scholarly journals Freeze-stable sialidase activity in human leucocytes: substrate specificity, inhibitor susceptibility, detergent requirements and subcellular localization

1994 ◽  
Vol 301 (3) ◽  
pp. 777-784 ◽  
Author(s):  
P J Waters ◽  
A P Corfield ◽  
R Eisenthal ◽  
C A Pennock
Keyword(s):  
Plasma Membrane ◽  
Heparan Sulphate ◽  
Subcellular Fractionation ◽  
Sodium Cholate ◽  
Acidic Ph ◽  
Sialidase Activity ◽  
Acetylneuraminic Acid ◽  
Mononuclear Leucocytes ◽  
Radioactive Assay ◽  
Assay Conditions

Human leucocytes contain a freeze-stable sialidase (neuraminidase; EC 3.2.1.18) activity in addition to the better-characterized lysosomal freeze-labile enzyme. In order to discriminate between the sialidase activities detected with the synthetic fluorimetric substrate 4-methylumbelliferyl-alpha-D-N-acetylneuraminic acid (MU-Neu5Ac), different tritiated sialoglycoconjugate substrates were prepared. Using this sensitive radioactive assay system, leucocyte sialidase activity towards glycoproteins was shown to be labile to repeated freeze-thawing, but a Triton-stimulated activity towards gangliosides was entirely freeze-stable. Assay conditions were optimized for this freeze-stable ganglioside sialidase activity. Subcellular fractionation of mononuclear leucocytes (MNLs) on Percoll-density gradients showed that this ganglioside sialidase activity was entirely associated with the plasma membrane. Study of the detergent requirements showed that MNLs also demonstrated ganglioside sialidase activity when sodium cholate was present in place of Triton. Cholate-stimulated ganglioside sialidase activity was found to be entirely freeze-stable and localized at the plasma membrane. Studies on whole homogenates of MNLs demonstrated that the Triton-stimulated and cholate-stimulated activities showed similar acidic pH optima at < or = 3.9 and were both strongly inhibited by 2-deoxy-2,3-didehydro-N-acetylneuraminic acid and Cu2+, but not by free N-acetylneuraminic acid, N-(4-nitrophenyl)oxamic acid or heparan sulphate. These results suggest that human MNLs contain, in addition to the lysosomal freeze-labile sialidase, a single sialidase activity which is freeze-stable, ganglioside-specific, plasma membrane-associated and stimulated both by Triton and by cholate.

scholarly journals Ganglioside GM3 sialidase activity in fibroblasts of normal individuals and of patients with sialidosis and mucolipidosis IV. Subcellular distribution and some properties

1989 ◽  
Vol 260 (1) ◽  
pp. 69-74 ◽  
Author(s):  
M Lieser ◽  
E Harms ◽  
H Kern ◽  
G Bach ◽  
M Cantz
Keyword(s):  
Plasma Membrane ◽  
External Surface ◽  
Human Fibroblasts ◽  
Sodium Cholate ◽  
Ganglioside Gm3 ◽  
Intact Cells ◽  
Sialidase Activity ◽  
Ionic Detergent ◽  
Mucolipidosis Iv ◽  
Triton X

