scholarly journals ISOLATION AND SOME CHARACTERISTICS OF CELL NUCLEI FROM BRAIN CORTEX OF ADULT CAT

1965 ◽  
Vol 26 (2) ◽  
pp. 383-393 ◽  
Author(s):  
A. A. Hadjiolov ◽  
Z. S. Tencheva ◽  
A. G. Bojadjieva-Mikhailova
Keyword(s):  
Glial Cells ◽  
High Speed ◽  
Brain Cortex ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Nuclear Fraction ◽  
Cell Nuclei ◽  
Sucrose Density Gradient Centrifugation ◽  
Ionic Detergent ◽  
Cat Brain

A rapid detergent method for the isolation of nuclei from cat brain cortex is described. It involves the homogenization of the tissue in buffered 0.34 M sucrose with the addition of the non-ionic detergent Cemulsol NPT 12 and the subsequent low speed centrifugal sieving of the nuclei through two layers of sucrose (0.68 M and 1.0 M). The final purification is achieved by high speed centrifugation (40,000 g) of the nuclear suspension layered over 1.8 M sucrose. Observations by light microscopy indicate that highly purified and well preserved nuclei are obtained from neurons and glial cells. Electron microscopy reveals some microsomal contaminants adhering to the nuclear membrane. According to an analysis of the nuclear size distribution, a considerable loss of smaller nuclei (10 to 20µ2), mainly from glial cells, occurs during the purification procedure. The action of different detergents is compared, the best results being obtained with Cemulsol NPT 12 or Triton X-100. Chemical analyses of the purified nuclear fraction give the following content expressed in picograms per nucleus: DNA, 6.54; RNA, 2.94; cholesterol, 1.50; and protein, 97.5. The sucrose density gradient centrifugation of nuclei isolated from cat brain cortex shows that their density is equal to or higher than that of 2.2 M sucrose and is thus similar to the density of nuclei from other tissues. The observation of a varying influence of different suspending media on the density of brain cell nuclei resolves the conflicting data in the literature on the density of these nuclei.

scholarly journals Uridine diphosphate glucose-sterol glucosyltransferase and nucleoside diphosphatase activities in etiolated pea seedlings

1978 ◽  
Vol 169 (2) ◽  
pp. 297-303 ◽  
Author(s):  
M J Staver ◽  
K Glick ◽  
D J Baisted
Keyword(s):  
Density Gradient ◽  
High Speed ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Particulate Fraction ◽  
Partial Recovery ◽  
Sucrose Density Gradient Centrifugation ◽  
Freeze Thaw ◽  
Pea Seedlings ◽  
Nucleoside Diphosphatase

1. UDP-glucose-sterol glucosyltransferase and nucleoside diphosphatases were isolated in a particulate fraction from 7-day-old etiolated pea seedlings. The glucosyltransferase and UDPase (uridine diphosphatase) are stimulated by Ca2+ cation, less so by Mg2+ cation, and inhibited by Zn2+. 2. Each activity has a pH optimum near 8. 3. The glucosyltransferase is specific for UDP-glucose as the glucosyl donor and is inhibited by UDP. Partial recovery from UDP inhibition is effected by preincubation of the enzyme. 4. Freeze-thaw treatment and subsequent sucrose-density-gradient centrifugation of the particulate fraction shows the glucosyltransferase to be widely distributed among cell fractions but to be most active in particles with a density of 1.15 g/ml. UDPase is most active in particulate material with a density of over 1.18 g/ml but an activity peak also appears at 1.15 g/ml. Of several nucleoside diphosphatase activities, UDPase activity is most enhanced by the freeze-thaw and sucrose-density-gradient-fractionation procedures. 5. Detergent treatment with 0.1% sodium deoxycholate allows the partial solubilization of the glucosyltransferase and UDPase. The two activities are similarly distributed between pellet and supernatant after high-speed centrifugation for two different time intervals. 6. A role for UDPase in the functioning of glucosylation reactions is discussed.

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 Effect of cations on structure-linked sedimentability of lysosomal hydrolases

1968 ◽  
Vol 109 (1) ◽  
pp. 149-154 ◽  
Author(s):  
M. Anthony Verity ◽  
R. Caper ◽  
W. Jann Brown
Keyword(s):  
High Speed ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Lysosomal Hydrolases ◽  
Freezing And Thawing ◽  
Sucrose Density Gradient Centrifugation ◽  
Bivalent Cation ◽  
Repeated Freezing ◽  
Triton X ◽  
Total Enzyme

1. A partially purified lysosomal preparation was obtained from mouse liver sucrose homogenates by differential and discontinuous gradient centrifugation. 2. Triton X-100 or repeated freezing and thawing of the lysosomal suspension (subfraction B) allowed comparison of free and activated values for acid phosphohydrolase, β-glucuronidase and N-acetylglucosaminidase in the presence and absence of ascorbate. 3. The distribution of hydrolase activities between supernatant and pellet after high-speed centrifugation was measured and the percentages of total enzyme found in the supernatant were: acid phosphohydrolase, 40·7; β-glucuronidase, 51; N-acetylglucosaminidase, 39·4. 4. Differential rates of elution of the three hydrolases from the membrane fraction occurred with increasing Na+ and K+ concentrations, whereas complex biphasic elution curves were obtained as a function of bivalent cation concentration with Ca2+ and Mg2+. 5. Sucrose-density-gradient centrifugation of frozen-and-thawed subfraction B demonstrated highly significant changes in the protein gradient profile in the presence of a low concentration of bivalent cation, indicating membrane aggregation and enzyme–membrane association. 6. The data provide further evidence for the nature of lysosomal enzyme binding and indicate the presence of different enzyme–membrane bonds conferring structure-linked latency upon individual lysosomal enzymes.

