scholarly journals Isolation and characterization of two distinct low-density, Triton-insoluble, complexes from porcine lung membranes

1996 ◽  
Vol 319 (3) ◽  
pp. 887-896 ◽  
Author(s):  
Edward T PARKIN ◽  
Anthony J TURNER ◽  
Nigel M HOOPER
Keyword(s):  
Light Scattering ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Annexin V ◽  
Density Fraction ◽  
Calcium Dependent ◽  
Membrane Complex ◽  
Isolation And Characterization ◽  
Insoluble Complex ◽  
Marker Proteins

The Triton-insoluble complex from porcine lung membranes has been separated into two distinct subfractions visible as discrete light-scattering bands following buoyant density-gradient centrifugation in sucrose. Both of these detergent-insoluble complexes were enriched in the glycosyl-phosphatidylinositol (GPI)-anchored ectoenzymes alkaline phosphatase, aminopeptidase P and 5´-nucleotidase, and both complexes excluded the polypeptide-anchored ectoenzymes angiotensin-converting enzyme, dipeptidyl peptidase IV and aminopeptidases A and N. The GPI-anchored proteins in both complexes were susceptible to release by phosphatidylinositol-specific phospholipase C. Both complexes were also enriched in cholesterol and glycosphingolipids, and in caveolin/VIP21, although only the higher-density fraction was enriched in the plasmalemmal caveolar marker proteins Ca2+-ATPase and the inositol 1,4,5-trisphosphate receptor. Among the annexin family of proteins, annexins I and IV were absent from the two detergent-insoluble complexes, annexin V was present in both, and annexins II and VI were only enriched in the higher-density fraction. When the metal chelator EGTA was present in the isolation buffers, annexins II and VI dissociated from the higher-density detergent-insoluble complex and only a single light-scattering band was observed on the sucrose gradient, at the same position as for the lower-density complex. In contrast, in the presence of excess calcium only a single detergent-insoluble complex was isolated from the sucrose gradients, at an intermediate density. Thus the detergent-insoluble membrane complex can be subfractionated on the basis of what appears to be calcium-dependent, annexin-mediated, vesicle aggregation into two distinct populations, only one of which is enriched in plasmalemmal caveolar marker proteins.

scholarly journals Isolation and characterization of high-buoyant-density proteoglycans from bovine femoral-head cartilage

1983 ◽  
Vol 213 (2) ◽  
pp. 355-362 ◽  
Author(s):  
M Lyon ◽  
J Greenwood ◽  
J K Sheehan ◽  
I A Nieduszynski
Keyword(s):  
Light Scattering ◽  
Femoral Head ◽  
Chondroitin Sulphate ◽  
Guanidine Hydrochloride ◽  
Buoyant Density ◽  
Radius Of Gyration ◽  
Gel Chromatography ◽  
Isolation And Characterization ◽  
Femoral Head Cartilage

Proteoglycans were extracted from bovine (15-18 months old) femoral-head cartilage. The heterogeneity of the A1D1 proteoglycan fraction was examined by gel chromatography, sedimentation velocity, sucrose rate-zonal centrifugation and CS2SO4 isopycnic centrifugation. In all cases polydisperse but unimodal distributions were obtained. Chemical analysis of the preparation yielded a galactosamine/glucosamine molar ratio of 7:1, and 13C n.m.r. spectroscopy showed that the chondroitin sulphate comprised equal proportions of the 4- and 6-sulphate isomers. Gel chromatography of a papain and Pronase digest of the proteoglycan indicated that the chondroitin sulphate chains had a Mn of approx. 10500. The mean buoyant density of the proteoglycan in pure CS2SO4 was 1.46 g/ml. Physical characterization of the proteoglycan preparation in 4M-guanidine hydrochloride, pH 7.4, by using conventional light-scattering gave a radius of gyration of 42 nm and a Mw of 0.96 × 10(6). Quasi-elastic light-scattering in the same solvent yielded a translational diffusion coefficient, D020, of 5.41 × 10(-8) cm2 × S-1, and ultracentrifugation gave a sedimentation coefficient, S020, of 12.0S. Thus from sedimentation-diffusion studies a Mw of 1.36 × 10(6) was calculated. The possible origins for the differences in the two molecular-weight estimates are discussed. It is concluded that the high-buoyant-density proteoglycans from bovine articular cartilage are significantly smaller than those from bovine nasal septum, and that this is largely due to the smaller size of their chondroitin sulphate chains.

scholarly journals Identification of the Raji cell membrane-derived C1q inhibitor as a receptor for human C1q. Purification and immunochemical characterization.

