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 Insertion of isolated insulin receptors into placental membrane vesicles

1992 ◽  
Vol 281 (2) ◽  
pp. 425-430 ◽  
Author(s):  
K Christiansen ◽  
J Carlsen
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Insulin Receptors ◽  
Membrane Vesicles ◽  
Insulin Binding ◽  
Scatchard Analysis ◽  
Plasma Membrane Vesicles ◽  
Intact Cells ◽  
Receptor Number ◽  
Triton X

Purified human insulin receptors were inserted into placental plasma-membrane vesicles by fusion of membranes with receptor-lysophosphatidylcholine micelles. Scatchard analysis of insulin binding showed that about 10-15% of the added receptors became inserted into the membrane. The receptor number could be increased about 3-fold, corresponding to approx. 5 pmol of receptor/mg of membrane protein. The receptors became firmly bound to the membrane, as they could not be removed by extensive wash. The insertion of exogenous receptors could be demonstrated by immunoblotting. The inserted insulin receptor had the same insulin-binding affinity as the isolated receptor and the endogenous receptor of the membrane. Insulin binding in the presence or absence of Triton X-100 revealed that more than 80% of the exogenous receptors had a right-side-out orientation. Function of the inserted receptors, as observed by insulin-stimulated autophosphorylation, could be demonstrated. About 80% of the added lysophospholipid, corresponding to approx. 160 nmol of lysophospholipid/mg of membrane protein, became integrated into the membrane and was partly metabolized to phospholipid and to non-esterified fatty acid. The method of insertion of isolated insulin receptors using the natural detergent, lysophospholipid, may be a method for insertion of receptors into intact cells, where the lysophospholipid, as in the plasma-membrane vesicles, will be acylated to phospholipid.

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 Membrane-associated phosphoproteins in Plasmodium berghei-infected murine erythrocytes.

1983 ◽  
Vol 97 (1) ◽  
pp. 196-201 ◽  
Author(s):  
M F Wiser ◽  
P A Wood ◽  
J W Eaton ◽  
J R Sheppard
Keyword(s):  
Plasma Membrane ◽  
Plasmodium Berghei ◽  
Plasma Membranes ◽  
Intact Cell ◽  
Erythrocyte Membranes ◽  
Amino Acid Residues ◽  
Two Dimensional Gel Electrophoresis ◽  
Intact Cells ◽  
Membrane Phosphorylation ◽  
Triton X

Normal and Plasmodium berghei (NYU-2 strain)-infected murine erythrocytes display substantially different patterns of plasma membrane phosphoproteins phosphorylation. Intact erythrocytes (normal and parasite infected) incubated with 32Pi and isolated washed erythrocyte plasma membranes incubated with gamma-32P-ATP were analyzed for phosphoproteins by SDS PAGE and autoradiography. Two new phosphoproteins of molecular weight 45,000 (pp45) and 68,000 (pp68), which are absent in normal erythrocyte membranes, are associated with the membranes of infected erythrocytes subjected to both intact-cell and isolated-membrane phosphorylation conditions. Two-dimensional gel electrophoresis indicates that pp45 and pp68 are of parasite origin. Partial or complete proteolytic digestion reveals that pp45 is phosphorylated at similar amino acid residues both in intact cells and in isolated membranes. The pp45 phosphoprotein can be detected at as low as 3% parasitemia and its phosphorylation is not affected by 10 microM cAMP, 1 mM Ca2+, or 5 mM EGTA. Extraction of isolated washed plasma membranes with 0.5% Triton X-100 or 0.1 M NaOH indicates that pp45 is detergent insoluble and only partially extractable with NaOH, suggesting that pp45 is closely associated with the host erythrocyte plasma membrane.

scholarly journals Ecto-ganglioside-sialidase activity of herpes simplex virus-transformed hamster embryo fibroblasts.

1976 ◽  
Vol 70 (3) ◽  
pp. 555-561 ◽  
Author(s):  
C L Schengrund ◽  
A Rosenberg ◽  
M A Repman
Keyword(s):  
Plasma Membrane ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Dehydrogenase Activity ◽  
Diazonium Salt ◽  
Intact Cells ◽  
Lactate Dehydrogenase Activity ◽  
Sialidase Activity ◽  
Embryo Fibroblasts ◽  
Simplex Virus

Cellular location of ganglioside-sialidase activity was determined in confluent hamster embryo fibroblasts transformed with herpes simplex virus type 2. Approximately equal specific activities of ganglioside-sialidase activity were found to be associated with the crude lysosomal and crude plasma membrane fractions isolated from whole cell homogenates. Whole transformed cells hydrolyzed exogenous ganglioside substrate, suggesting a partial location of the cellular sialidase on the outer surface of the plasma membrane of these cells. Intact cells were treated with the diazonium salt of sulfanilic acid, a nonpenetrating reagent inhibitory to ecto-enzymes (DePierre, J.W., and M. L. Karnovsky. 1974. J. Biol. Chem. 249:7111-7120). Cytoplasmic lactate dehydrogenase activity was not inhibited by this treatment, and mitochondrial succinate dehydrogenase activity was inhibited only 10%, indicating that intracellular enzymes were not affected. 5'-Nucleotidase activity was diminished 90%, and sialidase very rapidly lost 40% of its exogenously directed activity. These results show that, in herpes simplex virus-transformed fibroblasts, ganglioside-sialidase is both a lysosomal and a plasma membrane enzyme. The plasma membrane sialidase is capable of acting on endogenous plasma membrane sialolipids and also functions in the cultured transformed cell as an ecto-enzyme which can attack exogenous substrates.

