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 Cellular localization of neuraminidases in cultured human fibroblasts

1981 ◽  
Vol 198 (3) ◽  
pp. 505-508 ◽  
Author(s):  
M Zeigler ◽  
G Bach
Keyword(s):  
Cellular Localization ◽  
Plasma Membranes ◽  
Colloidal Silica ◽  
Human Fibroblasts ◽  
Lysosomal Hydrolase ◽  
Cell Disease ◽  
Cultured Fibroblasts ◽  
Cultured Human Fibroblasts ◽  
Triton X ◽  
Cellular Organelles

The cellular localization of glycoprotein and ganglioside sialidases in normal and I-cell-disease cultured fibroblasts has been investigated. Cellular organelles have been separated on a colloidal silica gradient. The subcellular distribution of these enzymes indicated that the glycoprotein sialidase is mainly a lysosomal hydrolase, whereas the ganglioside sialidase is primarily located in the plasma membranes. The latter isoenzymes is tightly bound to these membranes and thus could not be extracted by homogenization in the presence of Triton X-100. The interpretation of this finding and its relation to the pathochemistry of sialidase-deficient disorders is discussed.

Alloxan action on glucose metabolism in cultured fibroblasts. II. Effects on pentose-monophosphate shunt and tricarboxylic acid pathways

1981 ◽  
Vol 240 (6) ◽  
pp. E645-E648
Author(s):  
F. Ishibashi ◽  
P. H. Bennett ◽  
B. V. Howard
Keyword(s):  
Glucose Metabolism ◽  
Human Fibroblasts ◽  
Lactate Production ◽  
Tricarboxylic Acid ◽  
Cultured Fibroblasts ◽  
Cell Monolayers ◽  
Cell Extracts ◽  
Cultured Human Fibroblasts ◽  
Glucose Incorporation ◽  
14Co2 Production

The site of action of alloxan on glucose metabolism has been investigated using cultured human fibroblasts. Analysis of cell extracts after cell monolayers were exposed to D-[U-14C]glucose indicated that the initial stimulation of glucose incorporation by alloxan was observed primarily in the nucleotide fraction (ribose) with inhibition of lactate production. The subsequent inhibition of glucose incorporation was observed in the nucleotide fraction. Assay of 14CO2 production indicated that alloxan enhanced 14CO2 formation from D-[U-14C]glucose for approximately 10 min, followed by inhibition. To probe the site of alloxan action, rates of 14CO2 formation from 1- and 6-position labeled [14C]glucose, and [U-14C]pyruvate were compared. The initial stimulation was observed mainly in D-[1–14C]glucose oxidation, whereas inhibition was measurable with the 6-position tracer and [14C]pyruvate. The results suggest that alloxan initially stimulates the pentose-monophosphate shunt and then subsequently inhibits both the pentose-monophosphate shunt and tricarboxylic acid pathways.

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.

Immunogold labelling of neutral endopeptidase 24.11 (enkephalinase), on human neutrophils and neutrophil cytoplasts

1989 ◽  
Vol 47 ◽  
pp. 920-921
Author(s):  
V. Kriho ◽  
B. Wagner ◽  
E.G. Erdos ◽  
R.P. Becker
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Cell Surface ◽  
Plasma Membranes ◽  
Intact Cell ◽  
Neutral Endopeptidase ◽  
Immunogold Labelling ◽  
Human Neutrophils ◽  
Endopeptidase 24.11 ◽  
Cell Surface Structure

We have documented the presence of neutral endopeptidase 24.11 (NEP), on the surface of human neutrophils (PMN) and PMN cytoplasts. Cytoplasts are whole cell preparations which contain cytomatrix, but lack internal membranes and organelles ,such as nuclei and lysosomal granules. These structures have been extracted mechanically, leaving the plasma membrane “outside-out” topology intact. Cytoplasts are very useful in correlative studies of cell surface structure and function. Biochemically, the membrane component of cytoplasts is predominantly plasma membrane; structurally, chemical activity may be localized to domains of the intact cell surface. NEP is a membrane-bound metalloendopeptidase present in human PMN' s. We have marked NEP on the plasma membranes of PMNs and PMN cytoplasts via pre-embedding iramunocytochemistry. We used scanning electron microscopy (SEM) with backscattered electron imaging (BEI) to visualize Au labelled anti-NEP on the surface of a large number of cells. Transmission electron microscopy (TEM) was used to confirm the presence of the enzyme on PMN's and PMN cytoplasts.Suspensions of PMN or PMN cytoplasts (2 x 106 cells/ml) were fixed for 8 min at room temp. in 0.25% glutaraldehyde in phosphate buffered saline (PBS) pH 7.2 rinsed in PBS, treated with 0.1% glycine in PBS for 10 rain and then incubated for 15 min in 5% normal goat serum (NGS) in 0.1% bovine serum albumin dissolved in PBS (BSA/PBS). Following this step, cells were incubated for 20 min in anti- NEP antibody, rinsed in BSA/PBS, incubated in goat anti-rabbit IgG coupled to 15nm colloidal Au particles (GARG15) for 1 h and again rinsed in PBS. Postfixation for 30 min in 2.5% glutaraldehyde and PBS rinsing followed. For SEM a drop of cell suspension was put on a polylysine- treated Formvar-carbon-coated Au grid and cells were allowed to settle and attach for 30 min. The grid was rinsed in water, dehydrated and critical point dried. Cells were coated with carbon before viewing by SEM. For TEM, following immunolabelling, cells were post-fixed in OsO4, rinsed, dehydrated and embedded in Epon for sectioning.

scholarly journals Zymosterol is located in the plasma membrane of cultured human fibroblasts.

1990 ◽  
Vol 265 (15) ◽  
pp. 8484-8489 ◽  
Author(s):  
F Echevarria ◽  
R A Norton ◽  
W D Nes ◽  
Y Lange
Keyword(s):  
Plasma Membrane ◽  
Human Fibroblasts ◽  
Cultured Human Fibroblasts
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