1984 ◽  
Vol 160 (5) ◽  
pp. 1375-1389 ◽  
Author(s):  
B Ghebrehiwet ◽  
L Silvestri ◽  
C McDevitt
Keyword(s):  
Disulfide Bonds ◽  
Gradient Centrifugation ◽  
Raji Cell ◽  
Buoyant Density ◽  
Equilibrium Density ◽  
Macromolecular Complex ◽  
Density Fraction ◽  
Dependent Cell ◽  
Low Ionic Strength ◽  
Sepharose 4B

We have shown previously that an activity which is capable of precipitating purified C1q and inhibiting some of the C1q-dependent biologic reactions could be solubilized from the membranes of both normal human peripheral B lymphocytes and a B cell-derived lymphoblastoid cell line (Raji), both of which are known to possess receptors for human C1q. In this report we present evidence that this membrane-associated C1q inhibitor is a chondroitinase-insensitive macromolecule and is the receptor for human C1q. The receptor was solubilized from membranes of Raji cells with Nonidet P-40 and purified to homogeneity using C1q-Sepharose 4B affinity chromatography. Equilibrium density gradient centrifugation analysis revealed that the complex could be resolved into a protein-rich, low density fraction and a carbohydrate-rich, high density fraction. The large hydrodynamic size, coupled with the high buoyant density, suggests that a proteoglycan is a constituent of the complex and indicates that the receptor might be a macromolecular complex of a proteoglycan portion noncovalently linked to a 60-70 kD glycoprotein. The glycoprotein moiety, in turn, consists of two or more identical (70,000 mol wt) polypeptide chains held together by disulfide bonds and constitutes the C1q receptor (C1qR). Sucrose density ultracentrifugation analysis showed that the isolated receptor sediments with an apparent rate of 4.2 S. Immunochemical analyses demonstrated that a typical preparation of the C1qR complex consists of approximately 23% uronic acid and approximately 21% galactosamine with a galactosamine-to-glucosamine ratio of 3.2. Binding of C1q to the receptor was found to be optimal at low ionic strength and neutral or near-neutral pH (7-7.4). The isolated receptor was found to inhibit C1q hemolytic function, abrogate C1q-dependent rosette formation, and block the C1q-dependent, cell-mediated cytotoxicity, all of which are activities mediated by the receptor.

Lipid dynamics in the annexin V - membrane complex

1999 ◽  
Vol 96 (9/10) ◽  
pp. 1602-1607
Author(s):  
O. Saurel ◽  
P. Demange ◽  
A. Lopez ◽  
A. Milon
Keyword(s):  
Annexin V ◽  
Membrane Complex ◽  
Lipid Dynamics

scholarly journals Molecular heterogeneity of mouse duodenal alkaline phosphatase. Association of lipids and peptides

1974 ◽  
Vol 141 (1) ◽  
pp. 93-101 ◽  
Author(s):  
P. R. V. Nayudu ◽  
Fraser B. Hercus
Keyword(s):  
Alkaline Phosphatase ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Buoyant Density ◽  
Sucrose Density Gradient ◽  
Molar Ratio ◽  
Molecular Forms ◽  
Partial Specific Volume ◽  
Molecular Heterogeneity ◽  
Sucrose Density Gradient Centrifugation

Polyacrylamide-gel electrophoresis and Bio-Gel P-300 molecular-sieve chromatography of mouse duodenal alkaline phosphatase demonstrates its molecular heterogeneity, which, in a kinetic sense, is manifest also in the differential relative velocities of the heterogeneous forms of the enzyme with two substrates, phenylphosphate and β-glycerophosphate. Different treatments that eliminate most of the electrophoretic and chromatographic variability of the enzyme also decrease the velocities with both substrates so that the molar ratio of hydrolysis of one substrate relative to the other is also altered to a low but stable value. Concomitant with these changes, lipids and peptides are dissociated from the enzyme. The lipids are tentatively identified as a sterol and phospholipids. The peptides have an average composition of four to six amino acids and appear to be strongly electropositive. The conditions of dissociation suggest that their binding to the enzyme is non-covalent and predominantly based on hydrophobic and ionic bonding. The concept of lipid and peptide association would suggest prima facie differential molecular weights as a factor in the observed electrophoretic and chromatographic heterogeneity. However, the molecular forms of the enzyme with differences in elution volume equivalent to more than one-half the void volume of the Bio-Gel P-300 column, or even enzyme fractions dissociated from the lipids and peptides compared with undissociated portions, do not show any differences in sedimentation on sucrose-density-gradient centrifugation. This may be because the alterations in molecular weight owing to binding of small molecules are too small to be detected by this method. Alternatively, since lipids are involved, the binding may alter the partial specific volume in such a way that the buoyant density is not significantly altered.

