Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0027819 (neuroblastoma)
 27,800 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A trypsin-like enzyme has been purified to apparent homogeneity from neuroblastoma cell membranes by a procedure including extraction with Triton X-100, soybean trypsin inhibitor-immobilized Sepharose 4B affinity chromatography, and gel filtration. SDS-polyacrylamide gel electrophoresis under reducing conditions of the purified enzyme gave a single band corresponding to a molecular weight of 28,000. The molecular weight of the enzyme was also estimated to be 32,000 by gel filtration. The pH optimum of the activity was 8.5-9.0. The purified enzyme was inhibited by diisopropylphosphorofluoridate, p-aminobenzamidine, and leupeptin, and moderately by chymostatin, but not, or only scarcely, by bestatin, phosphoramidon, p-chloromercuribenzoate, and N-ethylmaleimide. The substrate subsite specificity of the purified enzyme was broad toward various peptidyl-arginine (or lysine) 4-methylcoumaryl-7-amides, but it cleaved dynorphin(1-17) only at two sites, i.e., between the Arg6-Arg7 and Lys11-Leu12 bonds, both of which correspond to the initial cleavage sites of dynorphin with a membrane preparation of neuroblastoma cells. A trypsin-like enzyme was also purified from a synaptic membrane preparation of rat brain, which shows almost the same properties as those of the enzyme from the neuroblastoma cell membrane. Thus, the trypsin-like enzyme present in the synaptic membrane would participate in the degradation of dynorphin.
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PMID:Membrane-bound trypsin-like enzyme functioning in degradation of dynorphin in neuroblastoma cells. Purification and characterization. 289 72

Both high and low affinity receptors for nerve growth factor (NGF) have been described, but only the former appear to mediate NGF actions and uptake. To specifically characterize the molecular identity of the high affinity site and to compare it with the low affinity site, the water-soluble carbodiimide EDC was used to cross-link 125I-NGF to NGF receptors on: rat PC12 cells, PC12nnr5 cells (PC12 mutants that have only low affinity NGF binding), SH-SY5Y human neuroblastoma cells (which have only high affinity binding sites), and cultured rat sympathetic ganglion cells. A variety of criteria were used to distinguish the two classes of affinity-labeled receptors: competition with unlabeled NGF, dissociation rate, and selective solubilization by 0.1% Triton X-100. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that cross-linking generated only a single Mr approximately 103,000 125I-NGF affinity-labeled species which represents both the low and high affinity forms of the receptor. The 125I-NGF X receptor complexes formed with both affinity classes of the receptor were quantitatively immunoprecipitated by the monoclonal anti-NGF-receptor antibody 192-IgG and both showed identical shifts in mobility when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. These findings indicate that both high and low affinity NGF receptors possess apparently identical NGF-binding moieties. The differences between the kinetic and functional properties of the two receptor types may therefore result from their interactions with other membrane components or with cytoplasmic proteins.
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PMID:A single Mr approximately 103,000 125I-beta-nerve growth factor-affinity-labeled species represents both the low and high affinity forms of the nerve growth factor receptor. 302 71

A membrane-bound enkephalin (ENK) -hydrolyzing amino-peptidase (AP) was partially purified from neuroblastoma (clone N1E-115) cell membranes; enzyme activity was assayed by determination of the leu-enkephalin (LENK) degradation product, tyrosine (Tyr), with HPLC. The enzyme was extracted with Triton X-100, resolved by anion-exchange chromatography and further purified by gel filtration. The overall purification was about 100-fold with a yield of 43%. The apparent Mr value of the AP by gel filtration in the presence of 0.3% Triton X-100 was approx. 400 kDa. In the absence of detergent the apparent Mr value was about 305 kDa. In the elution buffers, where Triton X-100 was omitted, the peptidase activity was lost. The enzyme had a Km of 0.13 mM and a Vmax of 450 nmoles per mg protein per min at 25 degrees C for LENK and exhibited little sensitivity to bestatin (IC50: 200 microM) and puromycin (IC50: 500 microM), but it was strongly inhibited by amastatin (IC50: 8 microM). The enzyme is an amphiphilic membrane protein; the native primary structure is preserved only in the 'detergent form'. It seems to be AP N (EC 3.4.11.2) with an optimum of pH 7.2 to 7.4. We assume that this AP plays the important role in inactivating ENKs on the neuronal level. The ENK-degrading AP, partially purified from primary rat brain astrocyte cell membranes, exhibited a smaller apparent Mr value (130 kDa) and a higher sensitivity to amastatin (IC50: 0.4 microM), bestatin (IC50: 90 nM) and puromycin (IC50: 5 microM) than did the N1E-115 enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization of the enkephalin-degrading aminopeptidase of neuroblastoma (N1E-115) cell membranes. 312 43

