scholarly journals Plasma membrane localization of alkaline phosphatase in HeLa cells.

1976 ◽  
Vol 24 (5) ◽  
pp. 659-667 ◽  
Author(s):  
C W Lin ◽  
M Sasaki ◽  
M L Orcutt ◽  
H Miyayama ◽  
R M Singer
Keyword(s):  
Alkaline Phosphatase ◽  
Plasma Membrane ◽  
Hela Cells ◽  
Dense Body ◽  
Enzyme Reaction ◽  
Subcellular Fractionation ◽  
Enzyme Characterization ◽  
Marker Enzyme ◽  
Electron Microscopic

The localization of alkaline phosphatase in HeLa cells was examined by electron microscopic histochemistry and subcellular fractionation techniques. Two monophenotypic sublines of HeLa cells which respectively produced Regan and non-Regan isoenzymes of alkaline phosphatase were used for this study. The electron microscopic histochemical results showed that in both sublines the major location of alkaline phosphatase is in the plasma membrane. The enzyme reaction was occasionally observed in some of the dense body lysosomes. This result was supported by data obtained from a subcellular fractionation study which showed that the microsomal fraction rich in plasma membrane fragments had the highest activity of alkaline phosphatase. The distribution of this enzyme among the subcellular fractions closely paralleled that of the 5'-nucleotidase, a plasma membrane marker enzyme. Characterization of the alkaline phosphatase present in each subcellular fraction showed identical enzyme properties, which suggests that a single isoenzyme exists among fractions obtained from each cell line. The results, therefore, confirm the reports suggesting that plasma membrane is the major site of alkaline phosphatase localization in HeLa cells. The absence of any enzyme reaction in the perimitochondrial space in these cultured tumor cells also indicates that the mitochondrial localization of the Regan isoenzyme reported in ovarian cancer may not be a common phenomenon in Regan-producing cancer cells.

scholarly journals Isolation and characterization of alkaline phosphatase-containing granules (phosphasomes) from human polymorphonuclear leucocytes

1985 ◽  
Vol 76 (1) ◽  
pp. 167-178
Author(s):  
G.P. Smith ◽  
G. Sharp ◽  
T.J. Peters
Keyword(s):  
Alkaline Phosphatase ◽  
Plasma Membrane ◽  
Sucrose Density Gradient ◽  
Subcellular Fractionation ◽  
Polymorphonuclear Leucocytes ◽  
Vesicle Membrane ◽  
Isolation And Characterization ◽  
Membrane Components ◽  
Counter Current

Human polymorphonuclear leucocytes were homogenized in isotonic sucrose and subjected to analytical subcellular fractionation by centrifugation on a discontinuous sucrose density gradient to produce a phosphasome-enriched fraction, contaminated primarily by plasma membrane. Experiments to separate these membrane fractions by centrifugation on gradients of Nycodenz or Percoll, or by toroidal coil counter current chromatography, were unsuccessful. Fractionation carried out on neutrophils previously suspended in isotonic sucrose containing a low concentration of digitonin resulted in the preparation of a highly purified phosphasome fraction, free of plasma membrane components. Electron-microscope cytochemistry of the purified fraction identified the phosphasomes as regular and irregular-shaped spheres and rods, the alkaline phosphatase being associated with the inner aspect of the vesicle membrane. These granule structures were very similar to phosphasomes observed in intact neutrophils. A proportion of the endoplasmic reticulum and Golgi membrane marker enzymes remained associated with the phosphasomes throughout the separation procedures.

Lipocortin 1 (annexin 1) in patches associated with the membrane of a lung adenocarcinoma cell line and in the cell cytoplasm

1998 ◽  
Vol 111 (10) ◽  
pp. 1405-1418 ◽  
Author(s):  
V. Traverso ◽  
J.F. Morris ◽  
R.J. Flower ◽  
J. Buckingham
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Lung Adenocarcinoma ◽  
Western Blotting ◽  
Signal Sequence ◽  
Membrane Surface ◽  
Integral Membrane Protein ◽  
Subcellular Fractionation ◽  
Cell Fractionation ◽  
Electron Microscopic