scholarly journals Impact of GADD34 on Apoptosis of Tonsillar Mononuclear Cells from IgA Nephropathy Patients by Regulating Eif2α Phosphorylation

2018 ◽  
Vol 50 (6) ◽  
pp. 2203-2215 ◽  
Author(s):  
Xiaofei Peng ◽  
Tao Yang ◽  
Liyu He ◽  
Xian Chen ◽  
Yafeng Jiang ◽  
...  
Keyword(s):  
Western Blotting ◽  
Cell Apoptosis ◽  
Mononuclear Cells ◽  
Gradient Centrifugation ◽  
Annexin V ◽  
Enzyme Linked Immunosorbent Assay ◽  
Eif2α Phosphorylation ◽  
Significant Difference ◽  
Mrna And Protein Expression ◽  
Sirna Interference

Background/Aims: Tonsillectomy may be an important method to achieve a long-term remission of IgAN, but patients’ physical status may limit their access to this surgery. We proposed an encouraging solution through inhibiting GADD34 expression in order to promote tonsillar mononuclear cells (TMCs) apoptosis and reduce nephropathic IgA secretion. Methods: A total of 12 IgAN and 9 non-IgAN patients were involved from March 2015 to May 2016. After TMCs were extracted by density gradient centrifugation and stimulated by inactivated hemolytic streptococcus, the mRNA and protein expression of GADD34, GRP78, CHOP, Bcl-2, Bcl-XL, AID, Iα-Cα, and cleaved caspase-3 were examined by fluorescent RT-PCR and Western blotting. Guanabenz treatment and siRNA interference were applied to downregulate GADD34 in tonsillar mononuclear cells from IgAN patients, and P-eIF2α expression was examined by Western Blotting. Cell apoptosis was evaluated by Annexin V FITC/PI flowcytometry, and IgA secretion in cultural supernatant was inspected by enzyme linked immunosorbent assay. Results: After stimulation, the expression of GADD34 was significantly increased in IgAN patients (P< 0.05). Cell apoptosis was mitigated and IgA secretion level was elevated (P< 0.05). To be noticed, CHOP expression had no significant difference between two groups. After guanabenz treatment and siRNA interference, a prolonged elevation of P-eIF2α expression was observed. Cell apoptosis was reinforced and IgA secretion level was decreased (P< 0.05). Conclusion: GADD34 may be a potential therapeutic target for IgAN treatment due to its effect on cell apoptosis.

scholarly journals Isolation and Characterization of Intracellular Protein Inclusions Produced by the Entomopathogenic BacteriumPhotorhabdus luminescens

2001 ◽  
Vol 67 (10) ◽  
pp. 4834-4841 ◽  
Author(s):  
David J. Bowen ◽  
Jerald C. Ensign
Keyword(s):  
Exponential Growth ◽  
Galleria Mellonella ◽  
Gradient Centrifugation ◽  
Sodium Dodecyl ◽  
Cellular Protein ◽  
Cross Reactivity ◽  
Crystalline Inclusion ◽  
Protein Subunit ◽  
Inclusion Type ◽  
Isolation And Characterization

ABSTRACT Cells of the entomopathogenic bacterium Photorhabdus luminescens contain two types of morphologically distinct crystalline inclusion proteins. The larger rectangular inclusion (type 1) and a smaller bipyramid-shaped inclusion (type 2) were purified from cell lysates by differential centrifugation and isopycnic density gradient centrifugation. Both structures are composed of protein and are readily soluble at pH 11 and 4 in 1% sodium dodecyl sulfate (SDS) and in 8 M urea. Electrophoretic analysis reveals that each inclusion is composed of a single protein subunit with a molecular mass of 11,000 Da. The proteins differ in amino acid composition, protease digestion pattern, and immunological cross-reactivity. The protein inclusions are first visible in the cells at the time of late exponential growth. Western blot analyses showed that the proteins appeared in cells during mid- to late exponential growth. When at maximum size in stationary-phase cells, the proteins constitute 40% of the total cellular protein. The protein inclusions are not used during long-term starvation of the cells and were not toxic when injected into or fed toGalleria mellonella larvae.

scholarly journals Analysis of the role of p200-containing vesicles in post-Golgi traffic.

