scholarly journals Iodinated fibroblast β-glucuronidase as a ligand for receptor-mediated endocytosis

1985 ◽  
Vol 229 (1) ◽  
pp. 213-219 ◽  
Author(s):  
M F Dean ◽  
S Diment ◽  
C Ostlünd ◽  
B M Jenne ◽  
S Contractor
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Peritoneal Macrophages ◽  
Affinity Column ◽  
3T3 Cells ◽  
Receptor Mediated Endocytosis ◽  
3T3 Fibroblasts ◽  
Different Types ◽  
Mannose Receptors ◽  
Mouse Peritoneal Macrophages

Antibodies raised to human placental beta-glucuronidase were shown to cross-react with the beta-glucuronidase secreted by mouse 3T3 fibroblasts, but did not react with other lysosomal enzymes. The beta-glucuronidase secreted by 3T3 cells was purified 15000-fold by chromatography on an affinity column made from this antibody and resolved into a single component, of Mr 68000, by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Iodinated samples of purified enzyme were taken up into mouse peritoneal macrophages by receptor-mediated endocytosis at a rate similar to that calculated previously for unlabelled enzyme, and uptake was competitively inhibited by yeast mannan. Binding of beta-glucuronidase to macrophages was saturable, with a Kd of 7 × 10(-9)l/mol, an affinity comparable with that calculated for the binding of mannosylated bovine serum albumin (Kd 1.3 × 10(-9)l/mol), a ligand specific for mannose receptors. Four times as many molecules of mannosylated albumin (12000) as of beta-glucuronidase (3000), however, bound to each cell. This purification and iodination procedure did not therefore have any adverse effect on the uptake properties of secreted beta-glucuronidase, and provides a ligand with which to investigate binding and specific endocytosis into a range of different types of cell.

scholarly journals Mouse macrophage elastase. Purification and characterization as a metalloproteinase

1981 ◽  
Vol 193 (2) ◽  
pp. 589-605 ◽  
Author(s):  
M J Banda ◽  
Z Werb
Keyword(s):  
Culture Medium ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Serine Proteinase ◽  
Peritoneal Macrophages ◽  
Serine Proteinases ◽  
Endogenous Inhibitor ◽  
Purification And Characterization ◽  
Predominant Form ◽  
Mouse Peritoneal Macrophages

Macrophage elastase was purified from tissue-culture medium conditioned by inflammatory mouse peritoneal macrophages. Characterized as a secreted neutral metalloproteinase, this enzyme was shown to be catalytically and immunochemically distinct from the mouse pancreatic and mouse granulocyte elastases, both of which are serine proteinases. Inhibition profiles, production of nascent N-terminal leucine residues and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of degraded elastin indicated that macrophage elastase is an endopeptidase, with properties of a metalloproteinase, rather than a serine proteinase. Macrophage elastase was inhibited by alpha 2-macroglobulin, but not by alpha 1-proteinase inhibitor. Macrophage elastase was resolved into three chromatographically distinct forms. The predominant form had mol.wt. 22 000 and was purified 4100-fold. Purification of biosynthetically radiolabelled elastase indicated that this form represented less than 0.5% of the secreted protein of macrophages. Approx. 800% of the starting activity was recovered after purification. Evidence was obtained for an excess of an endogenous inhibitor masking more than 80% of the secreted activity.

scholarly journals Modulation of apoprotein E secretion in response to receptor-mediated endocytosis in resident and inflammatory macrophages.

