scholarly journals Biochemical study of KB-cell receptor for adenovirus

1977 ◽  
Vol 166 (2) ◽  
pp. 237-247 ◽  
Author(s):  
Bernadette Hennache ◽  
Pierre Boulanger
Keyword(s):  
Specific Activity ◽  
Biochemical Study ◽  
Gradient Centrifugation ◽  
Sodium Dodecyl ◽  
Cell Receptor ◽  
Surface Proteins ◽  
Cross Linking ◽  
Affinity Column ◽  
Major Protein ◽  
Sucrose Density Gradient Centrifugation

Three different approaches were used in an attempt to characterize the KB-cell receptor for adenovirus: affinity chromatography, immunoadsorption and cross-linking with a cleavable bifunctional reagent. The first system used an affinity gel consisting of adenovirus-fibre projection linked to Sepharose matrix by an intermediate bis(aminopropyl)amine arm, the amino groups of the fibre ligand being preserved by prior citraconylation. The second system consisted of adenovirus complete penton capsomere attached to anti-(penton base) antibody and cross-linked to polyacrylamide particles with glutaraldehyde. In this latter affinity model, the penton-fibre projection was appropriately oriented outwards, as in the virus particle. Both affinity systems permitted isolation from a KB-cell plasma-membrane extract of fibre-binding and penton-fibre-binding protein material, which inhibited adenovirus attachment. The penton–immunoadsorbent appeared more efficient and more specific than the affinity column of fibre–bis(aminopropyl)amino-Sepharose gel in specific activity of inhibition of adenovirus attachment. The third method consisted of reversibly cross-linking KB-cell receptor proteins with adenovirus particles by means of a cleavable di-imidoester and isolation of the complexes by sucrose-density-gradient centrifugation. Polypeptide analysis on sodium dodecyl sulphate/polyacrylamide gel of labelled KB-cell surface proteins, selected by the different procedures, showed that three major protein subunits of 78000, 42000 and 34000mol.wt. were common to the three selection systems. A possible model for the structure and function of the KB-cell receptor for adenovirus is discussed.

scholarly journals Purification of the enzyme NADPH: protochlorophyllide oxidoreductase

1981 ◽  
Vol 195 (1) ◽  
pp. 83-92 ◽  
Author(s):  
N S Beer ◽  
W T Griffiths
Keyword(s):  
Specific Activity ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
High Specific Activity ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Triton X ◽  
Etioplast Membranes

A procedure for the purification of the enzyme NADPH:protochlorophyllide oxidoreductase is described. This involves fractionation of sonicated oat etioplast membranes by discontinuous-sucrose-density-gradient centrifugation, which gives membranes in which the enzyme is present at a high specific activity. The enzyme is solubilized from the membranes with Triton X-100, followed by gel filtration of the extract; enzyme activity is eluted in fractions corresponding to a mol.wt of approx. 35000. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the enzyme-containing fractions from gel filtration shows two peptides, of mol.wts. approx. 35000 and 37000.

scholarly journals Purification, properties and assay of d-ribulose 5-phosphate 3-epimerase from human erythrocytes

1983 ◽  
Vol 211 (3) ◽  
pp. 617-623 ◽  
Author(s):  
A Karmali ◽  
A F Drake ◽  
N Spencer
Keyword(s):  
Density Gradient ◽  
Specific Activity ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sodium Dodecyl ◽  
Minor Component ◽  
Sucrose Density Gradient ◽  
Human Erythrocytes ◽  
Sucrose Density Gradient Centrifugation ◽  
Dimeric Form

A direct assay procedure is described for D-ribulose 5-phosphate 3-epimerase (EC 5.1.3.1) which exploits differences in the c.d. spectra of substrate and product. The enzyme has been purified from human erythrocytes and was resolved by gel filtration and sucrose-density-gradient centrifugation into a major component of apparent Mr 45 000 and a minor component of Mr 23 000. Electrophoresis in sodium dodecyl sulphate gave a single component corresponding to Mr 23 000. Kinetic and sucrose-density-gradient centrifugation data indicate dissociation of the dimeric form of the enzyme into monomers of low specific activity; substrate favours the active dimeric form of the enzyme. At concentrations of the enzyme where both forms of the enzyme are present initial velocity data yielded a Hill plot with an interaction coefficient of approx. 2.0, indicating co-operative binding of substrate under these conditions.

scholarly journals Interaction of autoantibodies to thyrotropin receptor with a hydrophilic subunit of the thyrotropin receptor

1985 ◽  
Vol 228 (1) ◽  
pp. 111-117 ◽  
Author(s):  
E Davies Jones ◽  
F A Hashim ◽  
Y Kajita ◽  
F M Creagh ◽  
P R Buckland ◽  
...  
Keyword(s):  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Water Soluble ◽  
Tsh Receptor ◽  
Human Thyroid ◽  
Thyrotropin Receptor ◽  
Freezing And Thawing ◽  
Sucrose Density Gradient Centrifugation ◽  
A Subunit

