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 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.

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 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 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, characterization and identification of rat liver histidine-pyruvate aminotransferase isoenzymes

1976 ◽  
Vol 155 (1) ◽  
pp. 107-115 ◽  
Author(s):  
T Noguchi ◽  
E Okuno ◽  
Y Minatogawa ◽  
R Kido
Keyword(s):  
Density Gradient ◽  
Rat Liver ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Sucrose Density Gradient ◽  
Aminotransferase Activity ◽  
Sucrose Density Gradient Centrifugation ◽  
Ph Optimum

1. Histidine-pyruvate aminotransferase (isoenzyme 1) was purified to homogeneity from the mitochondrial and supernatant fractions of rat liver, as judged by polyacrylamide-gel electrophoresis and isolectric focusing. Both enzyme preparations were remarkably similar in physical and enzymic properties. Isoenzyme 1 had pI8.0 and a pH optimum of 9.0. The enzyme was active with pyruvate as amino acceptor but not with 2-oxoglutarate, and utilized various aromatic amino acids as amino donors in the following order of activity: phenylalanine greater than tyrosine greater than histidine. Very little activity was found with tryptophan and 5-hydroxytryptophan. The apparent Km values were about 2.6mM for histidine and 2.7 mM for phenylalanine. Km values for pyruvate were about 5.2mM with phenylalanine as amino donor and 1.1mM with histidine. The aminotransferase activity of the enzyme towards phenylalanine was inhibited by the addition of histidine. The mol.wt. determined by gel filtration and sucrose-density-gradient centrifugation was approx. 70000. The mitochondrial and supernatant isoenzyme 1 activities increased approximately 25-fold and 3.2-fold respectively in rats repeatedly injected with glucagon for 2 days. 2. An additional histidine-pyruvate aminotransferase (isoenzyme 2) was partially purified from both the mitochondrial and supernatant fractions of rat liver. Nearly identical properties were observed with both preparations. Isoenzyme 2 had pI5.2 and a pH optimum of 9.3. The enzyme was specific for pyruvate and did not function with 2-oxoglutarate. The order of effectiveness of amino donors was tyrosine = phenylalanine greater than histidine greater than tryptophan greater than 5-hydroxytryptophan. The apparent Km values for histidine and phenylalanine were about 0.51 and 1.8 mM respectively. Km values for pyruvate were about 3.5mM with phenylalanine and 4.7mM with histidine as amino donors. Histidine inhibited phenylalanine aminotransferase activity of the enzyme. Gel filtration and sucrose-density-gradient centrifugation yielded a mol.wt. of approx. 90000. Neither the mitochondrial nor the supernatant isoenzyme 2 activity was elevated by glucagon injection.

scholarly journals Purification of the constituent enzyme fractions of mycobacillin synthetase

1986 ◽  
Vol 235 (1) ◽  
pp. 81-85 ◽  
Author(s):  
S K Ghosh ◽  
N K Mukhopadhyay ◽  
S Majumder ◽  
S K Bose
Keyword(s):  
Gel Electrophoresis ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Sucrose Density Gradient ◽  
Polyacrylamide Gel ◽  
Subunit Structure ◽  
Sucrose Density Gradient Centrifugation ◽  
Final Purification

The final purification of the three-fraction enzyme complex mycobacillin synthetase was done by hydroxyapatite column chromatography and sucrose-density-gradient centrifugation; each of the fractions obtained migrates as a single component in SDS/polyacrylamide-gel electrophoresis and gel electrofocusing. The Mr of the enzyme fractions A, B and C by gel filtration is 260 000, 190 000 and 105 000, and that by SDS/polyacrylamide-gel electrophoresis is 252 000, 198 000 and 108 000 respectively. None of the enzyme fractions appears to possess subunit structure.