Sensitive assays for the determination of the ganglioside sialidase activity of fibroblast homogenates were established using ganglioside GM3, 3H-labelled in the sphingosine moiety, as a substrate. Ganglioside GM3 sialidase activity was greatly stimulated by the presence of the non-ionic detergent Triton X-100 and was further enhanced by salts such as NaCl; the optimal pH was 4.5. The subcellular localization of this activity was determined by fractionation using free-flow electrophoresis and found to be exclusively associated with the marker for the plasma membrane, but not with that for lysosomes. This Triton-stimulated ganglioside sialidase activity was selectively inhibited by preincubating intact cells in the presence of millimolar concentrations of Cu2+, suggesting that the activity resides on the external surface of the plasma membrane. In normal fibroblasts homogenates, ganglioside GM3 sialidase was also greatly stimulated by sodium cholate. In contrast to the Triton X-100-activated reaction, however, it was not diminished by prior incubation of intact cells in the presence of Cu2+. Only after cell lysis was Cu2+ inhibitory. the cholate-stimulated ganglioside sialidase activity thus paralleled the behaviour of the lysosomal 4-methylumbelliferyl-alpha-D-N-acetylneuraminic acid (4-MU-NeuAc) sialidase. In fibroblasts from sialidosis patients, the cholate-stimulated ganglioside GM3 sialidase activity, but not that of the Triton-activated enzyme, was profoundly diminished. In fibroblasts from patients with mucolipidosis IV (ML IV), both the Triton X-100- and the cholate-stimulated ganglioside GM3 sialidase activities were in the range of normal controls. The Triton-activated enzyme was associated with the plasma membrane in the same manner as in normal cells. Our findings suggest that, in human fibroblasts, there exist two sialidases that degrade ganglioside GM3: one on the external surface of the plasma membrane, and another that is localized in lysosomes and seems identical with the activity that acts on sialyloligosaccharides and 4-MU-NeuAc. As neither activity was found to be deficient in ML IV fibroblasts, our results argue against the hypothesis of a primary involvement of a ganglioside GM3 sialidase in the pathogenesis of ML IV.

scholarly journals CELL SURFACE IMMUNOGLOBULIN

1972 ◽  
Vol 135 (6) ◽  
pp. 1392-1405 ◽  
Author(s):  
Charles J. Sherr ◽  
Sonia Baur ◽  
Inge Grundke ◽  
Joseph Zeligs ◽  
Barbara Zeligs ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Noncovalent Interactions ◽  
Subcellular Fractionation ◽  
Splenic Lymphocytes ◽  
Surface Immunoglobulin ◽  
Established Line ◽  
Intracellular Proteins ◽  
Daudi Cells

Cells from an established line of Burkitt lymphoma (Daudi) were enzymatically radioiodinated, and labeled Ig from the cell surface was isolated and studied. Subcellular fractionation of labeled cells confirmed that intracellular proteins from the cytoplasm are not iodinated by this method. Radioactive Ig was identified as monomeric (8S) IgM, and an average of 105 Ig molecules was found per cell. Ig molecules could be released from the plasma membrane by detergent lysis under nonreducing conditions indicating that attachment of Ig to the plasma membrane occurs via noncovalent interactions. The ratio of µ/L radioactivity in surface Ig was the same as that of total cellular Ig radioiodinated in solution suggesting that a large portion of the Fc fragment is not buried within the membrane. In contrast to the results obtained with cell surface Ig, most intracellular Ig was found as "free" µ- and L chains regardless of whether lysates were labeled with 125I or cells were labeled with leucine-3H. The results indicate that only a small percentage of the total Ig of Daudi cells is associated with the cell surface and suggest that covalent assembly of Ig occurs at or near the time that the molecule becomes part of the plasma membrane. Similarities between cell surface Ig on normal splenic lymphocytes and Daudi cells suggest that the latter is a neoplasm of bone marrow-derived lymphocytes.

scholarly journals A cell-free assay for the insertion of a viral glycoprotein into the plasma membrane.

1986 ◽  
Vol 103 (5) ◽  
pp. 1829-1835 ◽  
Author(s):  
P G Woodman ◽  
J M Edwardson
Keyword(s):  
Influenza Virus ◽  
Plasma Membrane ◽  
Trichloroacetic Acid ◽  
Plasma Membranes ◽  
Semliki Forest Virus ◽  
Fusion Reaction ◽  
Viral Glycoprotein ◽  
Acetylneuraminic Acid ◽  
Donor And Acceptor ◽  
A Cell

