scholarly journals Similarities in the polypeptide composition of glyoxysomal and endoplasmic-reticulum membranes from castor-bean endosperm

1976 ◽  
Vol 154 (2) ◽  
pp. 491-499 ◽  
Author(s):  
L Bowden ◽  
J. M Lord
Keyword(s):  
Endoplasmic Reticulum ◽  
Castor Bean ◽  
Ricinus Communis ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Specific Radioactivity ◽  
Polypeptide Composition ◽  
Endosperm Tissue ◽  
Sucrose Density Gradient Centrifugation

Microsomal fractions, glyoxysomes and mitochondria were isolated from homogenates of germinating castor-bean (Ricinus communis) endosperm by sucrose-density-gradient centrifugation. Washed membrane preparations from these cellular fractions were examined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. At corresponding developmental stages the endoplasmic-reticulum and glyoxysomal membranes were strikingly similar in polypeptide composition, at least 16 polypeptides being present in membranes isolated from 3-day-old tissue. Supplying [35S]methionine to intact endosperm tissue resulted in the labelling of all membrane polypeptides, the specific radioactivity in the endoplasmic reticulum being greater than for equivalent polypeptides of the glyoxysomal membrane. Washing these membranes with sodium deoxycholate solution extensively solubilized protein components, with the exception of a predominant polypeptide of mol.wt. 55000. Mitochondrial membrane preparations differed from those of the endoplasmic reticulum and glyoxysomes in polypeptide molecular-weight distribution and the [35S]methionine-labelling pattern. The similarity in polypeptide composition between endoplasmic-reticulum and glyoxysomal membranes is discussed in relation to glyoxysome biogenesis.

scholarly journals Fractionation of the proteins of plant microbodies

1974 ◽  
Vol 144 (3) ◽  
pp. 559-566 ◽  
Author(s):  
R H Brown ◽  
J M Lord ◽  
M J Merrett
Keyword(s):  
Membrane Proteins ◽  
Ricinus Communis ◽  
Osmotic Shock ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Weight One ◽  
Protein Components

1. Glyoxysomes and peroxisomes have been isolated from dark- and light-grown seedlings of pumpkin (Cucurbita pepo) by sucrose-density-gradient centrifugation. 2. Pumpkin microbodies and castor-bean (Ricinus communis) glyoxysomes may be fractionated, by a combination of osmotic shock and treatment with KCl, into three distinct groups of proteins: readily soluble (matrix enzymes), solubilized in the presence of KCl (membrane-bound enzymes) and relatively insoluble (membrane ‘ghost’ proteins). 3. Sodium dodecyl sulphate–polyacrylamide-gel electrophoresis of ‘ghost’ fractions indicated that the membrane proteins were generally of low molecular weight; one gel band (mol.wt. 27000–28000) was common to all three microbodies. 4. Although there were major differences in the soluble protein components of pumpkin glyoxysomes and peroxisomes, electrophoresis of the pumpkin microbody ‘ghosts’ indicated that the membrane proteins were similar, four main components being common to each class of microbody (monomer molecular weights 42000, 34000, 27000 and 17000).

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 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 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 Protein and glycoprotein electrophoretic patterns of enriched fractions of primary and secondary granules from guinea pig polymorphonuclear leukocytes.

1975 ◽  
Vol 65 (3) ◽  
pp. 577-586 ◽  
Author(s):  
J Noseworthy ◽  
G H Smith ◽  
S R Himmelhoch ◽  
W H Evans
Keyword(s):  
Guinea Pig ◽  
Polymorphonuclear Leukocytes ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Species Difference ◽  
Sodium Dodecyl ◽  
Sucrose Density Gradient ◽  
Supernatant Fraction ◽  
Sucrose Density Gradient Centrifugation ◽  
Electrophoretic Patterns