1996 ◽  
Vol 7 (6) ◽  
pp. 961-974 ◽  
Author(s):  
E Ikonen ◽  
R G Parton ◽  
F Lafont ◽  
K Simons
Keyword(s):  
Gradient Centrifugation ◽  
Equilibrium Density ◽  
Apical Surface ◽  
Transport Pathways ◽  
Permeabilized Cells ◽  
Influenza Hemagglutinin ◽  
Streptolysin O ◽  
Marker Proteins ◽  
Viral Marker ◽  
Golgi Network

p200 is a cytoplasmic protein that associates with vesicles budding from the trans-golgi network (TGN). The protein was identified by a monoclonal antibody AD7. We have used this antibody to analyze whether p200 functions in exocytic transport from the TGN to the apical or basolateral plasma membrane in Madin-Darby canine kidney cells. We found that transport of the viral marker proteins, influenza hemagglutinin (HA) to the apical surface or vesicular stomatitis virus glycoprotein (VSV G) to the basolateral surface in streptolysin O-permeabilized cells was not affected when p200 was depleted from both the membranes and the cytosol. When vesicles isolated from perforated cells were analyzed by equilibrium density gradient centrifugation, the p200 immunoreactive membranes did not comigrate with either the apical vesicle marker HA or the basolateral vesicle marker VSV G. Immunoelectron microscopy of perforated and double-labeled cells showed that the p200 positive vesicular profiles were not labeled by antibodies to HA or VSV G when the viral proteins were accumulated in the TGN. Furthermore, the p200-decorated vesicles were more electron dense than those labeled with the viral antibodies. Together, these results suggest that p200 does not function in the transport pathways that carry HA from the TGN to the apical surface or VSV G from the TGN to the basolateral surface.

The isolation of plasma membrane from protoplasts of soybean suspension cultures

1977 ◽  
Vol 24 (1) ◽  
pp. 295-310
Author(s):  
D.W. Galbraith ◽  
D.H. Northcote
Keyword(s):  
Cell Wall ◽  
Plasma Membrane ◽  
Membrane Fraction ◽  
Sucrose Gradient ◽  
Plasma Membranes ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Membrane Systems ◽  
Enzymic Digestion ◽  
Nadh Cytochrome

A procedure for the isolation of plasma membranes from protoplasts of suspension-cultured soybean is described. Protoplasts were prepared by enzymic digestion of the cell wall and the plasma membrane was labelled with radioactive diazotized sulphanilic acid. The membrane systems from broken protoplasts were separated by continuous isopycnic sucrose gradient centrifugation. Radioactivity was localized in a band possessing a buoyant density of 1–14 g ml-1. The activities of NADPH- and NADH-cytochrome c reductase, fumarase, Mg2+-ATPase, IDPase and acid phosphodiesterase in the various regions of the density gradient were determined. A plasma membrane fraction was selected which was relatively uncontaminated with membranes derived from endoplasmic reticulum, tonoplasts and mitochondria. The results indicated that Mg2+-ATPase and possibly acid phosphodiesterase were associated with the plasma membrane.

Analysis of the intermediate size proteoglycans from the developing chick limb buds11Presented in part at 72nd Annual Meeting of the American Society of Biological Chemists, St Louis, Missouri, U.S.A. 31 May to 4 June, 1981.

Development ◽  
1982 ◽  
Vol 70 (1) ◽  
pp. 61-74
Author(s):  
Nagaswamisri Vasan
Keyword(s):  
Chondroitin Sulphate ◽  
Binding Capacity ◽  
Gradient Centrifugation ◽  
Buoyant Density ◽  
Limb Bud ◽  
Side Chain ◽  
Intermediate Size ◽  
Proteoglycan Aggregates ◽  
American Society ◽  
Hyaluronic Acid Binding

Limb-bud proteoglycans are heterogeneous molecules which vary in their chemical and physical properties with development. This report describes proteoglycan intermediates (PG-I) that predominate in stage-34 limbs, and compares them with proteoglycan aggregates (PG-A) in stage-38 limbs. We analysed proteoglycans and their components extracted with guanidinium chloride by subjecting them to density gradient centrifugation, molecular sieve chromatography, electrophoretic separation, and selective enzymatic degradation. PG-I and PG-A have similar chondroitin sulphate composition, amino sugars, chondroitin sulphate side-chain length, glycoprotein link factors, and hyaluronic acid binding capacity, and both cross react with antisera prepared against cartilage-specific chick sternal proteoglycans. However, PG-I has lower molecular weight, lower buoyant density, and fewer chondroitin sulphate side chains on the protein core. The PG-I in the developing limb can be considered a mixture of smaller aggregates and cartilage-specific large monomers in which the former predominate.

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