under conditions which prevent transport of proteins out of the ER. The efficiency and selectivity of degradation of newly synthesized proteins were also conserved in an isolated ER fraction. These data indicate that the machinery responsible for pre-Golgi degradation of newly synthesized proteins exists within the ER itself, and can operate independent of exogenously added ATP and cytosolic factors.

Zinc Enzymes in Crassostrea virginica

1970 ◽  
Vol 27 (1) ◽  
pp. 59-69 ◽  
Author(s):  
Douglas A. Wolfe
Keyword(s):  
Alkaline Phosphatase ◽  
Metal Binding ◽  
Zinc Enzymes ◽  
Tissue Extracts ◽  
Metabolic Functions ◽  
Binding Agents ◽  
High Molecular Weight Proteins ◽  
Cellular Components ◽  
Inhibition Studies

Nearly all the zinc in oysters is bound, either to soluble high-molecular weight proteins or to structural cellular components such as cell membranes. Oyster alkaline phosphatase is a zinc metalloenzyme, as indicated by in vitro inhibition studies with various metal-binding agents. Dialysis of soluble tissue extracts at pH 7–9 removes up to 96% of the total zinc without effect on alkaline phosphatase. If alkaline phosphatase is considered representative of the metabolic functions of zinc in oysters, most of the zinc accumulated by oysters must be superfluous to the animal's requirements.

Sign in / Sign up

Export Citation Format

Share Document