1984 ◽  
Vol 159 (1) ◽  
pp. 167-178 ◽  
Author(s):  
R Takemura ◽  
Z Werb
Keyword(s):  
Dextran Sulfate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Cholesterol Content ◽  
Peritoneal Macrophages ◽  
Dependent Manner ◽  
Activated Macrophages ◽  
Receptor Mediated Endocytosis ◽  
Inflammatory Macrophages ◽  
Dose Dependent ◽  
Mouse Peritoneal Macrophages

We have determined the effect of various endocytic ligands on the secretion of ApoE by macrophages. ApoE was a major secreted protein of resident macrophages, but BCG-activated macrophages secreted little ApoE and periodate-elicited macrophages secreted intermediate amounts of ApoE. Resident, periodate-elicited, and BCG-activated mouse peritoneal macrophages were incubated with AcLDL, EIgG, EIgMC, dextran sulfate, latex, or zymosan, and the resulting protein secretion patterns were analyzed by [35S]methionine labeling and SDS-polyacrylamide gel electrophoresis. AcLDL increased total [35S]methionine incorporation into secreted proteins. Although AcLDL increased the secretion of ApoE by resident macrophages less than or equal to fivefold in a dose-dependent manner, with maximal stimulation at 4.8 micrograms/ml, it decreased the secretion of ApoE by periodate-elicited macrophages to almost nothing and did not affect the low rate of secretion of ApoE by BCG-activated macrophages. However, EIgG, which increases cellular cholesterol content of macrophages as AcLDL does, did not increase ApoE secretion, and dextran sulfate, which is recognized by the same receptor as AcLDL, also did not increase ApoE secretion. The binding and uptake of EIgG, dextran sulfate, zymosan, latex, and EIgMC all decreased the secretion of ApoE. These endocytic ligands also altered the pattern of secreted and cellular proteins other than ApoE. The pattern of response was ligand-specific. However, increased secretion of polypeptides of Mr 62,000 and 68,000 was common to many stimuli. We conclude that receptor-mediated endocytosis modulates the secretion of ApoE and other proteins pleiotypically in resident, inflammatory, and activated macrophages.

Purification and characterization of a novel factor which stimulates rat ribosomal gene transcription in vitro by interacting with enhancer and core promoter elements

1990 ◽  
Vol 10 (10) ◽  
pp. 5177-5186
Author(s):  
J Zhang ◽  
S T Jacob
Keyword(s):  
Ribosomal Dna ◽  
Core Promoter ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Affinity Column ◽  
Mobility Shift ◽  
Enhancer Element ◽  
Molecular Weights ◽  
The Core ◽  
Pol I

Previous studies in our laboratory have characterized a 174-base-pair (bp) enhancer sequence in the rat ribosomal DNA spacer region that exhibits all of the characteristics of a polymerase (Pol) II enhancer. Further studies showed that at least half of the enhancer activity resides in a 37-bp motif (E1) within the 174-bp spacer sequence that is located between positions -2.183 and -2.219 kilobase pairs upstream of the initiation site. To identify the factor(s) that binds specifically to the 37-bp enhancer domain, we fractionated whole-cell extract from rat adenocarcinoma ascites cells by chromatography on a series of columns, including an oligodeoxynucleotide affinity column. The final preparation contained two polypeptides of molecular weights 79,400 and 89,100 and was completely devoid of RNA Pol I activity. Electrophoretic mobility shift analysis showed that the polypeptides in the purified preparation (designated E1BF) interacted with both the enhancer element and the core promoter. To determine whether each polypeptide can separately bind to the core promoter and the enhancer, the individual components were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, renatured, and subjected to gel retardation analysis. This experiment demonstrated that both polypeptides interacted with the two cis-acting sequences. The specificity of the binding was demonstrated by competition with unlabeled 37-bp and core promoter fragments and lack of competition with nonspecific DNAs in the mobility shift assay. The 37-bp enhancer as well as the downstream sequence of the core promoter were protected by E1BF in the DNase I footprinting assay. Addition of E1BF to limiting amounts of fraction DE-B, which contains all factors essential for Pol I-directed transcription, resulted in three- to fourfold stimulation of ribosomal DNA transcription. Comparison of molecular weights and footprinting profiles did not reveal any relationship between E1BF and other Pol I trans-acting factors.

