scholarly journals UDP-galactose-ceramide galactosyltransferase in rat brain myelin subfractions during development

1980 ◽  
Vol 186 (3) ◽  
pp. 959-969 ◽  
Author(s):  
O Koul ◽  
K H Chou ◽  
F B Jungalwala
Keyword(s):  
Rat Brain ◽  
Membrane Fraction ◽  
Specific Activity ◽  
Gradient Centrifugation ◽  
Proteolipid Protein ◽  
Myelin Proteins ◽  
Sucrose Density Gradient Centrifugation ◽  
Microsomal Membranes ◽  
Myelin Subfractions ◽  
Myelin Fraction

The localization and activity of the enzyme UDP-galactose-hydroxy fatty acid-containing ceramide galactosyltransferase is described in rat brain myelin subfractions during development. Other lipid-synthesizing enzymes, such as cerebroside sulphotransferase, UDP-glucose-ceramide glucosyltransferase and CDP-choline-1,2-diacylglycerol cholinephosphotransferase, were also studied for comparison in myelin subfractions and microsomal membranes. The purified myelin was subfractionated by isopycnic sucrose-density-gradient centrifugation. Four myelin subfractions, three floating respectively on 0.55 M- (light-myelin fraction), 0.75 M- (heavy-myelin fraction) and 0.85 M-sucrose (membrane fraction), and a pellet, were isolated and purified. At all ages, 70-75% of the total myelin proteins was found in the heavy-myelin fraction, whereas 2-5% of the protein was recovered in the light-myelin fraction, and about 7-12% in the membrane fraction. Most of the galactosyltransferase was associated with the heavy-myelin and membrane fractions. Other lipid-synthesizing enzymes studied appeared not to associate with purified myelin or myelin subfractions, but were enriched in the microsomal-membrane fraction. During development, the specific activity of the microsomal galactosyltransferase reached a maximum when the animals were about 20 days old and then declined. By contrast the specific activity of the galactosyltransferase in the heavy-myelin and membrane fractions was 3-4 times higher than that of the microsomal membranes in 16-day-old animals. The specific activity of the enzyme in the heavy-myelin fraction sharply declined with age. Chemical and enzymic analyses of the heavy-myelin and membrane myelin subfractions at various ages showed that the membrane fraction contained more proteins in relation to lipids than the heavy-myelin fraction. The membrane fraction was also enriched in phospholipids compared with cholesterol and contrined equivalent amounts of 2':3'-cyclic nucleotide 3'-phosphohydrolase compared with heavy- and light-myelin fractions. The membrane fraction was deficient in myelin basic protein and proteolipid protein and enriched in high-molecular-weight proteins. The specific localization of galactosyltransferase in heavy-myelin and membrane fractions at an early age when myelination is just beginning suggests that it may have some role in the myelination process.

scholarly journals Characterization of a cytoskeletal matrix associated with myelin from rat brain

1989 ◽  
Vol 260 (3) ◽  
pp. 689-696 ◽  
Author(s):  
C S Gillespie ◽  
R Wilson ◽  
A Davidson ◽  
P J Brophy
Keyword(s):  
Rat Brain ◽  
Soluble Fraction ◽  
Gradient Centrifugation ◽  
Proteolipid Protein ◽  
Immunoblot Analysis ◽  
Sucrose Density Gradient ◽  
Insoluble Residue ◽  
Sucrose Density Gradient Centrifugation ◽  
Specific Proteins ◽  
Cytoskeletal Elements

Extraction of rat brain myelin in a buffer containing Triton X-100 yielded a soluble fraction and an insoluble residue that was enriched in cytoskeletal elements. Immunoblot analysis of the detergent-soluble fraction and the insoluble cytoskeletal residue showed that all of the tubulin and more than half of the actin were found within the cytoskeletal fraction. The distribution of myelin-specific proteins was also examined, and revealed that 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNPase) I and most of the myelin basic proteins (MBPs) were equally distributed between both fractions. By contrast, the large MBP (21.5 kDa) and CNPase II (50 kDa) were observed to partition almost entirely with the cytoskeletal fraction. Proteolipid protein was found predominantly in the detergent-soluble fraction, as was DM-20 protein. Analysis of the cytoskeletal fraction by sucrose-density-gradient centrifugation demonstrated that a distinct subset of lipids was tightly bound to the cytoskeletal protein residue. The cytoskeleton-associated lipid was considerably enriched in cerebroside and sphingomyelin by comparison with total myelin lipids. These results indicate that a cytoskeletal matrix is associated with multilamellar myelin, and suggest that this structure may play a fundamental role in myelinogenesis.

