scholarly journals A comparison of methods for extracting ribonucleic acid polymerases from rat liver nuclei

1979 ◽  
Vol 183 (1) ◽  
pp. 43-54 ◽  
Author(s):  
T J C Beebee
Keyword(s):  
Molecular Weight ◽  
Ionic Strength ◽  
Rna Polymerase Ii ◽  
Rat Liver ◽  
High Ionic Strength ◽  
Rna Polymerases ◽  
Comparison Of Methods ◽  
Optimal Conditions ◽  
Liver Nuclei ◽  
Enzyme Molecules

Nuclei were prepared from rat liver after homogenization of the tissue in hyperosmotic sucrose and RNA polymerases (EC 2.7.7.6) extracted by two methods applied sequentially. Optimal conditions for washing loosely bound enzymes out of nuclei were determined first, and involved short (10 min) incubations at 0 degrees C in the presence of 5 mM-Mg2+ and 60 mM-(NH4)2SO4. Subsequent sonication of the residual nuclear pellet after resuspension and lysis at high ionic strength resulted in further release of RNA polymerases. The primary wash yielded about 2 × 10(4) molecules of RNA polymerases I and III (altogether) and 1 × 10(4) molecules of form-II enzymes per original nucleus, whereas subsequent sonication released 2 × 10(4)-2.5 × 10(4) form-I and -III enzyme molecules (altogether) and a further 7 × 10(3)-8 × 10(3) form-II enzyme molecules, as measured by end-labelling of nascent RNA. RNA polymerase II was partially purified from both types of extracts and shown to initiate very poorly on high-molecular-weight homologous DNA irrespective of the source of the enzyme.

scholarly journals Experimental conditions affecting ribonucleic acid polymerase in isolated rat liver nuclei. Effect of nucleoside triphosphate concentration, temperature, ammonium sulphate and heparin

1969 ◽  
Vol 112 (5) ◽  
pp. 721-727 ◽  
Author(s):  
F. Novello ◽  
F. Stirpe
Keyword(s):  
Ionic Strength ◽  
Rna Polymerase ◽  
Ammonium Sulphate ◽  
Rat Liver ◽  
High Ionic Strength ◽  
Isolated Rat Liver ◽  
Rat Liver Nuclei ◽  
Liver Nuclei ◽  
Low Ionic Strength ◽  
Isolated Rat

1. The conditions affecting the activity of RNA polymerase in isolated rat liver nuclei were studied with Mg2+ or Mn2+ as activating ions. 2. The enzyme assayed with Mg2+ and at low ionic strength is saturated by a lower concentration of nucleotide substrates than if assayed with Mn2+ at low ionic strength or with either ion at high ionic strength. 3. At low and at high ionic strength the incorporation of AMP is affected in a similar way by variations in the temperature of incubation. Preincubation at 37° impairs the AMP incorporation. 4. Heparin stimulates the RNA polymerase activity in the presence of Mn2+. 5. Both ammonium sulphate and heparin ‘restart’ the reaction if added after 15min., the effect being more marked with ammonium sulphate than with heparin, and also more marked in the presence of Mn2+ than of Mg2+. 6. α-Amanitin abolishes the effect of ammonium sulphate and of heparin.

scholarly journals Nucleic acid enzymology of extremely halophilic bacteria. Halobacterium cutirubrum polynucleotide phosphorylase

1971 ◽  
Vol 121 (4) ◽  
pp. 613-620 ◽  
Author(s):  
Pearl I. Peterkin ◽  
P. S. Fitt
Keyword(s):  
Molecular Weight ◽  
Ionic Strength ◽  
Nucleic Acid ◽  
Halophilic Bacteria ◽  
High Ionic Strength ◽  
Low Molecular Weight ◽  
Polynucleotide Phosphorylase

1. Polynucleotide phosphorylase was purified 200-fold from Halobacterium cutirubrum. 2. It is membrane-associated and can be solubilized by sonication. 3. The purified enzyme requires a high ionic strength for both stability and activity. 4. It is Mn2+-dependent, has all three typical polynucleotide phosphorylase activities and is specific for nucleoside diphosphates. 5. The enzyme is of low molecular weight.

scholarly journals The mitochondrial localization of coproporphyrinogen III oxidase

1978 ◽  
Vol 176 (1) ◽  
pp. 97-102 ◽  
Author(s):  
B Grandchamp ◽  
N Phung ◽  
Y Nordmann
Keyword(s):  
Ionic Strength ◽  
Rat Liver ◽  
Membrane Binding ◽  
High Ionic Strength ◽  
Alkaline Solutions ◽  
Intermembrane Space ◽  
Mitochondrial Localization ◽  
Inner Membrane ◽  
Membrane Matrix ◽  
Coproporphyrinogen Iii Oxidase

The location of coproporphyrinogen III oxidase in mitochondria was studied in rat liver by using the digitonin method or hypo-osmotic media for fractionation. The enzyme was found in the intermembrane space with a fraction loosely bound to the inner membrane. This fraction was released by washing the inner-membrane-matrix complex with alkaline solutions or solutions of high ionic strength. The enzyme in both fractions had the same Km (0.16 micrometer) for coproporphyrinogen III. When incubation was performed in a medium that avoided destruction of enzyme membrane binding, a dramatic increase in activity was observed after sonication of whole mitochondria or of the inner-membrane-matrix complex.

Dissociation of Factor VIII in the Presence of Proteolytic Inhibitors

1978 ◽  
Vol 40 (02) ◽  
pp. 316-325 ◽  
Author(s):  
Ira I Sussman ◽  
Harvey J Weiss
Keyword(s):  
Molecular Weight ◽  
Ionic Strength ◽  
Factor Viii ◽  
Direct Evidence ◽  
Gel Filtration ◽  
High Ionic Strength ◽  
Procoagulant Activity ◽  
Low Molecular Weight ◽  
Bean Trypsin Inhibitor ◽  
Benzamidine Hydrochloride

SummaryWhen gel filtration of factor VIII is performed with buffers of high ionic strength (1.0 M NaCl or 0.25 M CaCl2), the procoagulant activity elutes with proteins of relatively low molecular weight. It has been suggested that in the presence of proteolytic inhibitors, the procoagulant activity would appear at the void volume. To test this hypothesis, chromatography with buffers of high ionic strength was performed in the presence of benzamidine hydrochloride, soy bean trypsin inhibitor, heparin, DFP, and aprotinin. Under all of these conditions, the procoagulant activity continued to elute with proteins of low molecular weight. Similar findings were obtained after chromatographing cryoprecipitate prepared from the plasma of a normal subject who had received heparin. Thus, at present there is no direct evidence to suggest that proteolysis is involved in the dissociation of factor VIII by buffers of high ionic strength.

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