Lipocortin 1 (annexin I) is a calcium- and phospholipid-binding annexin protein which can be externalised from cells despite the lack of a signal sequence. To determine its cellular distribution lipocortin 1 in A549 human lung adenocarcinoma cells was localised by light- and electron-microscopic immunocytochemistry and by cell fractionation and western blotting. Lipocortin 1 immunoreactivity is concentrated in prominent patches associated with the plasma membrane. The intensity of these patches varied with the confluence and duration of the culture and was not detectably diminished by an EDTA wash before fixation. Tubulin and cytokeratin 8 were colocalized with lipocortin 1 in the patches. Within the cells lipocortin 1 was distributed throughout the cytoplasm. Electron microscopy revealed prominent immunoreactivity along the plasma membrane with occasional large clusters of gold particles in contact with the membrane surface of the cells; within the cytoplasm the membrane of some vesicle/vacuole structures and some small electron-dense bodies was immunoreactive, but no immunogold particles were associated with the multilamellar bodies. Subcellular fractionation, extraction and western blotting showed that lipocortin 1 in the membrane pellet was present as two distinct fractions; one, intimately associated with the lipid bilayer, which behaved like an integral membrane protein and one loosely attached which behaved like a peripheral membrane protein. The results show that a substantial amounts of lipocortin 1 is concentrated in focal structures associated with and immediately beneath the plasma membrane. These might form part of the mechanism by which lipocortin 1 is released from the cells.

Identification of intracellular sites of superoxide production in stimulated neutrophils

1998 ◽  
Vol 111 (1) ◽  
pp. 81-91 ◽  
Author(s):  
T. Kobayashi ◽  
J.M. Robinson ◽  
H. Seguchi
Keyword(s):  
Alkaline Phosphatase ◽  
Plasma Membrane ◽  
Phosphatase Activity ◽  
Alkaline Phosphatase Activity ◽  
Time Course ◽  
Superoxide Production ◽  
Human Neutrophils ◽  
Morphological Evidence ◽  
Marker Enzyme ◽  
Intracellular Compartments

In this study, we show that superoxide production is carried out within intracellular compartments of human neutrophils and not at the plasma membrane following stimulation with phorbol myristate acetate. Oxidant production was not observed in unstimulated cells. Stimulated cells exhibited superoxide production in two distinct types of intracellular organelles. Initially, activity was detected in slender rod-shaped granules and in spherical or elliptical granules. The oxidant-producing granules fused directly with the plasma membrane or fused to form larger intracellular vesicles which then became associated with the plasma membrane. Longer periods of stimulation with PMA resulted in a decrease in the number of vesicles containing oxidant reaction product only, and an increase in structures containing both the oxidant-reaction product and ferritin particles; the latter was used herein as a marker for endocytosis. Thus a complex pattern of intracellular vesicular trafficking occurs in stimulated neutrophils. Alkaline phosphatase activity, a marker enzyme for a type of intracellular neutrophil granule was co-localized in the oxidant reaction-positive intracellular compartments. The time course of up-regulation of alkaline phosphatase activity to the cell surface parallelled the release of superoxide from stimulated cells. Results from this study demonstrate for the first time cytochemical and morphological evidence that superoxide is released from stimulated neutrophils through exocytosis of an oxidant-producing intracellular granule.

scholarly journals ELECTRON MICROSCOPE STUDIES OF THE MICROVILLI OF HELA CELLS

1967 ◽  
Vol 34 (2) ◽  
pp. 569-576 ◽  
Author(s):  
Harold W. Fisher ◽  
T. W. Cooper
Keyword(s):  
Hela Cells ◽  
Electron Microscopic Examination ◽  
Spatial Arrangement ◽  
Bottom Surface ◽  
Electron Microscopic ◽  
Replica Technique ◽  
Embedding Technique ◽  
Surface Replica

Microvilli of HeLa cells cultured in vitro were preserved for electron microscopic examination at different stages of routine cultivation procedures. By a double-embedding technique, vertical sectioning for electron microscopy was possible. It revealed that, although the microvilli were present on all sides of the cell in the dispersed stage and in the attached stage, they were not present on the bottom of the cell when it was stretched on the surface of the dish. When the cells were grown in dense colonies, they were found on top of each other, and microvilli were present on all sides, except on the bottom surface of those cells in contact with the dish. We achieved a more dramatic demonstration of the microvilli by developing a surface-replica technique which retains their spatial arrangement and permits characterization of the distribution of their number, length, and diameter.

scholarly journals Placental Alkaline Phosphatase Expression at the Apical and Basal Plasma Membrane in Term Villous Trophoblasts