Identification and characterization of cDNA clones encoding two homologous proteins that are part of the asialoglycoprotein receptor

1987 ◽  
Vol 7 (5) ◽  
pp. 1841-1847
Author(s):  
M McPhaul ◽  
P Berg
Keyword(s):  
Rat Liver ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Asialoglycoprotein Receptor ◽  
Cdna Clones ◽  
Differential Splicing ◽  
Homologous Proteins ◽  
Different Types ◽  
Identification And Characterization

The asialoglycoprotein receptor (ASGP-R) from rat liver contains the following three distinct protein species when it is analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis: RHL1 (42 kilodaltons), RHL2 (49 kilodaltons), and RHL3 (54 kilodaltons). In this paper we describe the isolation of cDNA clones encoding RHL1 and RHL2 from a cDNA library constructed from rat liver mRNA. A comparison of the predicted coding sequence for RHL2 with that for RHL1 showed that these sequences are highly homologous. The library also contained numerous cDNA clones for both RHL1 and RHL2 that were derived from unspliced precursor mRNAs. Differential splicing at the 5' end of the RHL1 transcript was inferred from the finding that two different types of RHL1 cDNA were identified, each having a different 5' terminus.

scholarly journals Immunochemical evidence for an additional H-2 region closely linked to H-2D.

1977 ◽  
Vol 145 (2) ◽  
pp. 438-442 ◽  
Author(s):  
T H Hansen ◽  
S E Cullen ◽  
D H Sachs
Keyword(s):  
Sodium Dodecyl Sulfate ◽  
Spleen Cell ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Molecular Size ◽  
Migration Patterns ◽  
Different Types ◽  
D Region ◽  
Chemical Evidence

Anti-H-2 reagents have been tested on solubilized spleen cell preparations in combinations expected to be specific for D region products. Two different types of molecules were detected. One showed the expected reactivity with both antisera to private and antisera to public specificities. However, an additional molecule was detected which reacted only with antisera to public specificities. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis migration patterns indicated that both products have a similar molecular size of approximately 45,000 daltons. The data therefore present chemical evidence for the existence of a third H-2-associated gene product of 45,000 mol wt in addition to the classical H-2K and H-2D antigens.

scholarly journals Purification and characterization of a novel factor which stimulates rat ribosomal gene transcription in vitro by interacting with enhancer and core promoter elements.

1990 ◽  
Vol 10 (10) ◽  
pp. 5177-5186 ◽  
Author(s):  
J Zhang ◽  
S T Jacob
Keyword(s):  
Ribosomal Dna ◽  
Core Promoter ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Affinity Column ◽  
Mobility Shift ◽  
Enhancer Element ◽  
Molecular Weights ◽  
The Core ◽  
Pol I

Previous studies in our laboratory have characterized a 174-base-pair (bp) enhancer sequence in the rat ribosomal DNA spacer region that exhibits all of the characteristics of a polymerase (Pol) II enhancer. Further studies showed that at least half of the enhancer activity resides in a 37-bp motif (E1) within the 174-bp spacer sequence that is located between positions -2.183 and -2.219 kilobase pairs upstream of the initiation site. To identify the factor(s) that binds specifically to the 37-bp enhancer domain, we fractionated whole-cell extract from rat adenocarcinoma ascites cells by chromatography on a series of columns, including an oligodeoxynucleotide affinity column. The final preparation contained two polypeptides of molecular weights 79,400 and 89,100 and was completely devoid of RNA Pol I activity. Electrophoretic mobility shift analysis showed that the polypeptides in the purified preparation (designated E1BF) interacted with both the enhancer element and the core promoter. To determine whether each polypeptide can separately bind to the core promoter and the enhancer, the individual components were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, renatured, and subjected to gel retardation analysis. This experiment demonstrated that both polypeptides interacted with the two cis-acting sequences. The specificity of the binding was demonstrated by competition with unlabeled 37-bp and core promoter fragments and lack of competition with nonspecific DNAs in the mobility shift assay. The 37-bp enhancer as well as the downstream sequence of the core promoter were protected by E1BF in the DNase I footprinting assay. Addition of E1BF to limiting amounts of fraction DE-B, which contains all factors essential for Pol I-directed transcription, resulted in three- to fourfold stimulation of ribosomal DNA transcription. Comparison of molecular weights and footprinting profiles did not reveal any relationship between E1BF and other Pol I trans-acting factors.