A cell-free assay has been developed for the delivery of influenza virus neuraminidase to the plasma membrane. Two types of postnuclear supernatant, which acted as donor and acceptor of the enzyme, were prepared from baby hamster kidney cells. Donor preparations were obtained from cells infected with influenza virus and containing neuraminidase en route to the plasma membrane. Acceptor preparations were obtained from cells containing, bound to their plasma membranes, Semliki Forest virus with envelope glycoproteins bearing [3H]N-acetylneuraminic acid. Fusion between vesicles from these two preparations permits access of the enzyme to its substrate, which results in the release of free [3H]N-acetylneuraminic acid. This release was detected through the transfer of radioactivity from a trichloroacetic acid-insoluble to a trichloroacetic acid-soluble fraction. An ATP-dependent component of release was found, which appears to be a consequence of vesicle fusion. This component was enhanced when the donor was prepared from cells in which the enzyme had been concentrated in a compartment between the Golgi complex and the plasma membrane, which indicates that a specific exocytic fusion event has been reconstituted. The extent of fusion is greatly reduced by pre-treatment of donor and acceptor preparations with trypsin, which points to the involvement of proteins in the fusion reaction.

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.

scholarly journals The a-factor transporter (STE6 gene product) and cell polarity in the yeast Saccharomyces cerevisiae.

1993 ◽  
Vol 120 (5) ◽  
pp. 1203-1215 ◽  
Author(s):  
K Kuchler ◽  
H G Dohlman ◽  
J Thorner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Gene Product ◽  
Cell Polarity ◽  
Polyclonal Antibodies ◽  
Apparent Molecular Weight ◽  
Subcellular Fractionation ◽  
Mating Pheromone ◽  
Yeast Saccharomyces Cerevisiae ◽  
Factor Secretion

STE6 gene product is required for secretion of the lipopeptide mating pheromone a-factor by Saccharomyces cerevisiae MATa cells. Radiolabeling and immunoprecipitation, either with specific polyclonal antibodies raised against a TrpE-Ste6 fusion protein or with mAbs that recognize c-myc epitopes in fully functional epitope-tagged Ste6 derivatives, demonstrated that Ste6 is a 145-kD phosphoprotein. Subcellular fractionation, various extraction procedures, and immunoblotting showed that Ste6 is an intrinsic plasma membrane-associated protein. The apparent molecular weight of Ste6 was unaffected by tunicamycin treatment, and the radiolabeled protein did not bind to concanavalin A, indicating that Ste6 is not glycosylated and that glycosylation is not required either for its membrane delivery or its function. The amino acid sequence of Ste6 predicts two ATP-binding folds; correspondingly, Ste6 was photoaffinity-labeled specifically with 8-azido-[alpha-32P]ATP. Indirect immunofluorescence revealed that in exponentially growing MATa cells, the majority of Ste6 showed a patchy distribution within the plasma membrane, but a significant fraction was found concentrated in a number of vesicle-like bodies subtending the plasma membrane. In contrast, in MATa cells exposed to the mating pheromone alpha-factor, which markedly induced Ste6 production, the majority of Ste6 was incorporated into the plasma membrane within the growing tip of the elongating cells. The highly localized insertion of this transporter may establish pronounced anisotropy in a-factor secretion from the MATa cell, and thereby may contribute to the establishment of the cell polarity which restricts partner selection and cell fusion during mating to one MAT alpha cell.

Additive effect of contractions and insulin on GLUT-4 translocation into the sarcolemma

1994 ◽  
Vol 77 (4) ◽  
pp. 1597-1601 ◽  
Author(s):  
J. Gao ◽  
J. Ren ◽  
E. A. Gulve ◽  
J. O. Holloszy
Keyword(s):  
Plasma Membrane ◽  
Glucose Transport ◽  
Membrane Fraction ◽  
Glucose Transporter ◽  
Subcellular Fractionation ◽  
Muscle Group ◽  
Intracellular Membrane ◽  
Glut 4 ◽  
Incremental Effect ◽  
Glut 4 Translocation