Three novel components of neuromuscular junctions have been identified by use of monoclonal antibodies (McAb) against glycoproteins obtained from a mouse neuroblastoma X human dorsal root ganglion cell hybrid line. Antigen distribution was assessed by fluorescent immunohistochemistry on frozen sections of human intercostal muscle counterstained with labeled alpha-bungarotoxin to identify neuromuscular junctions. Antigen SOS 6 stained exclusively in the neuromuscular junction, whereas antigens SOS 5 and SOS 13 were highly enriched in the junction but also stained extrasynaptic regions. These antigens can be distinguished from previously described components of the neuromuscular junction by their molecular weights, insensitivity to collagenase treatment, and solubility in 0.1% Triton X-100. Indirect evidence suggests that these species-specific antigens are located in the postsynaptic muscle membrane, but location in the junctional basal lamina or subsarcolemmal region cannot be excluded.
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PMID:Novel antigens at the neuromuscular junction. 351 Feb 28

Antibodies were raised against a cytoskeleton-associated, nonphosphorylated, 230,000-dalton bovine lens polypeptide (designated p230), and rendered monospecific by using a novel immunoaffinity technique. In immunofluorescence and electron microscopy of cultured fibroblasts, as well as of various other cells (endothelial, epithelial, lenticular, monocytes, neuroblastoma cells) and tissues (human kidney and liver), p230 was localized as a distinct subplasmalemmal layer in the peripheral cytoplasm of the cells. It constituted less than 0.3% of the total cellular protein in cultured fibroblasts and was not extractable with Triton X-100. In detergent-extracted cytoskeletal preparations of cultured fibroblasts, p230 remained as an elaborate peripheral network that showed a distribution distinctly different from that of the major cytoskeletal structures, stress fibers, cortical myosin, vinculin, and intermediate filaments (IF). The distribution was not dependent on the presence of intact stress fibers or microtubules, as shown by double-fluorescence microscopy of cells exposed to cytochalasin B or cultured in the presence of monensin and of cold-treated cells. Upon demecolcine-induced reorganization of intermediate filaments, however, the localization of p230 was rapidly altered to a dense plaque underneath the perinuclear aggregate of intermediate filaments. On the other hand, p230 seemed to colocalize with the detergent-resistant cell surface lamina, visualized in fluorescence microscopy with fluorochrome-coupled wheat germ agglutinin-lectin. The results suggest that p230 is part of a cell surface- and cytoskeleton-associated subplasmalemmal structure that may play an important role in cell surface-cytoskeleton interaction in various cells both in vitro and in vivo.
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PMID:Immunolocalization of a novel, cytoskeleton-associated polypeptide of Mr 230,000 daltons (p230). 633 21

A cellular receptor for platelet-derived growth factor (PDGF) was demonstrated by incubation of 125I-labeled PDGF with human foreskin fibroblast cultures followed by liberation of cell-bound radioactivity with Triton X-100. The cellular binding of labeled PDGF in the presence of increasing amounts of unlabeled PDGF showed saturation; Scatchard analysis of binding data indicated a single class of receptors having kd = 1 X 10(-9) M. The number of PDGF binding sites was approximately 3 X 10(5)/cell. Labeled PDGF binding reached an apparent equilibrium after 3 hr at 4 degrees C. At 37 degrees C, it passed a maximum after 30 min and then decreased with time due to degradation of the tracer. A large excess of unlabeled PDGF reduced labeled PDGF binding by more than 90% whereas similar doses of epidermal growth factor, fibroblast growth factor, or insulin had no effect. It was concluded that PDGF did not share receptors with these factors. PDGF receptors were found on skin fibroblasts, normal and malignant glial cells, smooth muscle cells, and 3T3 cells but not on epithelial-derived cells, neuroblastoma cells, endothelial cells, or peripheral lymphocytes.l As only the receptor-positive cells--i.e., the connective tissue- and glia-derived cells--are responsive to stimulation with PDGF, these findings imply a functional significance of the PDGF receptor.
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PMID:Specific receptors for platelet-derived growth factor on cells derived from connective tissue and glia. 694 69

Choleragen, when bound to various cultured cells, resisted extraction by Triton X-100 under conditions which retained the cytoskeletal framework of the cells. The resistance (greater than 75% of the bound toxin) was observed in Friend erythroleukemic, mouse neuroblastoma N18 and NB41A and rat glioma C6 cells even though the different cells varied over 1000-fold in the number of toxin receptors. The extent of extraction did not depend on whether the cells were in monolayer culture of in suspension or whether choleragen was found at 0 or 37 degrees C. A similar resistance to extraction was also observed in membranes isolated from toxin-treated cells. Using more drastic conditions and other non-ionic detergents, 90% of the bound choleragen was solubilized from cells and membranes. When rat glioma C6 cells, which bind only small amounts of choleragen, were incubated with the ganglioside GM1, toxin binding was increased and the bound toxin was also resistant to extraction. When these cells were incubated with [3H]GM1, up to 70% of the cell-associated GM1 was extracted under the mild conditions. When the Gm1-labeled cells were incubated with choleragen or its B (binding) component, there was a significant reduction in the solubilization of GM1. Similar results were obtained with isolated membranes. When choleragen-receptor complexes were isolated from N18 cells labeled with [3H] galactose by immunoadsorption, only labeled GM1 was specifically recovered. These results suggest that it is the choleragen-ganglioside complex that is resistant to detergent extraction.
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PMID:Detergent extraction of cholera toxin and gangliosides from cultured cells and isolated membranes. 708 84