The postnuclear supernatant fraction of sucrose homogenates of guinea pig polymorphonuclear leukocytes (PMNL) was subjected to differential centrifugation to obtain a total particulate fraction, a particle-free supernatant fraction, highly enriched fractions of primary and secondary granules, and a membrane-rich fraction. The various fractions were solubilized in buffer containing sodium dodecyl sulfate (SDS) and analyzed for protein and glycoproteincomponents by SDS -polyacrylamide gel electrophoresis. The major glycoprotein components of the postnuclear supernatant fraction were found mainly associated with the enriched fraction of secondary granules and, to a lesser extent, with the membrane-rich fraction. No major glycoprotein components were visible in the polypeptide electrophoretic patterns of the primary granule fraction or of the particle-free supernate. Attempts at separation of guinea pig granules by zonal sucrose density gradient centrifugation were only partially successful. Data supporting a species difference in this regard between rabbit and guinea pig PMNL granules are presented.

scholarly journals Immunoaffinity chromatography as a means of purifying legumin from Pisum (pea) seeds

1979 ◽  
Vol 177 (2) ◽  
pp. 509-520 ◽  
Author(s):  
R Casey
Keyword(s):  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Immunoaffinity Chromatography ◽  
Exchange Chromatography ◽  
Sucrose Density Gradient Centrifugation ◽  
Salting Out ◽  
Wide Range ◽  
Gel Isoelectric Focusing

The potential of immunoaffinity chromatography as a means of purifying legumin from a wide range of Pisum (pea) types was assessed. The method required small amounts of highly purified legumin from a single Pisum type, and this was obtained by salting out with (NH4)2SO4 followed by zonal isoelectric precipitation, ion-exchange chromatography on DEAE-cellulose and sucrose-density-gradient centrifugation. Some physiocochemical properties of purified legumin were determined, a number of which (Strokes radius, subunit molecular weights, subunit N-terminal residues and subunit molar ratios) have not previously been reported for Pisum legumin. Examination of Pisum legumin by two-dimensional gel isoelectric focusing/electrophoresis indicated the existence of extensive subunit heterogeneity, and polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate showed apparent variation in the nature of this heterogeneity from one Pisum variety to another. Despite this variation, immunoaffinity chromatography on immobilized anti-legumin (which was prepared by affinity chromatography on the immubolized purified legumin from the single Pisum type) was shown to be a generally applicable method for the purification of undegraded legumin from a range of pisum types, including two primate lines.

scholarly journals Solubilization of pig lymphocyte plasma membrane and fractionation of some of the components

1971 ◽  
Vol 123 (5) ◽  
pp. 967-975 ◽  
Author(s):  
D. Allan ◽  
M. J. Crumpton
Keyword(s):  
Plasma Membrane ◽  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Biological Activities ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sucrose Density Gradient ◽  
Similar Distribution ◽  
Sucrose Density Gradient Centrifugation

The degree of solubilization of pig lymphocyte plasma membrane by sodium deoxycholate was determined at a variety of temperatures and detergent concentrations. Approx. 95% of the membrane protein was soluble in 2% deoxycholate at 23°C. Some of the biological activities of the membrane survived this treatment. The leucine β-naphthylamidase activity was more readily soluble than the 5′-nucleotidase and these enzymes could be separated by extraction with 0.5% deoxycholate at 0°C. Membrane solubilized in 2% deoxycholate at 23°C was fractionated by sucrose-density-gradient centrifugation in 1% deoxycholate. The phospholipid was separated from the protein, which formed a fairly symmetrical peak that sedimented slightly slower than ovalbumin; the leucine naphthylamidase and 5′-nucleotidase activities were resolved from each other and from the main protein peak. Similar separations were achieved by elution from Sephadex G-200 and Sepharose 6B in 1% deoxycholate. The main proteins, however, appeared to possess much higher molecular weights than those indicated by sucrose-density-gradient centrifugation. This disparity suggests that many of the membrane proteins have a rod-like shape, especially since the results of experiments with [14C]deoxycholate revealed that the proteins did not bind significant amounts of deoxycholate. In contrast, 5′-nucleotidase and leucine naphthylamidase appeared to be globular. Polyacrylamide-gel electrophoresis of membrane solubilized in sodium dodecyl sulphate gave a similar distribution of protein to that achieved by sucrose-density-gradient centrifugation. Trace amounts only of polypeptides of molecular weight less than 10000 were detected.