2001 ◽  
Vol 49 (9) ◽  
pp. 1155-1164 ◽  
Author(s):  
Karl Leitner ◽  
Roman Szlauer ◽  
Isabella Ellinger ◽  
Adolf Ellinger ◽  
Klaus-Peter Zimmer ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Plasma Membrane ◽  
Marker Enzyme ◽  
Placental Alkaline Phosphatase ◽  
Membrane Expression ◽  
Morphological Marker ◽  
Basal Plasma Membrane ◽  
Human Placental Alkaline Phosphatase ◽  
Basal Plasma

Human placental alkaline phosphatase (PLAP) was localized at the apical and basal plasma membrane of syncytiotrophoblasts and at the surface of cytotrophoblasts in term chorionic villi using immunoelectron microscopy. Similarly, apical and basolateral PLAP expression was found in polarized trophoblast-derived BeWo cells. Trophoblasts isolated from term placentas exhibited mainly vesicular PLAP immunofluorescence staining immediately after isolation. After in vitro differentiation into syncytia, PLAP plasma membrane expression was upregulated and exceeded that observed in mononuclear trophoblasts. These data call for caution in using PLAP as a morphological marker to differentiate syncytiotrophoblasts from cytotrophoblasts or as a marker enzyme for placental brush-border membranes. (J Histochem Cytochem 49:1155–1164, 2001)

scholarly journals Isolation and characterization of sea urchin egg cortical granules.

1982 ◽  
Vol 95 (3) ◽  
pp. 924-932 ◽  
Author(s):  
G S Kopf ◽  
G W Moy ◽  
V D Vacquier
Keyword(s):  
Plasma Membrane ◽  
Sea Urchin ◽  
Specific Activity ◽  
Specific Radioactivity ◽  
Marker Enzyme ◽  
Cortical Granules ◽  
Electrophoretic Patterns ◽  
Isolation And Characterization ◽  
Sea Urchin Egg

A method has been developed to isolate cortical granules (CG) free in suspension. It involves the mechanical disruption of the CG from CG lawns (CGL; Dev. Biol. 43:62-74, 1975) and concentration of the CG by low speed centrifugation. The isolated CG are intact and are a relatively pure population as judged by electron microscopy. Granule integrity is confirmed by the fact that isolated intact CG are radioiodinated to only 0.05% of the specific activity of hypotonically lysed CG. Purity of the CG preparation is assessed by the enrichment (four- to sevenfold) of CG marker enzymes and the absence or low activity of plasma membrane, mitochondrial, cytoplasmic, and yolk platelet marker enzyme activities. CG isolated from 125I-surface-labeled eggs have a very low specific radioactivity, demonstrating that CG contamination by the plasma membrane-vitelline layer (PM-VL) is minimal. CG yield is approximately 1% of the starting egg protein. The CG isolation method is simple and rapid, 4 mg of CG protein being obtained in 1 h. Isolated CG and PM-VL display distinct electrophoretic patterns on SDS gels. Actin is localized to the PM-VL, and all bands present in the CGL are accounted for in the CG and PM-VL. Calmodulin is associated with the CGL, CG, and PM-VL fractions, but is not specifically enriched in these fractions as compared with whole egg homogenates. This method of isolating intact CG from unfertilized sea urchin eggs may be useful for exploring the mechanism of Ca2+-mediated CG exocytosis.

scholarly journals Immunohistochemical characterization of a differentiation-specific carbohydrate epitope in the plasma membrane of the epithelial cells of rat small intestine.

1993 ◽  
Vol 41 (1) ◽  
pp. 71-79 ◽  
Author(s):  
H Schiechl
Keyword(s):  
Small Intestine ◽  
Plasma Membrane ◽  
Epithelial Cells ◽  
Basolateral Membrane ◽  
Microscopic Level ◽  
Electron Microscopic Level ◽  
Lateral Part ◽  
Rat Small Intestine ◽  
Electron Microscopic

The monoclonal antibody (MAb) SI/EC1 was produced by immunization of Balb/c mice with an antigen prepared from the isolated basolateral membrane (BLM) of rat small intestine epithelial cells by trypsin cleavage. Immunohistochemical labeling at the light and electron microscopic level shows that the SI/EC1 epitope is localized in the plasma membrane (PM) of the small intestine epithelial cells and is expressed around Day 21 after birth (weaning time). There are, however, differences in the labeling between crypt and villous cells. In the crypt cells, the microvillous membrane (MVM) and the lateral part of the BLM are strongly labeled, whereas the basal part of the BLM is unlabeled. In the villous cells, both the MVM and the basal and lateral part of the BLM are labeled, but the labeling is not as intense as in the crypts. In immunoblotting experiments with the isolated BLM, three protein bands (125 KD, 110 KD, and 90 KD) were labeled specifically with the MAb. Enzymic cleaving of the BLM with exo- and endoglycosidases and subsequent immunoblotting, as well as other findings, suggest that the specific structure of the SI/EC1 epitope consists mainly of carbohydrates (CH) (oligosaccharides). This finding points out the possibility that this epitope may have something to do with the variable adhesion of the small intestine epithelial cells along the crypt-villus axis.