Tritiated 2-deoxy-D-glucose (dGlc) was rapidly taken up into cultured mouse neuroblastoma C1300 cells (clone 41A3). Upon perfusion the preloaded cultures slowly released radioactivity as [3H] 2-deoxy-D-glucose-6-phosphate ([3H]dGlc-6-P) (rate const. = 0.017 min-1) from a pool corresponding to 74% (t1/2 = 41 min) of the total radioactivity incorporated. Destruction of the plasma membrane of the cells by means of Triton X-100 (1.0%) resulted in a rapid and total release of the radioactivity. CH3HgCl, HgCl2, (C2H5)3SnCl and K2Cr2O7 all caused an increase in the passive cell membrane permeability to [3H]dGlc-6-P. A membrane toxic concentration (MTC) was defined as the concentration of the tested metal compound giving rise to an increase in the relative efflux from 1.0 to 1.2 during 60 min perfusion. Using this MTC-value, the membrane toxicity of the compounds could be ranked in the following order: CH3HgCl (MTC = 9 x 10(-7) M) greater than HgCl2 (MTC = 6 x 10(-6) M) greater than (C2H5)3SnCl (MTC = 3 x 10(-4) M) greater than K2Cr2O7 (MTC = 7 x 10(-4) M). Since this differential toxicity is in accordance with other reports it is concluded that 2-deoxy-D-glucose (dGlc) may be used together with 41A3 cells to screen metal compounds for their membrane toxicity.
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PMID:Membrane lesions in cultured mouse neuroblastoma cells exposed to metal compounds. 715 93

Studies were carried out on the polymorphism of acetylcholinesterase (AChE, EC 3.1.1.7) in a neuroblastoma x sympathetic ganglion cell hybrid cell line (T28) and its parental clone (N18TG2). These cells contain the tetrameric (G4, 10S), dimeric (G2, 6.5S) and monomeric (G1, 4S) forms of AchE, but not the collagen-tailed A12(16S) form of the sympathetic ganglion. Three variants of these forms could be distinguished on the basis of their solubility properties: (i) secreted forms which do not interact with the detergent Triton X-100; (ii) cellular forms which may be solubilized in detergent-free buffer and which interact reversibly with Triton X-100; (iii) cellular forms which require detergent for solubility, and aggregate in its absence. By using a nonpenetrating inhibitor, we demonstrated that, in T28 stationary cells, the cellular G4 form is associated with the plasma membrane, whereas the G1 form is intracellular. During induction of AChE activity in T28 cells, the relative proportion of the G4 form increases, suggesting, in agreement with previous observations, that G1 is a metabolic precursor of G4. The evolution of AChE molecular forms released into the culture medium closely resembles that of the cellular forms. The preferential accumulation of the G4 molecules does not simply depend on the cellular level of G1. It is favoured by culture conditions which promote morphological differentiation, but does not require the actual extension of neurites. T28 cells as well as other neuroblastoma-derived cells appear to be useful experimental materials to investigate the regulatory mechanisms underlying the maturation of AChE globular forms.
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PMID:Modulation of the distribution of acetylcholinesterase molecular forms in a murine neuroblastoma x sympathetic ganglion cell hybrid cell line. 745 5

It has been known for a number of years that glycosyl-phosphatidylinositol (GPI)-anchored proteins, in contrast to many transmembrane proteins, are insoluble at 4 degrees C in nonionic detergents such as Triton X-100. Recently, it has been proposed that this behavior reflects the incorporation of GPI-linked proteins into large aggregates that are rich in sphingolipids and cholesterol, as well as in cytoplasmic signaling molecules such as heterotrimeric G proteins and src-family tyrosine kinases. It has been suggested that these lipid-protein complexes are derived from caveolae, non-clathrin-coated invaginations of the plasmalemma that are abundant in endothelial cells, smooth muscle, and lung. Caveolin, a proposed coat protein of caveolae, has been hypothesized to be essential for formation of the complexes. To further investigate the relationship between the detergent-resistant complexes and caveolae, we have characterized the behavior of GPI-anchored proteins in lysates of N2a neuroblastoma cells, which lack morphologically identifiable caveolae, and which do not express caveolin (Shyng, S.-L., J. E. Heuser, and D. A. Harris. 1994. J. Cell Biol. 125:1239-1250). We report here that the complexes prepared from N2a cells display the large size and low buoyant density characteristic of complexes isolated from sources that are rich in caveolae, and contain the same major constituents, including multiple GPI-anchored proteins, alpha and beta subunits of heterotrimeric G proteins, and the tyrosine kinases fyn and yes. Our results argue strongly that detergent-resistant complexes are not equivalent to caveolae in all cell types, and that in neuronal cells caveolin is not essential for the integrity of these complexes.
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PMID:Glycolipid-anchored proteins in neuroblastoma cells form detergent-resistant complexes without caveolin. 753 73


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