Reduction of human thyroid membranes with dithiothreitol caused the release of a water-soluble glycoprotein which neutralized the thyrotropin (TSH) receptor-binding and thyroid-stimulating activities of Graves‘ serum. Analysis of the protein by gel filtration and sucrose density gradient centrifugation allowed estimates of 3.45 nm for the Stokes’ radius, 3.6 S for the s20,w and 47 000 +/- 5000 (mean +/- S.D.; n = 4) for the Mr. The material released by dithiothreitol treatment could be crosslinked to 125I-labelled TSH coupled to N-hydroxysuccinimidyl 4-azidobenzoate (125I-HSAB-TSH), suggesting that it contained a component of the TSH receptor. Furthermore, analysis of the crosslinked material by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated that it contained the TSH receptor A subunit (Mr 50 000). Several factors suggested therefore that the glycoprotein released by dithiothreitol treatment of human thyroid membranes was the TSH receptor A subunit. In particular, (a) both preparations were hydrophilic and were released from membranes by reduction, (b) they had similar Mr values and (c) both preparations crosslinked to 125I-HSAB-TSH. Material similar to the TSH receptor A subunit was released from thyroid membranes by treatment with papain, probably as a result of cleavage of the receptor A subunit at a site close to the interchain disulphide bridge. A similar mechanism, involving thyroid proteinases, was probably involved in release of material with similar properties to the TSH receptor A subunit during freezing and thawing of human thyroid homogenates.

scholarly journals A glycoprotein associated with the membrane fraction of human B but not T lymphocytes.

1975 ◽  
Vol 142 (6) ◽  
pp. 1416-1424 ◽  
Author(s):  
S Fujita ◽  
S D Litwin ◽  
N Hartman
Keyword(s):  
Membrane Fraction ◽  
Gradient Centrifugation ◽  
Human Lymphocytes ◽  
Sodium Dodecyl ◽  
Protein Band ◽  
Sucrose Density Gradient ◽  
Peripheral Blood Lymphocytes ◽  
Sucrose Density Gradient Centrifugation ◽  
Lymphoid Lines

A method is described which employs differential centrifugation and sucrose density gradient centrifugation to isolate a membrane fraction from human lymphocytes. Membrane preparations from long-term human cultured B- and T-lymphoid lines, peripheral blood lymphocytes, tonsillar lymphocytes, and thymocytes were analyzed on 0.5% sodium dodecyl sulfate-7.5% polyacrylamide gels stained for protein and carbohydrate. The most important finding was a major glycoprotein of approximately 30,000 daltons associated with the membrane preparations from B lymphocytes. T-lymphocyte preparations did not contain readily detectable amounts of this membrane-associated component. The T-cell lymphoid line MOLT-4 was unique in that it had a narrow protein band at approximately 30,000 daltons which did not contain carbohydrate.

scholarly journals Separation and Paired Proteome Profiling of Plant Chloroplast and Cytoplasmic Ribosomes

Plants ◽  
2020 ◽  
Vol 9 (7) ◽  
pp. 892
Author(s):  
Alexandre Augusto Pereira Firmino ◽  
Michal Gorka ◽  
Alexander Graf ◽  
Aleksandra Skirycz ◽  
Federico Martinez-Seidel ◽  
...  
Keyword(s):  
Ribosome Biogenesis ◽  
Gradient Centrifugation ◽  
Leaf Tissue ◽  
Proteome Analysis ◽  
Sucrose Density Gradient ◽  
Cross Linking ◽  
Sucrose Density Gradient Centrifugation ◽  
Cytoplasmic Ribosome ◽  
Plant Chloroplast

Conventional preparation methods of plant ribosomes fail to resolve non-translating chloroplast or cytoplasmic ribosome subunits from translating fractions. We established preparation of these ribosome complexes from Arabidopsis thaliana leaf, root, and seed tissues by optimized sucrose density gradient centrifugation of protease protected plant extracts. The method co-purified non-translating 30S and 40S ribosome subunits separated non-translating 50S from 60S subunits, and resolved assembled monosomes from low oligomeric polysomes. Combining ribosome fractionation with microfluidic rRNA analysis and proteomics, we characterized the rRNA and ribosomal protein (RP) composition. The identity of cytoplasmic and chloroplast ribosome complexes and the presence of ribosome biogenesis factors in the 60S-80S sedimentation interval were verified. In vivo cross-linking of leaf tissue stabilized ribosome biogenesis complexes, but induced polysome run-off. Omitting cross-linking, the established paired fractionation and proteome analysis monitored relative abundances of plant chloroplast and cytoplasmic ribosome fractions and enabled analysis of RP composition and ribosome associated proteins including transiently associated biogenesis factors.