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 Turnover of myelin and other structural proteins in the developing rat brain

1974 ◽  
Vol 142 (3) ◽  
pp. 499-507 ◽  
Author(s):  
M. I. Sabri ◽  
A. H. Bone ◽  
A. N. Davison
Keyword(s):  
Rat Brain ◽  
Slow Component ◽  
Proteolipid Protein ◽  
Myelin Proteins ◽  
Turnover Rates ◽  
Basic Proteins ◽  
Glucose Injection ◽  
Total Brain ◽  
The Difference ◽  
Developing Rat

1. Protein metabolism of myelin and other subcellular components from developing rat brain was studied for periods from 5h to 210 days after intraperitoneal injection of [3H]lysine and [14C]glucose. 2. Half-lives for total brain proteins (t0.5) were 27 days after [3H]lysine and 4 days after [14C]glucose injection. 3. Factors accounting for the difference in the turnover rates obtained with different precursors, and the problem of reutilization of the label were investigated. 4. The catabolism of purified myelin proteins was studied and the half-lives of individual myelin proteins were calculated. 5. Myelin basic proteins turned over at two different rates. Half-life of the fast component of myelin basic proteins was 19–22 days and the slow component exhibited a high degree of metabolic stability. 6. Proteolipid protein underwent slow turnover. High-molecular-weight Wolfgram (1966) proteins underwent (relatively) fast metabolism (t0.5 of 17–22 days).

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.

5-ENE-3β-HYDROXYSTEROID DEHYDROGENASE OF HUMAN AND BOVINE ADRENOCORTICAL ENDOPLASMIC RETICULUM: SOLUBILIZATION AND FRACTIONATION

1977 ◽  
Vol 72 (2) ◽  
pp. 225-233 ◽  
Author(s):  
A. R. EASTMAN ◽  
A. M. NEVILLE
Keyword(s):  
Molecular Weight ◽  
Adrenal Glands ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Hydroxysteroid Dehydrogenase ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Microsomal Membranes ◽  
Molecular Sizes ◽  
Triton X

SUMMARY Protein moieties of various molecular sizes and possessing 5-ene-3β-hydroxysteroid dehydrogenase activity have been successfully solubilized from the microsomal membranes of both bovine and human adrenal glands using a combination of Triton X-100 and sonication. These moieties have been studied by gel filtration, sucrose density gradient centrifugation and isoelectric focusing, and were shown to possess a minimum molecular weight of about 118000, with an isoelectric point between 7·2 and 7·4. The molecular weight was dependent upon the concentration of Triton X-100 used during fractionation. No separation of dehydrogenase activities toward the three steroid substrates, pregnenolone, 17α-hydroxypregnenolone and dehydroisoandrosterone, was observed. Changes in the relative activities for the steroid substrates during fractionation were observed, but have been attributed to the formation of allotypes rather than to the existence of separate dehydrogenases with restricted substrate specificity.

scholarly journals Membrane-associated actin from the microvillar membranes of ascites tumor cells.

1982 ◽  
Vol 94 (3) ◽  
pp. 624-630 ◽  
Author(s):  
K L Carraway ◽  
R F Cerra ◽  
G Jung ◽  
C A Carraway
Keyword(s):  
Membrane Fraction ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Gel Filtration ◽  
Sucrose Density Gradient ◽  
Membrane Preparation ◽  
Intermediate Form ◽  
Sucrose Density Gradient Centrifugation ◽  
Transmission Electron ◽  
Triton X

A membrane fraction (MF2) has been purified from isolated microvilli of the MAT-C1 subline of the 13762 rat mammary ascites adenocarcinoma under conditions which cause F-actin depolymerization. This membrane preparation contains actin as a major component, although no filamentous structures are observed by transmission electron microscopy. Membranes were extracted with a Triton X-100-containing actin-stabilizing buffer (S buffer) or actin-destabilizing buffer (D buffer). In D buffer greater than 90% of metabolically labeled protein and glycoprotein was extracted, and 80-90% of these labeled species was extracted in S buffer. When S buffer extracts of MF2 were fractionated by either gel filtration on Sepharose 6 B or rate-zonal sucrose density gradient centrifugation, most of the actin was found to be intermediate in size between G- and F-actin. In D buffer most of the MF2 actin behaved as G-actin. Extraction and gel filtration of intact microvilli in S buffer also showed the presence of the intermediate form of actin, indicating that it did not arise during membrane preparation. When [35S]methionine-labeled G-actin from ascites cells was added to S buffer extracts of MF2 and chromatographed, all of the radioactivity chromatographed as G-actin, indicating that the intermediate form of actin did not result from an association of G-actin molecules during extraction or chromatography. The results of this study suggest that the microvillar membrane fraction is enriched in an intermediate form of actin smaller than F-actin and larger than G-actin.

scholarly journals The intracellular localization of glycollate oxidoreductase in Euglena gracilis