Purification and properties of human placental NADPH–cytochrome P-450 reductase

1986 ◽  
Vol 64 (3) ◽  
pp. 184-193 ◽  
Author(s):  
Norio Muto ◽  
Liat Tan
Keyword(s):  
Salt Concentration ◽  
High Performance ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Visible Region ◽  
Relative Mass ◽  
Affinity Column ◽  
Nadph Cytochrome ◽  
Cytochrome P 450

Human placental NADPH–cytochrome P-450 reductase (EC 1.6.2.4) was purified to electrophoretic homogeneity in two chromatographic steps with a high retention of bioactivity. After solubilization with 1% sodium cholate in a protective medium containing 20% glycerol, 10 μM 4-androstene-3, 17-dione, 1 mM dithiothreitol, and 0.2 mM EDTA, a 35–60% ammonium sulfate precipitate was prepared. The crude protein mixture was then applied to a 2′, 5′-ADP–Sepharose 4B affinity column, followed by high-performance anion-exchange chromatography (Pharmacia Mono-Q column). Two forms of the reductase were isolated. One was eluted at higher salt concentration and had a relative mass (Mr) of 79 kdaltons (kDa) as estimated by sodium dodecyl sulfate – polyacrylamide gel electrophoresis and high-performance gel permeation chromatography. A smaller size reductase with a Mr of 70 kDa, eluting at lower salt concentration, was also formed by trypsinolyis of the 79-kDa reductase. It must therefore be regarded as a proteolytic artifact. The absolute spectra in the visible region of the two reductases were identical with maxima at 376 and 452 nm, typical of a flavoprotein. They also had the same specific activity of 24.7 ± 0.7 μmol/min per milligram protein towards cytochrome c. However, only the 79-kDa reductase showed aromatase-reconstitution activity. The homogeneity of these reductases was further confirmed by the appearance of a single peak when subjected to gradient, reversed-phase high-performance liquid chromatography. According to its amino acid composition, the 79-kDa reductase is a highly acidic and hydrophobic protein, composed of 695 residues.

scholarly journals Characterization of the cholera toxin receptor on Balb/c 3T3 cells as a ganglioside similar to, or identical with, ganglioside GM1. No evidence for galactoproteins with receptor activity

1982 ◽  
Vol 204 (1) ◽  
pp. 209-219 ◽  
Author(s):  
D R Critchley ◽  
C H Streuli ◽  
S Kellie ◽  
S Ansell ◽  
B Patel
Keyword(s):  
Cholera Toxin ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Receptor Activity ◽  
Galactose Oxidase ◽  
3T3 Cells ◽  
Ganglioside Gm1 ◽  
Intact Cells ◽  
Terminal Galactose ◽  
Toxin Receptor

Balb/c 3T3 cells contain a large number [(0.8-1.6) x 10(6)] of high-affinity (half-maximal binding at 0.2 nM) binding sites for cholera toxin that are resistant to proteolysis, but are quantitatively extracted with chloroform/methanol. The following evidence rigorously establishes that the receptor is a ganglioside similar to, or identical with, ganglioside GM1 by the galactose oxidase/NaB3H4 technique on intact cells was inhibited by cholera toxin. (2) Ganglioside GM1 was specifically adsorbed from Nonidet P40 extracts of both surface- (galactose oxidase/NaB3H4 technique) and metabolically ([1-14C]palmitate) labelled cells in the presence of cholera toxin, anti-toxin and Staphylococcus aureus. (3) Ganglioside GM1 was the only ganglioside labelled when total cellular gangliosides separated on silica-gel sheets were overlayed with 125I-labelled cholera toxin, although GM3 and GD1a were the major gangliosides present. In contrast no evidence for a galactoprotein with receptor activity was obtained. Cholera toxin did not protect the terminal galactose residues of cell-surface glycoproteins from labelling by the galactose oxidase/NaB3H4 technique. No toxin-binding proteins could be identified in Nonidet P40 extracts of [35S]-methionine-labelled cells by immunochemical means. After sodium dodecyl sulphate/polyacrylamide-gel electrophoresis none of the major cellular galactoproteins identified by overlaying gels with 125I-labelled ricin were able to bind 125I-labelled cholera toxin. It is concluded that the cholera toxin receptor on Balb/c 3T3 cells is exclusively ganglioside GM1 (or a related species), and that cholera toxin can therefore be used to probe the function and organisation of gangliosides in these cells as previously outlined [Critchley, Ansell, Perkins, Dilks & Ingram (1979) J. Supramol. Struct. 12, 273-291].