Plasma membrane production of ceramide from ganglioside GM3 in human fibroblasts

2006 ◽  
Vol 20 (8) ◽  
pp. 1227-1229 ◽  
Author(s):  
Rea Valaperta ◽  
Vanna Chigorno ◽  
Luisa Basso ◽  
Alessandro Prinetti ◽  
Roberto Bresciani ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Human Fibroblasts ◽  
Ganglioside Gm3

scholarly journals Neutral endopeptidase-24.11 (enkephalinase). Biosynthesis and localization in human fibroblasts

1987 ◽  
Vol 248 (2) ◽  
pp. 345-350 ◽  
Author(s):  
G Lorkowski ◽  
J E Zijderhand-Bleekemolen ◽  
E G Erdös ◽  
K von Figura ◽  
A Hasilik
Keyword(s):  
Plasma Membrane ◽  
Subcellular Localization ◽  
Half Life ◽  
Human Fibroblasts ◽  
Neutral Endopeptidase ◽  
Cultured Fibroblasts ◽  
Endopeptidase 24.11 ◽  
Cultured Human Fibroblasts ◽  
Triton X

The biosynthesis, glycosylation and subcellular localization of the neutral endopeptidase-24.11 were studied in cultured human fibroblasts. The enzyme was synthesized as a precursor (Mr 88,000) containing four or five N-linked oligosaccharides. Within 1 h the synthesis-mature (Mr 94,000) endopeptidase-24.11 was formed and contained sialylated oligosaccharides. The half-life of endopeptidase-24.11 was 3.7 days and in the presence of 10 mM-NH4Cl it increased to 6 days. Mature endopeptidase-24.11 was solubilized with 0.2% saponin and partitioned into Triton X-114. In intact fibroblasts, endopeptidase-24.11 was accessible to antibodies and to neuraminidase even when the treatment was performed at 4 degrees C. The localization of endopeptidase-24.11 to the plasma membrane in cultured fibroblasts was further demonstrated by immunocytochemistry.

scholarly journals The incorporation of solubilized choline-transport activity into liposomes

1982 ◽  
Vol 204 (2) ◽  
pp. 565-576 ◽  
Author(s):  
R G King ◽  
R M Marchbanks
Keyword(s):  
Plasma Membrane ◽  
Transport System ◽  
Potential Gradient ◽  
Sodium Cholate ◽  
Synaptic Plasma Membrane ◽  
Transport Activity ◽  
Choline Transport ◽  
Triton X ◽  
Stimulation Of

The choline-transport system has been solubilized from synaptic plasma membrane by using either sodium cholate or Triton X-100, and re-incorporated into unilamellar liposomes by using the technique of cholate dialysis. The criteria of choline-transport activity were saturability by excess choline, inhibition by hemicholinium-3, and trans-activation (i.e. stimulation of the uptake of [3H]choline into liposomes by preloading them with non-radioactive choline). Liposomes prepared from detergent extracts of synaptic plasma membrane and added lipid showed uptake of [3H]choline fulfilling these three criteria. Data on choline-transport activity of liposomes at various choline concentrations could be interpreted as implying that the transport system has two apparent Km values (2-5 microM and 50-100 microM), or alternatively that the system is composed of two or more negatively co-operating subunits (or units). It was shown by t.l.c. that the transported radioactivity was choline and that it was not significantly acetylated. Replacing Na+ by K+ on the outside of these liposomes partially inhibited uptake, and the formation of a potential gradient (inside negative) with valinomycin increased the total but not the saturable components of uptake when liposomes were prepared in a K+ medium, and transferred to an Na+ medium.

scholarly journals A cell surface integral membrane glycoprotein of 85,000 mol wt (gp85) associated with triton X-100-insoluble cell skeleton.

1984 ◽  
Vol 99 (2) ◽  
pp. 512-519 ◽  
Author(s):  
G Tarone ◽  
R Ferracini ◽  
G Galetto ◽  
P Comoglio
Keyword(s):  
Monoclonal Antibody ◽  
Extracellular Matrix ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Membrane Glycoprotein ◽  
Surface Integral ◽  
Intact Cells ◽  
Bhk Cells ◽  
Triton X ◽  
Cell Skeleton

The Triton X-100-insoluble skeleton of baby hamster kidney BHK cells consists of the nucleus, intermediate-size filaments, and actin fibers. By transmission electron microscopy, membrane fragments were found to be associated with these insoluble structures. When radioiodinated or [3H]glucosamine-labeled cells were extracted with 0.5% Triton, most plasma membrane glycoproteins were solubilized except for a glycoprotein with a molecular weight of 85,000 (gp85) that remained associated with the insoluble skeletons. Immunoprecipitation with a specific antiserum indicated that the gp85 is not a proteolytic degradation product of fibronectin, an extracellular matrix glycoprotein insoluble in detergent. A monoclonal antibody of BHK cells specific for gp85 was produced. Immunofluorescence analysis with this monoclonal antibody indicated that gp85 is not associated with the extracellular matrix, but is confined to the cell membrane. Both in fixed and unfixed intact cells, fluorescence was concentrated in dots preferentially aligned in streaks on the cell surface. Gp85 was found to behave as an integral membrane protein interacting with the hydrophobic core of the lipid bilayer since it was extracted from membrane preparations by ionic detergents such as SDS, but not by 0.1 N NaOH (pH 12) in the absence of detergents, a condition known to release peripheral molecules. Association of gp85 with the cell skeleton was unaffected by increasing the Triton concentration up to 5%, but it was affected when actin filaments were dissociated or when a protein-denaturing agent (6 M urea) was used in the presence of Triton, suggesting that protein-protein interactions are involved in the association of gp85 with the cell skeleton. We conclude that gp85 is an integral plasma membrane glycoprotein that might have a role in cell surface-cytoskeleton interaction.

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