1971 ◽  
Vol 124 (2) ◽  
pp. 275-281 ◽  
Author(s):  
J. M. Lord ◽  
M. J. Merrett
Keyword(s):  
De Novo ◽  
Specific Activity ◽  
Gradient Centrifugation ◽  
Intracellular Localization ◽  
Sucrose Density Gradient ◽  
Enzyme Synthesis ◽  
Sucrose Density Gradient Centrifugation ◽  
Cell Extracts ◽  
Ionic Detergent ◽  
Triton X

1. Lowering of the concentration of carbon dioxide in air available to phototrophically growing Euglena cultures from 5% to the normal value (0.03%) resulted in an increased specific activity of glycollate oxidoreductase. 2. The effects of chloramphenicol and cycloheximide suggested that this increase in activity was due to enzyme synthesis de novo on cytoplasmic ribosomes. 3. The Km for glycollate oxidation by the enzyme in crude cell extracts was 3.0×10−3m. 4. Differential centrifugation established that glycollate oxidoreductase present in phototrophically grown Euglena is a particulate enzyme. The enzyme was partially solubilized by the non-ionic detergent Triton X-100. 5. Sucrose-density-gradient centrifugation achieved the separation of the particulate glycollate oxidoreductase from chloroplasts and mitochondria. 6. Glutamate–glyoxylate aminotransferase was associated with particulate glycollate oxidoreductase.

scholarly journals Purification of the G-protein G13 from rat brain membranes

1994 ◽  
Vol 303 (1) ◽  
pp. 135-140 ◽  
Author(s):  
R Harhammer ◽  
B Nürnberg ◽  
K Spicher ◽  
G Schultz
Keyword(s):  
Rat Brain ◽  
G Protein ◽  
G Proteins ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Sucrose Density Gradient Centrifugation ◽  
Brain Membranes ◽  
Rat Brain Membranes

Significant amounts of G13, a member of the recently described G12-subfamily of heterotrimeric G-proteins, have been detected in rat brain membranes by specific antisera. The alpha-subunits of G13 (G alpha 13) were purified by using a combination of conventional and subunit-exchange chromatography. Purification was facilitated by the fact that the initial anion-exchange chromatography separated G13 from most of the other G-proteins, including Gq/11. Moreover, G alpha 13-enriched fractions obtained from this chromatographic step were devoid of beta gamma-dimers, despite the absence of G-protein-activating agents. Nevertheless, the purified G alpha 13 retained its ability to interact with beta gamma-dimers under appropriate conditions, i.e. the addition of Lubrol PX instead of cholate as detergent and the omission of ethylene glycol routinely used as a protecting additive. The interaction was demonstrated by (i) the binding of G alpha 13 to immobilized beta gamma-complexes and (ii) the formation of stable heterotrimers during sucrose-density-gradient centrifugation. Furthermore, our studies on G alpha 13 provide evidence for an extremely slow basal GDP/GTP exchange rate. The purified protein showed negligible binding of guanosine 5′-[gamma-[35S]thio]triphosphate (GTP[35S]). Accordingly, dissociation of G alpha 13 from immobilized beta gamma-complexes was achieved by AlF4-/Mg2+, but not by GTP[S]. These data indicate that G13 exhibits properties highly distinct from those of other G-proteins.

scholarly journals SELECTIVE RELEASE OF CONTENT FROM MICROSOMAL VESICLES WITHOUT MEMBRANE DISASSEMBLY

1973 ◽  
Vol 58 (2) ◽  
pp. 436-462 ◽  
Author(s):  
Gert Kreibich ◽  
Pascale Debey ◽  
David D. Sabatini
Keyword(s):  
Gradient Centrifugation ◽  
Membrane Vesicles ◽  
Sucrose Density Gradient ◽  
Transport Systems ◽  
Secretory Proteins ◽  
Sucrose Density Gradient Centrifugation ◽  
Detergent Concentration ◽  
Doc Concentration ◽  
Microsomal Membranes

Rat liver rough microsomes treated with a series of desoxycholate (DOC) concentrations from 0.003 to 0.4% were analyzed by isopycnic sucrose density gradient centrifugation in media containing high or low salt concentrations. Tritium-labeled precursors administered in vivo were used as markers for ribosomes (orotic acid, 40 h), phospholipids (choline, 4 h), membrane proteins (leucine, 3 days), and completed secretory proteins of the vesicular cavity (leucine, 30 min). Within a narrow range of DOC concentrations (0.025–0.05%), the vesicular polypeptides were selectively released from the microsomes, while ribosomes, nascent polypeptides, and microsomal enzymes of the electron transport systems were unaffected. The detergent concentration which led to leakage of content was a function of the ionic strength and of the microsome concentration. At the lowest effective DOC concentration the microsomal membranes became reversibly permeable to macromoles as shown by changes in the density of the vesicles in Dextran gradients and by the extent of proteolysis by added proteases. Incubation of rough microsomes with proteases in the presence of 0.025% DOC also led to digestion of proteins from both faces of the microsomal membranes and to a lighter isopycnic density of the membrane vesicles.

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