scholarly journals Identification and characterization of cDNA clones encoding two homologous proteins that are part of the asialoglycoprotein receptor.

1987 ◽  
Vol 7 (5) ◽  
pp. 1841-1847 ◽  
Author(s):  
M McPhaul ◽  
P Berg
Keyword(s):  
Rat Liver ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Asialoglycoprotein Receptor ◽  
Cdna Clones ◽  
Differential Splicing ◽  
Homologous Proteins ◽  
Different Types ◽  
Identification And Characterization

The asialoglycoprotein receptor (ASGP-R) from rat liver contains the following three distinct protein species when it is analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis: RHL1 (42 kilodaltons), RHL2 (49 kilodaltons), and RHL3 (54 kilodaltons). In this paper we describe the isolation of cDNA clones encoding RHL1 and RHL2 from a cDNA library constructed from rat liver mRNA. A comparison of the predicted coding sequence for RHL2 with that for RHL1 showed that these sequences are highly homologous. The library also contained numerous cDNA clones for both RHL1 and RHL2 that were derived from unspliced precursor mRNAs. Differential splicing at the 5' end of the RHL1 transcript was inferred from the finding that two different types of RHL1 cDNA were identified, each having a different 5' terminus.

scholarly journals Identification of macrophage external membrane proteins and their possible role in cell adhesion.

1978 ◽  
Vol 79 (1) ◽  
pp. 263-267 ◽  
Author(s):  
E Pearlstein ◽  
S R Dienstman ◽  
V Defendi
Keyword(s):  
Cell Adhesion ◽  
Sodium Dodecyl ◽  
Surface Protein ◽  
Peritoneal Macrophages ◽  
Immunofluorescent Staining ◽  
Adhesive Properties ◽  
Adhesive Matrix ◽  
Mouse Peritoneal Macrophages ◽  
Adhesion Factor ◽  
Morphological Phenotype

Starch-activated mouse peritoneal macrophages (STpMAC) plated on plastic demonstrate the adhesive properties typical for activated pMAC: attaching as round cells and, within 15 min, spreading out with marginal membrane ruffles. These attached STpMAC were labeled by lactoperoxidase-catalysed 125I surface iodination, sodium dodecyl-sulfate-lysed, and the lysates electrophoresed on polyacrylamide gels which were examined by autoradiography. The STpMAC morphological phenotype correlates with the labeling of a particular protein (195,000, estimated mol wt). Normal pMAC (NpMAC), from unstimulated mice, do not spread and do not display the 195,000 band. Both pMAC band patterns, including the 195,000 band, are relatively resistant to trypsin digestion, as is pMAC adhesion itself trypsin-resistant. Neither class of pMAC exhibits fibronectin (Cell Adhesion Factor, LETS protein) which is a component in the adhesive matrix of cells forming trypsin-sensitive monolayers. When pMAC are tested against antifibronectin antibody, these cells do not give immunofluorescent staining. In summary, two functions in pMAC adhesion, enzyme resistance and the ability to spread, appear related to molecular properties distinctive for pMAC surface protein.

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