scholarly journals Characterization of neutral endopeptidase 24.11 in dog glomeruli

1993 ◽  
Vol 291 (3) ◽  
pp. 773-779 ◽  
Author(s):  
C Landry ◽  
P Santagata ◽  
W Bawab ◽  
M C Fournié-Zaluski ◽  
B P Roques ◽  
...  
Keyword(s):  
Cell Surface ◽  
Brush Border ◽  
Neutral Endopeptidase ◽  
Glycosylation Pattern ◽  
Electrophoretic Mobilities ◽  
Mammalian Tissues ◽  
Dog Kidney ◽  
A Cell ◽  
Membrane Bound ◽  
Triton X

Neutral endopeptidase (NEP; also known as neprilysin and enkephalinase; EC 3.4.24.11) is a cell-surface metallopeptidase that is present in many mammalian tissues. It is particularly abundant on the brush-border membranes of the kidney proximal tubule. In this paper, the presence of NEP in purified glomeruli from dog kidney was assessed by measuring phosphoramidon- and thiorphan-sensitive [D-Ala2,Leu5]enkephalin-degrading activity. Using this assay, the Km and kcat. of the glomerular enzyme were found to be identical to those of the tubular enzyme. By Western blotting the apparent M(r) of the glomerular enzyme was found to be 104,000, compared with 94,000 for the tubular enzyme. This might be due to a different glycosylation pattern, since endoglycosidase F treatment of NEP obtained from both tissues yielded deglycosylated enzymes with similar electrophoretic mobilities. The glomerular enzyme also appears to be membrane-bound, since it was retained in the detergent-rich phase after phase separation with Triton X-114. Autoradiography experiments performed with RB104, a new highly selective and potent NEP inhibitor, showed that NEP was expressed in both glomeruli and proximal tubules. The presence in glomeruli of NEP and some other brush-border peptidases (dipeptidyl-dipeptidase IV, aminopeptidase N and angiotensin I-converting enzyme) suggests that cell-surface peptidases might play an important role as regulators of plasma-derived peptides in this part of the nephron.

scholarly journals Cytoskeletal-membrane interactions: a stable interaction between cell surface glycoconjugates and doublet microtubules of the photoreceptor connecting cilium.

1987 ◽  
Vol 105 (6) ◽  
pp. 2973-2987 ◽  
Author(s):  
C J Horst ◽  
D M Forestner ◽  
J C Besharse
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Molecular Mass ◽  
Lipid Bilayer ◽  
Neonatal Rat ◽  
Sds Page ◽  
A Cell ◽  
The Cross ◽  
Triton X ◽  
Cell Surface Glycoconjugates

The ciliary base is marked by a transition zone in which Y-shaped cross-linkers extend from doublet microtubules to the plasma membrane. Our goal was to investigate the hypothesis that the cross-linkers form a stable interaction between membrane or cell surface components and the underlying microtubule cytoskeleton. We have combined Triton X-100 extraction with lectin cytochemistry in the photoreceptor sensory cilium to investigate the relationship between cell surface glycoconjugates and the underlying cytoskeleton, and to identify the cell surface components involved. Wheat germ agglutinin (WGA) binds heavily to the cell surface in the region of the Y-shaped cross-linkers of the neonatal rat photoreceptor cilium. WGA binding is not removed by prior digestion with neuraminidase and succinyl-WGA also binds the proximal cilium, suggesting a predominance of N-acetylglucosamine containing glycoconjugates. Extraction of the photoreceptor plasma membrane with Triton X-100 removes the lipid bilayer, leaving the Y-shaped crosslinkers associated with the axoneme. WGA-binding sites are found at the distal ends of the crosslinkers after Triton X-100 extraction, indicating that the microtubule-membrane cross-linkers retain both a transmembrane and a cell surface component after removal of the lipid bilayer. To identify glycoconjugate components of the cross-linkers we used a subcellular fraction enriched in axonemes from adult bovine retinas. Isolated, detergent-extracted bovine axonemes show WGA binding at the distal ends of the cross-linkers similar to that seen in the neonatal rat. Proteins of the axoneme fraction were separated by SDS-PAGE and electrophoretically transferred to nitrocellulose. WGA labeling of the nitrocellulose transblots reveals three glycoconjugates, all of molecular mass greater than 400 kD. The major WGA-binding glycoconjugate has an apparent molecular mass of approximately 600 kD and is insensitive to prior digestion with neuraminidase. This glycoconjugate may correspond to the dominant WGA-binding component seen in cytochemical experiments.