Characteristics of the outer membrane of selected oral Bacteroides species

1985 ◽  
Vol 31 (3) ◽  
pp. 238-250 ◽  
Author(s):  
G. D. Williams ◽  
S. C. Holt
Keyword(s):  
Outer Membrane ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Specific Pattern ◽  
Sucrose Density Gradient Centrifugation ◽  
Outer Membranes ◽  
Bacteroides Species ◽  
Polypeptide Patterns ◽  
Sds Page

The outer membranes from selected oral Bacteroides species were isolated and characterized morphologically, chemically, and physically. Both sucrose density gradient centrifugation and sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE) revealed outer membranes which varied slightly with the species, as well as showing complex polypeptide patterns after SDS–PAGE. The polypeptide distribution revealed a species-specific pattern; however, there was often a variation within a given species in many minor polypeptide bands, with heat-modifiable minor peptides occurring in nearly all species. Protein-associated carbohydrates occurred in several species. Outer membrane fragments (blebs) recovered by ultracentrifugation of the growth supernatants were either nearly identical to the outer membrane polypeptide patterns, or contained reduced amounts of specific polypeptides, again varying with the species. l25I-labelling of whole cells indicated possible surface exposure for several of the major polypeptides.

scholarly journals Matrix proteins bound to associatively prepared proteoglycans from bovine cartilage

1979 ◽  
Vol 183 (3) ◽  
pp. 539-545 ◽  
Author(s):  
M Paulsson ◽  
D Heinegård
Keyword(s):  
High Speed ◽  
Matrix Protein ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sucrose Density Gradient ◽  
Exchange Chromatography ◽  
Cartilage Matrix ◽  
Gel Chromatography ◽  
Sucrose Density Gradient Centrifugation

Proteoglycans were extracted from bovine tracheal cartilage by high-speed homogenization, the use of dissociative solvents being avoided. The homogenate was fractionated by gel chromatography, sucrose-density-gradient centrifugation and ion-exchange chromatography. A previously unrecognized protein, cartilage matrix protein, was identified by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. It cofractionated with the proteoglycans in all systems, indicating an interaction. The cartilage matrix protein-proteoglycan complex was dissociated by treatment with 4M-guanidinium chloride. The complex again formed when the guanidine was removed. The cartilage matrix protein has a mol.wt. of more than 200000. On reduction it yields subunits with a mol.wt. of approx. 60000.

scholarly journals The heterogeneity of cytoplasmic deoxyribonucleic acid polymerase from Physarum polycephalum

1978 ◽  
Vol 171 (2) ◽  
pp. 445-451 ◽  
Author(s):  
K S Zänker ◽  
W Schiebel
Keyword(s):  
Dna Polymerase ◽  
Physarum Polycephalum ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Dimensional Structure ◽  
Sucrose Density Gradient Centrifugation ◽  
Molecular Weights

Cytoplasmic DNA polymerase (DNA deoxynucleotidyltransferase, EC 2.7.7.7) was partially purified from Physarum polycephalum. The first step of the purification procedure utilized the fact that the enzyme on gel filtration behaves in anomalous fashion. The second step was either ion-exchange chromatography or sucrose-density-gradient centrifugation. The partially purified DNA polymerase was heterogeneous and at least four species with different sedimentation coefficients (5.5S, 7.2S, 8.6S and 11.5S) were detected. Calculated molecular weights indicated a tendency for stoicheiometric polypeptide aggregation, accompanied by an alteration of the three-dimensional structure froma compact spheroid to a more open elliptical form. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and computed molecular weights suggest an active protomer in the range of 113000 daltons; all data pertain to I 0.045, which was maintained during the whole procedure.

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