cAMP increases synthesis of surfactant-associated protein A by perfused rat lung

1991 ◽  
Vol 260 (4) ◽  
pp. L226-L233 ◽  
Author(s):  
A. B. Fisher ◽  
I. Arad ◽  
C. Dodia ◽  
A. Chander ◽  
S. I. Feinstein
Keyword(s):  
Protein Fraction ◽  
Specific Activity ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Synthetic Medium ◽  
Protein A ◽  
Sodium Dodecyl ◽  
Lamellar Body ◽  
Surfactant Protein ◽  
Lag Phase

Synthesis and secretion of surfactant-associated protein were studied in isolated rat lungs perfused with [3H]phenylalanine or [35S]methionine in synthetic medium. Surfactant was isolated by lung lavage and density-gradient centrifugation followed by dialysis to remove unincorporated amino acid and extraction with ethanol-ether to yield a delipidated protein fraction. Incorporation of [3H]phenylalanine into the delipidated surfactant protein fraction showed a lag phase of approximately 3 h followed by progressive increase over the next 3 h at a rate of 1.6 nmol.mg protein-1.h-1. With 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP, 0.1 mM) added to the perfusate, the incorporation rate between 3 and 6 h was increased by 75%. 3H specific activity in a delipidated lamellar body-rich fraction isolated from lung homogenates was unchanged by 8-BrcAMP at 3 h but was increased by 45% at 6 h. The major peak of radioactivity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of surfactant and lamellar bodies corresponded to proteins of 27–36 kDa that were identified as surfactant protein A (SP-A) by immunoblot. In the presence of 8-BrcAMP during 6 h of perfusion, specific activity of 35S-labeled SP-A in immunoprecipitated protein was increased by 93% and the SP-A mRNA content of lung was increased 145%. These results show that isolated perfused lungs synthesize and secrete surfactant-associated proteins and that the presence of a permeable cAMP analogue in the lung perfusate leads to increased secretion followed by induction of synthesis for SP-A.

scholarly journals Isolation of 10-nm filaments from astrocytes in the mouse optic nerve.

1981 ◽  
Vol 88 (1) ◽  
pp. 245-250 ◽  
Author(s):  
S Tsukita ◽  
H Ishikawa ◽  
M Kurokawa
Keyword(s):  
Optic Nerve ◽  
Peptide Mapping ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sucrose Density Gradient ◽  
Molar Ratio ◽  
Sucrose Density Gradient Centrifugation ◽  
Optic Nerves ◽  
10 Nm Filaments

Astroglial filaments approximately 10 nm in diameter were isolated from degenerated mouse optic nerves by Triton X-100 and DNase I treatments followed by sucrose density gradient centrifugation. 2-4 wk after bilateral enucleation, optic nerves contained virtually a single population of 10-nm filaments (astroglial filaments), free from neurofilaments. In negative-staining and thin-section electron microscopy, the isolated filaments were seen as nonbranching linear structures with smooth contour, and were morphologically identical to those in situ. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed the isolated filaments to be composed of two major polypeptides with molecular weights of 45,000 and 55,000, present in an approximate molar ratio of 1:1. These findings, together with the results of one-dimensional peptide mapping and solubility study, indicate that the astroglial filaments in the mouse optic nerve are primarily composed of these two polypeptides.

scholarly journals The lanthanide-enhanced affinity chromatography of clostridial collagenase

1985 ◽  
Vol 225 (2) ◽  
pp. 553-556 ◽  
Author(s):  
C H Evans
Keyword(s):  
Periodic Acid ◽  
Sodium Dodecyl ◽  
Minor Component ◽  
Electrophoretic Analysis ◽  
Lanthanide Ions ◽  
Affinity Column ◽  
Major Protein ◽  
Periodic Acid Schiff ◽  
Highly Active ◽  
A Minor

Clostridiopeptidase A (EC 3.4.24.3) did not bind to a collagen affinity column in the absence of Ca2+, but did so in the presence of lanthanide ions (Ln3+). The sequestered enzyme could be eluted with EGTA. For the four Ln3+ ions tested, the order of efficiency in promoting enzyme binding, Sm3+ greater than Lu3+ greater than Er3+ much greater than La3+, reflected their relative abilities to inhibit clostridiopeptidase A. By using Sm3+ as an adjunct, it proved possible to separate a highly active preparation of collagenase from crude clostridial collagenase. Sodium dodecyl sulphate/polyacrylamide-gel-electrophoretic analysis of the preparation revealed a major protein of Mr 95000 and a minor component of Mr 82000. As both were stained by periodic acid/Schiff reagent, they were probably glycoproteins.