scholarly journals Characterization of calcium-ion-activated adenosine triphosphatase in the plasma membrane of rat mast cells

1976 ◽  
Vol 156 (3) ◽  
pp. 691-700 ◽  
Author(s):  
P H Cooper ◽  
D R Stanworth
Keyword(s):  
Plasma Membrane ◽  
Mast Cells ◽  
Adenosine Triphosphatase ◽  
Gradient Centrifugation ◽  
Subcellular Fractionation ◽  
Ph Optimum ◽  
Peritoneal Mast Cells ◽  
Triton X ◽  
Calcium Ion Activated

The properties of a Ca2+ activated adenosine triphosphatase shown to be present in homogenates of purified rat peritoneal mast cells were investigated. The enzyme was activated by Ca2+, Mg2+, and to a lesser extent by Mn2+ and Co2+. Ca2+ alone was necessary for full activity and the further addition of Mg2+ did not have any effect. The chelating agents EGTA (ethanedioxybis(ethylamine)tetra-acetate) and EDTA completely inhibited the reaction. The pH optimum was 7.8. Reduced glutathione, cysteine, dithiothreitol, N-ethylmaleimide, urea, ADP, NaF, increasing ionic strength and Triton X-100 all inhibited the reaction. On subcellular fractionation of mast-cell homogenates by density-gradient centrifugation, the distribution of Ca2+ activated adenosine triphosphatase resembled that of 5′-nucleotidase, but differed from that of the other markers used, suggesting localization in the plasma membrane. Further experiments indicated that the enzyme is present on the external surface of the plasma membrane.

scholarly journals An Electron Microscopic Study of Erythrophagocytosis

1960 ◽  
Vol 7 (2) ◽  
pp. 329-334 ◽  
Author(s):  
Edward Essner
Keyword(s):  
Plasma Membrane ◽  
Acid Phosphatase ◽  
Phosphatase Activity ◽  
Microscopic Study ◽  
Hela Cells ◽  
Acid Phosphatase Activity ◽  
Ascites Hepatoma ◽  
Electron Microscopic ◽  
Intracellular Digestion ◽  
Physicochemical State

The present study describes a submicroscopic surface fragmentation of erythrocytes which occurs in the ascitic fluid of rats bearing the Novikoff ascites hepatoma. The resulting fragments attach to the surface of macrophages and are phagocytized by pseudopod formation. Plasma membrane in the region of these phagocytosis vacuoles appears to condense into electron-opaque material, suggesting an alteration in its physicochemical state. Stages in intracellular digestion of intact erythrocytes or small fragments within the phagocytosis vacuoles are illustrated; no particles resembling ferritin are observed. The phagocytosis vacuoles possess high levels of acid phosphatase activity. They may be called phagosomes, a type of lysosome. There is no indication of a connection between phagosomes and other formed cytoplasmic organelles. Small vacuoles of the order of 80 mµ in diameter, which may represent pinocytosis vacuoles, are present in the cytoplasm and some appear to be in contact with the phagosome membrane, reminiscent of observations of Rose with HeLa cells.

Electron Microscopic Detection and Characterization of Nonhuman Primate Enteric Viruses

1982 ◽  
Vol 40 ◽  
pp. 306-307
Author(s):  
G. C. Smith ◽  
R. L. Heberling ◽  
S. S. Kalter
Keyword(s):  
Electron Microscopy ◽  
Animal Model ◽  
Nonhuman Primate ◽  
Clinical Condition ◽  
Intestinal Tract ◽  
Enteric Viruses ◽  
Electron Microscopic ◽  
Microscopic Detection

A number of viral agents are recognized as and suspected of causing the clinical condition “gastroenteritis.” In our attempts to establish an animal model for studies of this entity, we have been examining the nonhuman primate to ascertain what viruses may be found in the intestinal tract of “normal” animals as well as animals with diarrhea. Several virus types including coronavirus, adenovirus, herpesvirus, and picornavirus (Table I) were detected in our colony; however, rotavirus, astrovirus, and calicivirus have not yet been observed. Fecal specimens were prepared for electron microscopy by procedures reported previously.

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