The maximal effects of insulin and muscle contractions on glucose transport are additive. GLUT-4 is the major glucose transporter isoform expressed in skeletal muscle. Muscle contraction and insulin each induce translocation of GLUT-4 from intracellular sites into the plasma membrane. The purpose of this study was to test the hypothesis that the incremental effect of contractions and insulin on glucose transport is mediated by additivity of the maximal effects of these stimuli on GLUT-4 translocation into the sarcolemma. Anesthetized rats were given insulin by intravenous infusion to raise plasma insulin to 2,635 +/- 638 microU/ml. The gastrocnemius-plantaris-soleus group was stimulated to contract via the sciatic nerve by using a protocol that maximally activates glucose transport. After treatment with insulin, contractions, or insulin plus contractions or no treatment, the gastrocnemius-plantaris-soleus muscle group was dissected out and was subjected to subcellular fractionation to separate the plasma membrane and intracellular membrane fractions. Insulin induced a 70% increase and contractions induced a 113% increase in the GLUT-4 content of the plasma membrane fraction. The effects of insulin and contractions were additive, as evidenced by a 185% increase in the GLUT-4 content of the sarcolemmal fraction. This finding provides evidence that the incremental effect of maximally effective insulin and contractile stimuli on glucose transport is mediated by additivity of their effects on GLUT-4 translocation into the sarcolemma.

scholarly journals The organelles of the trans domain of the cell. Ultrastructural localization of sialoglycoconjugates using Limax flavus agglutinin.

1986 ◽  
Vol 34 (8) ◽  
pp. 1069-1077 ◽  
Author(s):  
K Hedman ◽  
I Pastan ◽  
M C Willingham
Keyword(s):  
Plasma Membrane ◽  
Ultrastructural Localization ◽  
Ultrastructural Level ◽  
Coated Pits ◽  
Golgi Stack ◽  
Acetylneuraminic Acid ◽  
Secretory Glycoproteins ◽  
Double Label ◽  
Endocytic Vesicles ◽  
Exocytic Pathway

The subcellular distribution of sialic acid was determined at the ultrastructural level using Limax flavus agglutinin (LFA). This lectin, which is specific for N-acetylneuraminic acid and N-glycolylneuraminic acid, was covalently conjugated to horseradish peroxidase (HRP). The conjugates (LFA-HRP) were applied to aldehyde-fixed, saponin-permeabilized 3T3 cells in pre-embedding labeling electron microscopy. Peroxidase label was detected in a patchy distribution at the cell surface, and in plasma-membrane-coated pits, endocytic vesicles (receptosomes), multivesicular bodies, and lysosomes. Smooth-surfaced tubular and vesicular structures, similar to those that participate in membrane recycling, were labeled. In the Golgi complex, more than half of the cisternae contained label--typically only one cisterna on the cis side was unlabeled. Heavily labeled structures of the trans Golgi included a reticular membranous system with coated regions--50-80 nm diameter vesicular or pit-like profiles and larger coated vacuoles. Smooth 200-300 nm vacuoles were labeled on the trans side of the Golgi stack. Similar structures have been previously shown to participate in the exocytosis of plasma membrane and secretory glycoproteins from the Golgi stacks. These findings identify those intracellular organelles that are functionally at the level of, or distal to, the sialyltransferase-containing membranes of the Golgi, and distinguish them from the pre-Golgi membranous structures. The LFA-HRP conjugate is an indicator for this functional trans domain of the cell, and should be applicable for ultrastructural double-label experiments as a cis versus trans marker of the exocytic pathway.

scholarly journals Association of protein kinase-C-alpha with cytoplasmic vesicles in melanoma cells.