Purification and regulation of an AMP-specific cytosolic 5'-nucleotidase from dog heart

1993 ◽  
Vol 264 (5) ◽  
pp. H1528-H1534 ◽  
Author(s):  
A. Darvish ◽  
P. J. Metting
Keyword(s):  
Adenine Nucleotides ◽  
Gradient Centrifugation ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Maximal Activity ◽  
Maximal Velocity ◽  
Sucrose Density Gradient Centrifugation ◽  
Dog Heart ◽  
Myocardial Hypoxia

The major enzyme responsible for adenosine production during myocardial hypoxia or ischemia is 5'-nucleotidase. We purified an AMP-specific 5'-nucleotidase to homogeneity from the 150,000-g supernatant of dog heart homogenate using phosphocellulose, DEAE-cellulose, and ADP-agarose affinity chromatography. Sodium dodecyl sulfate-poly-acrylamide gel electrophoresis of the purified enzyme yielded a single protein band of 43 kDa. The molecular mass of the holoenzyme, determined by gel filtration and sucrose density-gradient centrifugation, was approximately 166 kDa, suggesting a tetrameric structure. Dog heart cytosolic 5'-nucleotidase was active at physiological pH (6.8-7.8) and demonstrated a preference for AMP over IMP as substrate. The enzyme exhibited sigmoidal saturation kinetics, with half-maximal activity at 2.6 mM AMP in the absence of ADP. ADP (0-250 microM) activated cytosolic 5'-nucleotidase by increasing maximal velocity and affinity for AMP. The enzyme was inhibited by 4 mM ATP, but 5'-nucleotidase activity increased as [ATP] was reduced. Mg2+ was required for activity, with maximal activation at approximately 3.5 mM free Mg2+. These data suggest that the regulation of AMP-specific cytosolic 5'-nucleotidase by adenine nucleotides and free Mg2+ may be important in the production of adenosine during conditions promoting ATP hydrolysis, such as myocardial hypoxia or ischemia.

Rat intestinal maltase–glucoamylase. Purification of the detergent-solubilized enzyme in the presence of protease inhibitors: properties and identification of a protease-sensitive subunit

1984 ◽  
Vol 62 (1) ◽  
pp. 36-43 ◽  
Author(s):  
Lilian Lee ◽  
Gordon Forstner
Keyword(s):  
Protease Inhibitors ◽  
Gradient Centrifugation ◽  
Sodium Dodecyl ◽  
Prolonged Storage ◽  
Affinity Column ◽  
Molecular Weights ◽  
Clear Cut ◽  
Maltase Activity ◽  
Continuous Presence ◽  
Monomeric Proteins

Failure to develop clear-cut, distinguishing characteristics for hydrophobic and hydrophilic forms of maltase–glucoamylase led us to attempt the purification of the detergent-extracted enzyme in the continuous presence of protease inhibitors (phenylmethylsulfonyl fluoride and N-ethylmaleimide). The enzyme was purified by molecular exclusion, anion-exchange, and affinity column chromatography to a final specific maltase activity of 80 U/mg protein, comparable to previously solubilized enzymes. Both detergent (d-maltase) and proteolytically (p-maltase) solubilized enzymes had identical Km's for maltose and similar glycogenase activity. d-Maltase was clearly amphipathic. Whereas 95% of p-maltase was eluted with aqueous buffer from an octyl-Sepharose CL-4B column, the elution of d-maltase required solutions containing Triton X-100 and ethylene glycol. On density gradient centrifugation and sodium dodecyl sulfate (SDS) – polyacrylamide gels, p-maltase migrated as one high molecular weight species of 500 000. In contrast d-maltase migrated heterogeneously and the smallest maltase-active forms delineated by these two techniques, as well as by high pressure liquid chromatography, had molecular weights which ranged from 120 000 to 150 000. Both p- and d-maltase were dissociated by heat in SDS, forming five prominent species as we have previously described. In contrast to p-maltase, in which the smallest species, band 1, equalled 36.7% of the total mass, band 1 of d-maltase accounted for 66.5%. Band 1 was separable when smaller amounts of enzyme were applied to slab gels and stained with silver, into two proteins of 130 000 and 145 000 daltons. The 145 000 dalton protein was absent in p-maltase and was replaced by a faint band of 140 000 daltons. The 140 000 dalton band, plus a new N-terminal glycine, were also generated from d-maltase during prolonged storage at −20 °C. These data suggest that rat intestinal maltase–glucoamylase contains two monomeric proteins. The largest monomer contains an additional peptide segment at the N-terminus, which is removed by proteolysis and presumably anchors the enzyme to the microvillus membrane. After removal from the membrane, the two monomers of the d-enzyme are apparently partially dissociated to account for maltase activity within the 120 000 to 150 000 dalton range. Conversely, removal of the anchor segment favours a polymeric structure.

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