scholarly journals Role of glycosylation in transport and enzymic activity of neutral endopeptidase-24.11

1994 ◽  
Vol 302 (2) ◽  
pp. 451-454 ◽  
Author(s):  
M H Lafrance ◽  
C Vézina ◽  
Q Wang ◽  
G Boileau ◽  
P Crine ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Cell Surface ◽  
Intracellular Transport ◽  
Neutral Endopeptidase ◽  
Soluble Form ◽  
Cell Extracts ◽  
Glycosylation Sites ◽  
Sugar Addition ◽  
Membrane Bound

Neutral endopeptidase (NEP, EC 3.4.24.11) is a major ectoenzyme of the brush-border membrane. The ectodomain of NEP contains five putative N-glycosylation sites. In order to determine the role of the addition of sugar moieties on the activity and intracellular transport of NEP, we have used site-directed mutagenesis to remove all or some of the five potential sites of sugar addition in membrane-bound and secreted forms of the enzyme. Expression of NEP glycosylation mutants in COS-1 cells showed that all five sites are used for sugar addition. Immunoblotting of NEP in COS-1 cell extracts or culture media indicated that total expression of normal membrane-bound NEP was not affected by mutations at glycosylation sites, whereas this expression level appeared to be strictly dependent on the number of glycosylation sites retained on the soluble form. The transport to the cell surface was also reduced by decreased glycosylation, but again the phenomenon appeared more drastic in the case of the soluble form than for the membrane-bound enzyme. Enzyme activity was decreased by deglycosylation. However, the presence of either of two crucial sites (sites 1 and 5; numbered from the N-terminus of the protein) was sufficient to recover close-to-normal enzymic activities. Transport to the cell surface and enzyme activity of NEP are thus both dependent on sugar residues, probably through different conformational constraints. These constraints seem to be local for enzyme activity but more global for transport to the cell surface.

Regulation of Rho protein binding to membranes by rhoGDI: inhibition of releasing activity by physiological ionic conditions

1999 ◽  
Vol 77 (1) ◽  
pp. 59-69 ◽  
Author(s):  
Diane Bilodeau ◽  
Sylvie Lamy ◽  
Richard R Desrosiers ◽  
Denis Gingras ◽  
Richard Béliveau
Keyword(s):  
Ionic Strength ◽  
Brush Border ◽  
Rat Kidney ◽  
Regulatory Protein ◽  
Rho Proteins ◽  
Glutathione S Transferase ◽  
Free System ◽  
Brush Border Membranes ◽  
A Cell ◽  
Membrane Bound

The Rho GDP dissociation inhibitor (GDI) is an ubiquitously expressed regulatory protein involved in the cycling of Rho proteins between membrane-bound and soluble forms. Here, we characterized the Rho solubilization activity of a glutathione S-transferase (GST) - GDI fusion protein in a cell-free system derived from rat kidney. Addition of GST-GDI to kidney brush border membranes resulted in the specific release of Cdc42 and RhoA from the membranes, while RhoB and Ras were not extracted. The release of Cdc42 and RhoA by GST-GDI was dose dependent and saturable with about 50% of both RhoA and Cdc42 extracted. The unextracted Rho proteins were tightly bound to membranes and could not be solubilized by repeated GST-GDI treatment. These results demonstrated that kidney brush border membranes contained two populations of RhoA and Cdc42. Furthermore, the GST-GDI solubilizing activity on membrane-bound Cdc42 and RhoA was abolished at physiological conditions of salt and temperature in all tissues examined. When using bead-immobilized GST-GDI, KCl did not reduced the binding of Rho proteins. However, washing brush border membranes with KCl prior treatment by GST-GDI inhibited the extraction of Rho proteins. Taken together, these results suggest that the binding of GDI to membrane-bound Cdc42 and RhoA occurs easily under physiological ionic strength conditions, but a complementary factor is required to extract these proteins from membranes. These observations suggest that the shuttling activity of GDI upon Rho proteins could be normally downregulated under physiological conditions.Key words: rhoGDI, rho proteins, ionic strength, kidney.

scholarly journals Polarized distribution of neutral endopeptidase 24.11 at the cell surface of cultured human intestinal epithelial Caco-2 cells