1996 ◽  
Vol 44 (2) ◽  
pp. 177-182 ◽  
Author(s):  
J Timar ◽  
B Liu ◽  
R Bazaz ◽  
K V Honn
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Fluid Phase ◽  
Subcellular Fractionation ◽  
Melanoma Cells ◽  
Kinase C ◽  
Cytoplasmic Vesicles ◽  
Protein Kinase C Alpha ◽  
Pkc Alpha

In B16a melanoma cells, protein kinase-C-alpha (PKC alpha) is immunomorphologically associated with cytoplasmic vesicles in addition to the previously observed locations (plasma membrane, cytoskeleton, nucleus), as detected with monoclonal antibody (MAb) MC3a. Subcellular fractionation indicated that the authentic 80-KD protein as well as PKC activity can be detected in several particulate fractions except for L2, which contains dense lysosomes. The highest PKC activity is associated with the cytosol-ultralight vesicles and the L1 fraction (containing plasma membrane, endosomes, and the Golgi apparatus). Both of these fractions contained the fluid-phase endocytosis marker peroxidase, indicating that PKC alpha, in addition to other subcellular structures, is most probably associated with endosomal membranes in B16a melanoma cells.

scholarly journals The R-SNARE Endobrevin/VAMP-8 Mediates Homotypic Fusion of Early Endosomes and Late Endosomes

2000 ◽  
Vol 11 (10) ◽  
pp. 3289-3298 ◽  
Author(s):  
Wolfram Antonin ◽  
Claudia Holroyd ◽  
Ritva Tikkanen ◽  
Stefan Höning ◽  
Reinhard Jahn
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Subcellular Fractionation ◽  
Immunogold Electron Microscopy ◽  
Fusion Reactions ◽  
Coated Pits ◽  
Late Endosomes ◽  
Early Endosomes ◽  
Golgi Network

Endobrevin/VAMP-8 is an R-SNARE localized to endosomes, but it is unknown in which intracellular fusion step it operates. Using subcellular fractionation and quantitative immunogold electron microscopy, we found that endobrevin/VAMP-8 is present on all membranes known to communicate with early endosomes, including the plasma membrane, clathrin-coated pits, late endosomes, and membranes of thetrans-Golgi network. Affinity-purified antibodies that block the ability of endobrevin/VAMP-8 to form SNARE core complexes potently inhibit homotypic fusion of both early and late endosomes in vitro. Fab fragments were as active as intact immunoglobulin Gs. Recombinant endobrevin/VAMP-8 inhibited both fusion reactions with similar potency. We conclude that endobrevin/VAMP-8 operates as an R-SNARE in the homotypic fusion of early and late endosomes.

Identification and expression of NEU3, a novel human sialidase associated to the plasma membrane

2000 ◽  
Vol 349 (1) ◽  
pp. 343-351 ◽  
Author(s):  
Eugenio MONTI ◽  
Maria T. BASSI ◽  
Nadia PAPINI ◽  
Mirko RIBONI ◽  
Marta MANZONI ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Northern Blot Analysis ◽  
Expressed Sequence Tag ◽  
Bos Taurus ◽  
Transmembrane Helix ◽  
High Sequence Identity ◽  
Ph Optimum ◽  
Sialidase Activity ◽  
Ganglioside Content ◽  
Expressed Sequence

Several mammalian sialidases have been described so far, suggesting the existence of numerous polypeptides with different tissue distributions, subcellular localizations and substrate specificities. Among these enzymes, plasma-membrane-associated sialidase(s) have a pivotal role in modulating the ganglioside content of the lipid bilayer, suggesting their involvement in the complex mechanisms governing cell-surface biological functions. Here we describe the identification and expression of a human plasma-membrane-associated sialidase, NEU3, isolated starting from an expressed sequence tag (EST) clone. The cDNA for this sialidase encodes a 428-residue protein containing a putative transmembrane helix, a YRIP (single-letter amino acid codes) motif and three Asp boxes characteristic of sialidases. The polypeptide shows high sequence identity (78%) with the membrane-associated sialidase recently purified and cloned from Bos taurus. Northern blot analysis showed a wide pattern of expression of the gene, in both adult and fetal human tissues. Transient expression in COS7 cells permitted the detection of a sialidase activity with high activity towards ganglioside substrates at a pH optimum of 3.8. Immunofluorescence staining of the transfected COS7 cells demonstrated the protein's localization in the plasma membrane.

Sign in / Sign up

Export Citation Format

Share Document