Purification and properties of mycoferritin from Mortierella alpina

1972 ◽  
Vol 18 (5) ◽  
pp. 619-622 ◽  
Author(s):  
R. F. Bozarth ◽  
A. Goenaga
Keyword(s):  
High Speed ◽  
Analytical Ultracentrifugation ◽  
Gradient Centrifugation ◽  
Potassium Phosphate ◽  
Sucrose Density Gradient ◽  
Mortierella Alpina ◽  
Protein Subunit ◽  
High Concentration ◽  
Sucrose Density Gradient Centrifugation ◽  
Purification And Properties

A culture of Mortierella alpina Peyroud isolated from soil and grown on Czapek-Dox medium was found to contain a high concentration of mycoferritin (MF). The MF was extracted from lyophilized mycelium by grinding with 0.1 M, pH 7.0, potassium-phosphate buffer and chloroform followed by differential centrifugation. Sucrose density-gradient centrifugation of the concentrated MF resulted in a brownish-yellow band in the region of 60–70 S. Following dialysis and concentration by high-speed centrifugation, the MF was compared to horse-spleen ferritin (HF). The ultraviolet (uv.) and infrared (i.r.) spectra of MF and HF were identical. Negatively stained preparations examined in the electron microscope showed particles of about 10 nm diameter. Sedimentation rates of S20,w = 66 for MF and S20,w = 56 for HF were obtained by analytical ultracentrifugation. The MF preparation contained 83% protein and 17% iron. The molecular weight of the protein subunit was determined by gel electrophoresis to be about 19 300 daltons.

A significant soluble keratin fraction in ‘simple’ epithelial cells. Lack of an apparent phosphorylation and glycosylation role in keratin solubility

1993 ◽  
Vol 105 (2) ◽  
pp. 433-444 ◽  
Author(s):  
C.F. Chou ◽  
C.L. Riopel ◽  
L.S. Rott ◽  
M.B. Omary
Keyword(s):  
Cell Cycle ◽  
High Speed ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Cytosolic Fraction ◽  
Epithelial Cell Line ◽  
Sucrose Density Gradient Centrifugation ◽  
Alternate Mechanism ◽  
10 Nm Filaments

We studied the solubility of keratin polypeptides 8 and 18 (K8/18), which are the predominant intermediate filaments in the human colonic epithelial cell line HT29. We find that asynchronously growing cells (G0/G1 stage of the cell cycle) have a substantial pool of soluble keratin that constitutes approx. 5% of total cellular keratin. This soluble keratin pool was observed after immunoprecipitation of K8/18 from the cytosolic fraction of cells disrupted using three detergent-free methods. Several other cell lines showed a similar significant soluble cytosolic K8/18 pool. Arrest of HT29 cells in G2/M stage of the cell cycle was associated with a concurrent increase in keratin solubility. Comparison of K8/18 obtained from the soluble cytosolic fraction and the insoluble high-speed pellet fraction showed similar levels of phosphorylation and glycosylation and similar tryptic radiolabeled phospho- and glycopeptide patterns. Soluble K8/18 can form characteristic 10 nm filaments in vitro as determined by electron microscopy. Cross-linking of soluble K8/18 followed by immunoprecipitation resulted in dimeric and tetrameric forms, based on migration in SDS-polyacrylamide gels. In addition, cross-linked and native soluble K8/18 showed similar migration on nondenaturing gels and similar sedimentation after sucrose density gradient centrifugation. Our results indicate that simple epithelial keratins are appreciably more soluble than previously recognized. The soluble keratin form is assembly competent and appears to be primarily tetrameric. Although K8/18 solubility was found to increase during mitotic arrest, glycosylation and phosphorylation did not play an obvious role in generating the soluble fraction, suggesting an alternate mechanism for keratin solubility.

scholarly journals Human placental coated vesicles contain receptor-bound transferrin

1981 ◽  
Vol 196 (1) ◽  
pp. 355-362 ◽  
Author(s):  
A G Booth ◽  
M J Wilson
Keyword(s):  
Wheat Germ ◽  
Serum Proteins ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Coated Vesicles ◽  
Coated Vesicle ◽  
Sucrose Density Gradient Centrifugation ◽  
Crossed Immunoelectrophoresis ◽  
Ionic Detergent

Human placental coated vesicles have been purified by a method involving sucrose-density-gradient centrifugation and treatment with wheat-germ agglutinin. These preparations were free of contamination by placental microvillus fragments. Crossed immunoelectrophoresis demonstrated that the coated vesicles contained a single serum protein, which was identified as transferrin. This transferrin was only observed after the vesicles were treated with a non-ionic detergent, and its behaviour during crossed hydrophobic-interaction immunoelectrophoresis suggested that a large proportion of it was receptor-bound. No other serum proteins, including immunoglobulin G, could be detected in these preparations. Receptor-bound transferrin was the only antigen common to placental coated vesicles and microvilli, implying that other plasma-membrane proteins are excluded from the region of membrane involved in coated-vesicle formation.

