scholarly journals The rapid preparation of peroxisomes from rat liver by using a vertical rotor

1979 ◽  
Vol 180 (2) ◽  
pp. 445-448 ◽  
Author(s):  
C E Neat ◽  
H Osmundsen
Keyword(s):  
Fatty Acid ◽  
Rat Liver ◽  
Fatty Acid Metabolism ◽  
Acid Metabolism ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Rapid Preparation ◽  
Vertical Rotor

A method is described for the rapid preparation of peroxisomes from rat liver by using sucrose-density-gradient centrifugation in a vertical rotor. The preparation, shown to be virtually free of mitochondrial and microsomal contamination, can be used to study fatty acid metabolism by isolated peroxisomes.

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 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°.

scholarly journals GOLGI FRACTIONS PREPARED FROM RAT LIVER HOMOGENATES

1973 ◽  
Vol 59 (1) ◽  
pp. 45-72 ◽  
Author(s):  
J. H. Ehrenreich ◽  
J. J. M. Bergeron ◽  
P. Siekevitz ◽  
G. E. Palade
Keyword(s):  
Rat Liver ◽  
Microsomal Fraction ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation ◽  
Liver Homogenates ◽  
Main Component

In devising a new procedure for the isolation of Golgi fractions from rat liver homogenates, we have taken advantage of the overloading with very low density lipoprotein (VLDL) particles that occurs in the Golgi elements of hepatocytes ∼90 min after ethanol is administered (0.6 g/100 g body weight) by stomach tube to the animals. The VLDLs act as morphological markers as well as density modifiers of these elements. The starting preparation is a total microsomal fraction prepared from liver homogenized (1:5) in 0.25 M sucrose. This fraction is resuspended in 1.15 M sucrose and loaded at the bottom of a discontinuous sucrose density gradient. Centrifugation at ∼13 x 106 g·min yields by flotation three Golgi fractions of density >1.041 and <1.173. The light and intermediate fractions consist essentially of VLDL-loaded Golgi vacuoles and cisternae. Nearly empty, often collapsed, Golgi cisternae are the main component of the heavy fraction. A procedure which subjects the Golgi fractions to hypotonic shock and shearing in a French press at pH 8.5 allows the extraction of the content of the Golgi elements and the subsequent isolation of their membranes by differential centrifugation.

The Identification of Proinsulin-Synthesizing Polysomes in Fetal Bovine Pancreas

1974 ◽  
Vol 52 (11) ◽  
pp. 959-965 ◽  
Author(s):  
Choy-L. Hew ◽  
C. C. Yip
Keyword(s):  
Density Gradient ◽  
Rat Liver ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Rnase A ◽  
Sucrose Density Gradient Centrifugation ◽  
Fetal Bovine ◽  
Pancreatic Rnase ◽  
Bovine Pancreas

Antiserum against bovine pancreatic RNase A was produced in the rabbit. (Fab′)2 fragments were prepared from the antiserum and were found to inhibit the degradation of rat liver polysomes by fetal bovine pancreatic supernatant which contained RNase activity. Using this anti-RNase preparation, we were able to obtain fetal bovine pancreatic polysomes which appeared intact. Radioimmunoassay of the polysomal fractions after sucrose density gradient centrifugation showed that insulin immunoreactivity was associated mainly with tetrasomes, pentasomes, and some larger polysomal aggregates, suggesting the participation of these polysomes in the synthesis of proinsulin. The possibility that a precursor larger than proinsulin might be involved in the synthesis of insulin was discussed.

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.

3 ß-Hydroxysteroid Oxidoreductase in Suspension Cultures of Digitalis lanata EH RH

1990 ◽  
Vol 45 (9-10) ◽  
pp. 963-972 ◽  
Author(s):  
Hildegard Maria Warneck ◽  
Hanns Ulrich Seitz
Keyword(s):  
Incubation Temperature ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Cell Compartment ◽  
Sucrose Density Gradient Centrifugation ◽  
Ph Optimum ◽  
Digitalis Lanata ◽  
Substrate Kinetics ◽  
Soluble Part

Abstract A 3 β-hydroxysteroid oxidoreductase was isolated and characterized in the microsomes of Digitalis lanata cell cultures. The enzyme catalyzes the conversion of 5α-pregnane-3,20-dione to 5a-pregnan-3 β-ol-20-one and requires NAD(P)H2. The enzyme was found to have a pH optimum of 80. The reaction had an optimum incubation temperature of 25 °C with linear reduction for the first 4 h, reaching maximum enzyme activity after 7 h. Substrate kinetics for 5a-pregnane-3,20-dione and NADPH2 resulted in apparent Km-values of 18.5-20 (µM for 5a-pregnane-3,20-dione and 50-120 µM for the co-substrate NADPH2. In order to localize 3β-hydroxysteroid oxidoreductase differential centrifugation as well as linear sucrose density gradient centrifugation were performed. The results obtained lead to the conclusion that 3β-hydroxysteroid oxidoreductase is not associated with a single cell compartment, but consists of a major soluble part and a markedly smaller part of endoplasmic reticulum-associated activity

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