Molecular characteristics of cytostatic factors in amphibian egg cytosols

Development ◽  
1989 ◽  
Vol 106 (4) ◽  
pp. 799-808
Author(s):  
E.K. Shibuya ◽  
Y. Masui
Keyword(s):  
Density Gradient ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Sedimentation Coefficient ◽  
Sucrose Density Gradient ◽  
Molecular Characteristics ◽  
Small Peptides ◽  
Sucrose Density Gradient Centrifugation ◽  
Cytostatic Factor

In amphibians, zygotes microinjected with cytosol of unactivated eggs are arrested at metaphase of mitosis. The factor responsible for this effect has been designated ‘cytostatic factor, (CSF)’. CSF is inactivated by Ca2+ addition to cytosols. During storage of the Ca(2+)-containing cytosols, a stable CSF activity develops. Therefore, the first Ca(2+)-sensitive CSF and the second Ca(2+)-insensitive CSF have been referred to as primary CSF (CSF-1) and secondary CSF (CSF-2), respectively. We have partially purified CSF-1, which had been stabilized with NaF and ATP, and CSF-2 from cytosols of Rana pipiens eggs by ammonium sulphate (AmS) precipitation and sucrose density gradient centrifugation or gel filtration, and investigated their molecular characteristics. CSF-1 was sensitive to protease, but resistant to RNAse, and inactivated within 2 h at 25 degrees C. CSF-1 could be sedimented in a sucrose density gradient from a fresh cytosol or its crude fraction precipitated at 20–30% saturation of AmS, showing the sedimentation coefficient 3S. When analyzed by SDS-polyacrylamide gel electrophoresis (PAGE), all the proteins in partially purified CSF-1 samples entered the gel and were separated into numerous peptide bands. In contrast, CSF-2 was an extremely large molecule, being eluted from Sepharose columns as molecules larger than 2 × 10(6), and failed to enter the gel when analyzed by SDS-PAGE. It could be purified 40 times from cytosols. CSF-2 was a highly stable molecule, being neither inactivated nor dissociated at pH 11.5 or by 4M-NaCl and LiCl and 8 M-urea. It was also resistant to RNAse treatment. However, CSF-2 could be broken down into small peptides of variable sizes by trypsin, alpha-chymotrypsin, and papain, but not by S. aureus V8 protease, although it was less sensitive to proteases than CSF-1. The dose-dependency test showed that the activity of CSF-2 is independent of its concentration and that an amount of CSF-2 could cause cleavage arrest earlier when injected into a blastomere in a larger volume.

Isolation and antigen analysis of surface tegument membranes from schistosomula ofSchistosoma mansoni

Parasitology ◽  
1984 ◽  
Vol 89 (3) ◽  
pp. 495-510 ◽  
Author(s):  
D. W. Taylor ◽  
Pamela Z. Wells
Keyword(s):  
Specific Activity ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sucrose Density Gradient ◽  
Two Dimensional ◽  
Sucrose Density Gradient Centrifugation ◽  
Western Blots ◽  
Peptide Composition ◽  
Membrane Antigens ◽  
Compare And Contrast

SUMMARYThe outer tegument membranes ofSchistosoma mansonischistosomula have been removed by mechanical disruption in a hypotonic salt solution and partially purified by differential and sucrose density gradient centrifugation. Fractionation was monitored by measuring increase in specific activity of bound [125I] wheat-germ agglutinin (WGA), alkaline phosphatase and calcium-stimulated ATPase. Two-dimensional IEF/SDS polyacrylamide gel electrophoresis was used to analyse the peptide composition of the isolated membranes and to compare and contrast with lactoperoxidase/glucose oxidase surface labelled peptides. At least 35 surface-labelled peptides were resolved on the two-dimensional maps: all were also present in the membrane material recovered from the sucrose gradient. Western blot analysis demonstrated a marked heterogeneity in the antibody response of infected human patients to individual membrane antigens. The antigenic profile of membranes isolated from cercariae, 18 and 96 h schistosomula were compared using Western blots: some minor differences were observed between the three preparations.

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.

Sign in / Sign up

Export Citation Format

Share Document