1992 ◽  
Vol 288 (3) ◽  
pp. 945-951 ◽  
Author(s):  
F Jalal ◽  
C Jumarie ◽  
W Bawab ◽  
D Corbeil ◽  
C Malo ◽  
...  
Keyword(s):  
Cell Surface ◽  
Brush Border ◽  
Flow Cytometric Analysis ◽  
Neutral Endopeptidase ◽  
Rabbit Kidney ◽  
Human Colon Cancer ◽  
Cell Extracts ◽  
Endopeptidase 24.11 ◽  
Dpp Iv ◽  
Apical Side

The human colon cancer cell line Caco-2 undergoes spontaneous enterocytic differentiation during growth, and expresses a number of brush-border-membrane-associated hydrolases typical of a differentiated phenotype. Among these are alkaline phosphatase, dipeptidyl peptidase IV and sucrase-isomaltase (sucrase, EC 3.2.1.48). Neutral endopeptidase 24.11 [EC 3.4.24.11, neprilysin (NEP)] is another abundant protease of normal enterocytes but its presence in Caco-2 cells has not been fully documented yet. In this paper, we show that Caco-2 cell extracts hydrolyse tritiated [D-Ala2Leu5]enkephalin with a Km of 180 microM, very close to the value obtained for the NEP present in the rabbit kidney (118 microM). Western-blot analysis of brush-border membranes purified from post-confluent cells revealed a protein with an apparent molecular mass of 94000 Da similar to that of the rabbit kidney NEP. The amount of enzyme in cell extracts increased as a function of the age of the culture, indicating that NEP expression is correlated with the degree of cell differentiation as is also the case for sucrase and dipeptidylpeptidase IV (DPP-IV). Binding of a radiolabelled antibody to Caco-2 cell monolayers grown on semi-permeable filters indicated that 95% of NEP molecules present at the cell surface are on the apical side. Immunocytochemical and flow cytometric analysis of intact and permeabilized cells were also used to investigate the presence of NEP and DPP-IV at the surface of Caco-2 cells. Whereas DPP-IV staining appeared to be homogeneous throughout the entire cell population, NEP-related fluorescence exhibited a bimodal distribution which indicates an uneven expression of the protein at the cell surface. Permeabilization of monolayers with saponin before staining restored a labelling pattern for NEP similar to the one obtained for DPP-IV. This suggests that although DPP-IV and NEP follow similar patterns of expression when enzymic activities are measured on whole-cell extracts, targeting of these brush-border proteins to the cell surface appears to be regulated in different ways.

scholarly journals Lactotransferrin receptor of mouse small-intestinal brush border. Binding characteristics of membrane-bound and triton X-100-solubilized forms

1988 ◽  
Vol 249 (2) ◽  
pp. 435-441 ◽  
Author(s):  
W L Hu ◽  
J Mazurier ◽  
G Sawatzki ◽  
J Montreuil ◽  
G Spik
Keyword(s):  
Brush Border ◽  
Binding Activity ◽  
Binding Characteristics ◽  
Intestinal Brush Border Membrane ◽  
Intestinal Brush Border ◽  
Number Of Binding Sites ◽  
Mouse Milk ◽  
Membrane Bound ◽  
Small Intestinal ◽  
Triton X

A specific lactotransferrin receptor was identified in the mouse small-intestinal brush-border membrane and the binding features were investigated in homologous and heterologous systems. The receptor was found to be specific for lactotransferrins isolated from milk of various species, but the affinity was higher toward the homologous ligand (Ka = 3.5 x 10(6) M-1 compared with 2.6 x 10(6) M-1 for both human and bovine lactotransferrins). However, the number of binding sites (n) was the same for the three lactotransferrins, namely 0.53 x 10(12)/micrograms of membrane protein. The binding of mouse lactotransferrin to its receptor was found to be pH-dependent, with an optimal binding at pH 5.5, and seemed unlikely to be carbohydrate-mediated. The receptor was demonstrated to be devoid of any affinity for human and mouse serotransferrins or for a ‘serotransferrin-like’ protein isolated from mouse milk. The receptor was solubilized with 1% Triton X-100 with good yield. The solubilized receptor was found to retain lactotransferrin-binding activity and sensitivity to pH.

scholarly journals A neutral endopeptidase in the microvillar membrane of pig intestine. Partial purification and properties

