scholarly journals A simple method of characterizing mitochondrial ribonucleic acid.

1975 ◽  
Vol 147 (3) ◽  
pp. 621-623
Author(s):  
D Haldar ◽  
B K Waters ◽  
D B Freeman
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Ribonucleic Acid ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sodium Dodecyl ◽  
Sucrose Density Gradient ◽  
Mitochondrial Fraction ◽  
Proteinase K ◽  
Sucrose Density Gradient Centrifugation ◽  
Simple Method

A simple method of isolating and characterizing RNA from L-cell mitochondria is described. The mitochondrial fraction is lysed by sodium dodecyl sulphate, and the RNA fractionated by sucrose-density-gradient centrifugation. The efficacy of proteinase K in preventing ribonuclease activity is also demonstrated.

scholarly journals Appearance of a methylated ribonucleic acid on illumination of Euglena gracillis cells grown in the dark

1971 ◽  
Vol 125 (2) ◽  
pp. 401-405 ◽  
Author(s):  
M. Perl
Keyword(s):  
Molecular Weight ◽  
Nucleic Acid ◽  
High Molecular Weight ◽  
Ribonucleic Acid ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Acid Fraction ◽  
Sucrose Density Gradient Centrifugation ◽  
Exclusion Chromatography

Investigation of the methylation of nucleic acids by [Me-3H]methionine after illumination of Euglena cells grown in the dark has shown that a high-molecular-weight nucleic acid fraction undergoes methylation after exposure to light for 60–120min. This methylated nucleic acid fraction was isolated both by sucrose-density-gradient centrifugation and exclusion chromatography on Sephadex G-200. The fraction was shown to consist of a preformed RNA that is present in cells grown in the dark and which on illumination is transmethylated by methionine.

scholarly journals FORMATION OF MITOCHONDRIA IN NEUROSPORA CRASSA

1963 ◽  
Vol 16 (3) ◽  
pp. 483-499 ◽  
Author(s):  
David J. L. Luck
Keyword(s):  
Neurospora Crassa ◽  
Density Gradient ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Continuous Process ◽  
Sucrose Density Gradient ◽  
Mitochondrial Fraction ◽  
Sucrose Density Gradient Centrifugation ◽  
Mitochondrial Mass ◽  
Logarithmic Growth

Cells of a choline-requiring mutant of Neurospora crassa, labeled with radioactive choline, were transferred to unlabeled medium. At various times during their subsequent logarithmic growth, a highly purified mitochondrial fraction was prepared by sucrose density gradient centrifugation, and the distribution of label among individual mitochondria was determined by quantitative autoradiography. Preliminary experiments indicated that, under the conditions of this "washout" experiment, choline served as a stable mitochondrial label. Radioautographic analysis showed that, in fully labeled mycelia and for three mass doubling cycles in the unlabeled medium, radioactivity was randomly distributed among all mitochondria; i.e., the distribution of autographic grains among individual mitochondria followed a Poisson distribution. In experiments in which pulse labeling for 10 minutes was used, the label was randomly distributed among all mitochondria. The data suggest that the mitochondrial mass is increased by a continuous process of addition of new lecithin units to the already existing mitochondrial framework.

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.

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 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.

scholarly journals Studies on a nucleoprotein prepared from rat liver polysomes by digestion with T1 ribonuclease

1969 ◽  
Vol 113 (4) ◽  
pp. 643-650 ◽  
Author(s):  
A. O. Hawtrey
Keyword(s):  
Density Gradient ◽  
Rat Liver ◽  
Magnesium Chloride ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sodium Dodecyl ◽  
Sucrose Density Gradient ◽  
Transfer Rna ◽  
Sucrose Density Gradient Centrifugation ◽  
Transfer Factors

1. Treatment of rat liver polysomes in a buffer containing 2·5mm-magnesium chloride with T1 ribonuclease at a concentration of 330units/ml. of reaction medium at 37° for 2hr. leads to the production of an insoluble nucleoprotein. 2. On the bases of analysis for protein and RNA and of u.v.-absorption spectra the nucleoprotein appears to have lost approx. 60% of the structural RNA originally present in the ribosome. Degradation of 3H-labelled polysomes (structural RNA labelled with orotic acid) with T1 ribonuclease leads to nucleoprotein preparations retaining approx. 30% of the radioactivity originally present in the polysomes. By means of sucrose-density-gradient centrifugation it is shown that the nucleoprotein preparations are free of single 73s ribosomes and ribosomal subunits. No evidence for the presence of 28s and 18s structural RNA was obtained on examination of extracted nucleoprotein-particle RNA by means of sucrose-density-gradient centrifugation. 3. Digestion of washed polysomes carrying 14C-labelled nascent peptide chains with T1 ribonuclease gives a nucleoprotein particle that retains approx. 70% of the original labelled chains. Treatment of labelled nucleoprotein particles with 1mm-puromycin in the absence of transfer factors releases 20% of the labelled chains. Addition of GTP (0·48μmole) increases this release to 37%. 4. Treatment of nucleoprotein particles carrying 14C-labelled peptide chains with either EDTA (50mm) or ammonium chloride (0·5m) brings about a small release of labelled material (approx. 15%). 5. Disruption of nucleoprotein particles carrying 14C-labelled peptide chains with either sodium dodecyl sulphate or 2m-lithium chloride, followed by addition of transfer RNA as marker and chromatography on Sephadex G-200, show in both cases that considerable amounts of labelled peptide material move well ahead of the added transfer RNA marker. Further, if nucleoprotein particles carrying labelled peptide chains are treated with 0·3m-potassium hydroxide at 20° for 24 hr., neutralized to pH7·6, and then chromatographed on Sephadex G-200, the labelled peptide material moves much closer to the added transfer RNA marker. These results suggest that a proportion of the nascent 14C-labelled peptides on the nucleoprotein are attached to transfer RNA or large fragments of transfer RNA. 6. [3H]Polyuridylic acid binds to nucleoprotein particles in 1mm-magnesium chloride. The rate of binding is rapid when measured at 20°.

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.

scholarly journals Alteration of hepatic glucocorticoid receptor stability and nuclear binding in vitro by citrate

1983 ◽  
Vol 210 (1) ◽  
pp. 259-263 ◽  
Author(s):  
J Hubbard ◽  
M Kalimi
Keyword(s):  
Glucocorticoid Receptor ◽  
Glucocorticoid Receptors ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Deae Cellulose ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Nuclear Binding ◽  
Wide Range

Citrate greatly stabilized rat hepatic unbound glucocorticoid receptors in cell-free conditions at 4 degrees C with optimal effectiveness at 5-15 mM. Control receptors were inactivated at 4 degrees C with a half-life of less than 12 h. However, in the presence of 10 mM-citrate, unbound receptors were almost completely stabilized for 48 h at 4 degrees C. Citrate at a concentration of 1-2 mM yielded half-maximal stabilization. The stabilizing effect of citrate was rather specific, as succinate, alpha-oxoglutarate, oxaloacetate, malate and pyruvate had no apparent stabilizing action. Citrate stabilized receptors over a wide range of H+ concentrations, with complete protection between pH 6.5 and 8.5. In addition, citrate appeared to have a significant effect on glucocorticoid-receptor complex activation into a nuclear binding form. Thus 5-10 mM-citrate enhanced nuclear binding, with optimal activation achieved at 10 mM concentration. As analysed by sucrose-density-gradient centrifugation and DEAE-cellulose chromatography, no apparent change was observed in the physical characteristics of the glucocorticoid receptor in the presence of citrate.

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.

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