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 Fractionation of rat ventricular nuclei

1980 ◽  
Vol 188 (2) ◽  
pp. 363-373 ◽  
Author(s):  
G Jackowski ◽  
C C Liew
Keyword(s):  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sodium Dodecyl ◽  
Sucrose Density Gradient ◽  
Myocardial Cell ◽  
Myocardial Cells ◽  
Cell Nuclei ◽  
Sucrose Density Gradient Centrifugation ◽  
Ventricular Tissue

Myocardial cells were isolated after treatment with collagenase (0.05%) and hyaluronidase (0.1%) by discontinuous-gradient centrifugation on 3% Ficoll. Nuclei derived from these myocardial cells were then fractionated on a discontinuous sucrose density gradient with the following steps: (I) 2.0M/2.3M, (II) 2.3M/2.4M, (III) 2.4M/2.5M, (IV) 2.5M/2.6M, and (V) 2.6M/2.85M. The myocardial nuclei were sedimented in the interfaces of gradient fractions (II) and (III). Nuclei from whole ventricles that had been treated with the enzymes before isolation sedimented into five major subsets of nuclei. These findings suggest that nuclei sedimented in the isopycnic gradient at fractions (II) and (III) are most probably derived from myocardial cells. However, this procedure is laborious and lengthy, and the recovery of myocardial-cell nuclei is low. An alternative method was developed to isolate an enriched fraction of myocardial-cell nuclei from whole ventricular tissue without exposing the tissues to enzyme digestion. These ventricular nuclei could be fractionated into five nuclear subsets by using the same discontinuous sucrose density gradient as that described above. The content of DNA, RNA and protein per nucleus for each band was determined. Although the DNA content per nucleus was constant (10pg), that of RNA varied from 1.5 to 4.5pg and that of protein from 16 to 24pg. Nuclei from each band were examined by light-microscopy: large nuclei occurred in the ligher regions whereas smaller nuclei were found in the denser regions of the gradient. From the size distribution pattern of myocardial-cell nuclei compared with that of total ventricular nuclei, it was found that nuclear subsets (II), (III), and (IV) were similar to myocardial nuclei. Electrophoretic analyses of the proteins solubilized in sodium dodecyl sulphate/phenol or Tris/EDTA/2-mercaptoethanol/phenol obtained from each nuclear subset indicate that these fractions are similar, with limited qualitative differences. These findings indicate that isolation of an enriched fraction of myocardial-cell nuclei could be achieved by discontinuous-sucrose-density-gradient centrifugation.

The Combining Ability of Glycoproteins IIb, IIIa and Ca2+ in EDTA-Treated Nonaggregable Platelets

1983 ◽  
Vol 50 (04) ◽  
pp. 848-851 ◽  
Author(s):  
Marjorie B Zucker ◽  
David Varon ◽  
Nicholas C Masiello ◽  
Simon Karpatkin
Keyword(s):  
Density Gradient ◽  
Combining Ability ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Electrophoretic Pattern ◽  
Sucrose Density Gradient ◽  
Irreversible Loss ◽  
Sucrose Density Gradient Centrifugation ◽  
Crossed Immunoelectrophoresis ◽  
Triton X

SummaryPlatelets deprived of calcium and incubated at 37° C for 10 min lose their ability to bind fibrinogen or aggregate with ADP when adequate concentrations of calcium are restored. Since the calcium complex of glycoproteins (GP) IIb and IIIa is the presumed receptor for fibrinogen, it seemed appropriate to examine the behavior of these glycoproteins in incubated non-aggregable platelets. No differences were noted in the electrophoretic pattern of nonaggregable EDTA-treated and aggregable control CaEDTA-treated platelets when SDS gels of Triton X- 114 fractions were stained with silver. GP IIb and IIIa were extracted from either nonaggregable EDTA-treated platelets or aggregable control platelets with calcium-Tris-Triton buffer and subjected to sucrose density gradient centrifugation or crossed immunoelectrophoresis. With both types of platelets, these glycoproteins formed a complex in the presence of calcium. If the glycoproteins were extracted with EDTA-Tris-Triton buffer, or if Triton-solubilized platelet membranes were incubated with EGTA at 37° C for 30 min, GP IIb and IIIa were unable to form a complex in the presence of calcium. We conclude that inability of extracted GP IIb and IIIa to combine in the presence of calcium is not responsible for the irreversible loss of aggregability that occurs when whole platelets are incubated with EDTA at 37° C.

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