1980 ◽  
Vol 191 (2) ◽  
pp. 645-648 ◽  
Author(s):  
E M Danielsen ◽  
J P Vyas ◽  
A J Kenny
Keyword(s):  
Brush Border ◽  
Chelating Agents ◽  
Neutral Endopeptidase ◽  
Partial Purification ◽  
Immunological Properties ◽  
Neutral Proteinase ◽  
Purification And Properties ◽  
Intestinal Enzyme ◽  
Triton X

An enzyme hydrolysing [125I]iodo-insulin B chain was enriched in preparations of intestinal microvilli. The activity could be solubilized by Triton X-100 and was partially (76-fold) purified. It was very sensitive to inhibition by phosphoramidon and was also inhibited by chelating agents. In its enzymic, molecular and immunological properties the intestinal enzyme closely resembled kidney microvillar neutral endopeptidase (kidney-brush-border neutral proteinase, EC 3.4.24.11).

Hypoxia decreases lung neprilysin expression and increases pulmonary vascular leak

2001 ◽  
Vol 281 (4) ◽  
pp. L941-L948 ◽  
Author(s):  
Todd C. Carpenter ◽  
Kurt R. Stenmark
Keyword(s):  
Enzyme Activity ◽  
Substance P ◽  
Cell Surface ◽  
Neutral Endopeptidase ◽  
Vascular Leak ◽  
Pulmonary Vascular Permeability ◽  
Hypoxia Exposure ◽  
Vasoactive Peptides ◽  
Weanling Rats ◽  
A Cell

Although prior studies suggest that hypoxia may increase pulmonary vascular permeability, the mechanisms responsible for that effect remain uncertain. Neprilysin (neutral endopeptidase) is a cell surface metallopeptidase that degrades several vasoactive peptides including substance P and bradykinin. We hypothesized that hypoxia could reduce lung neprilysin expression, leading to increased vascular leak. Weanling rats were exposed to normobaric hypoxia (inspired O2 fraction = 0.1). Lung neprilysin activity was significantly decreased after 24 and 48 h of hypoxia ( P < 0.006). The decrease in enzyme activity was associated with decreased lung neprilysin protein content and decreased lung neprilysin mRNA expression. Immunohistochemistry showed a predominantly perivascular distribution of neprilysin, with clear reductions in neprilysin immunoreactivity after exposure to hypoxia. Exposure to hypoxia for 24 h also caused marked increases in vascular leak ( P = 0.008), which were reversed by the administration of recombinant neprilysin. The hypoxia-induced increase in leak was also reversed by substance P and bradykinin receptor antagonists. We conclude that in young rats hypoxia decreases lung neprilysin expression, which contributes to increased pulmonary vascular leak via substance P and bradykinin receptors.

Electrophoretic variants of alanine aminopeptidase, alkaline phosphatase, and gamma-glutamyltransferase in urine.

1984 ◽  
Vol 30 (6) ◽  
pp. 856-859 ◽  
Author(s):  
K Jung ◽  
U W Wischke
Keyword(s):  
Alkaline Phosphatase ◽  
Electrophoretic Mobility ◽  
Brush Border ◽  
Neuraminidase Treatment ◽  
Electrophoretic Variants ◽  
Brush Border Enzymes ◽  
Gamma Glutamyltransferase ◽  
Electrophoretic Mobilities ◽  
Alanine Aminopeptidase ◽  
Triton X

Abstract The three brush-border enzymes--alanine aminopeptidase (EC 3.4.11.2), alkaline phosphatase (EC 3.1.3.1), and gamma-glutamyltransferase (EC 2.3.2.2)--are present in the urine of healthy persons in two variants, a particulate form and a soluble one. They can be separated by electrophoresis in agarose gel and by ultracentrifugation. The particulate forms exhibit similar electrophoretic mobility, but the soluble forms of these brush-border enzymes differ in their electrophoretic mobilities. The enzyme components of the particulate activity can be mobilized by Triton X-100 and trypsin. The electrophoretic mobility of the soluble forms of alanine aminopeptidase and gamma-glutamyltransferase is slowed by neuraminidase treatment. Both forms of gamma-glutamyltransferase are influenced in their electrophoretic mobility by treatment with n-butanol/diisopropyl ether, showing their lipid dependence. These findings enhance our knowledge of the biochemical nature of brush-border enzymes in urine.

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