scholarly journals Effects of α-amanitin on the stimulation of prostatic ribonucleic acid polymerase by prostatic steroid–protein receptor complexes

1974 ◽  
Vol 140 (3) ◽  
pp. 565-567 ◽  
Author(s):  
P. Davies ◽  
K. Griffiths
Keyword(s):  
Ionic Strength ◽  
Rna Polymerase ◽  
Ribonucleic Acid ◽  
Selective Inhibitor ◽  
High Ionic Strength ◽  
Receptor Complexes ◽  
Protein Receptor ◽  
Stimulation Of

Stimulation of prostatic RNA polymerase in vitro by prostatic 17β-hydroxy-5α-androstan-3-one (5α-dihydrotestosterone)–receptor complexes has been previously reported. By use of the selective inhibitor, α-amanitin, we have shown that both nucleolar and extranucleolar RNA polymerase activities may be stimulated, but stimulation is abolished at high ionic strength.

scholarly journals Stimulation of ribonucleic acid polymerase activity in vitro by prostatic steroid–protein receptor complexes

1973 ◽  
Vol 136 (3) ◽  
pp. 611-622 ◽  
Author(s):  
P. Davies ◽  
K. Griffiths
Keyword(s):  
Ionic Strength ◽  
Rna Polymerase ◽  
Polymerase Activity ◽  
Supernatant Fraction ◽  
Nuclear Fraction ◽  
Receptor Complexes ◽  
Protein Receptor ◽  
Ammonium Sulphate Fractionation ◽  
Stimulation Of

A system has been developed which allows the stimulation in vitro of prostatic RNA polymerase by prostatic 5α-dihydrotestosterone–protein receptor complexes prepared from the tissues of castrated rats. The reconstitution in vitro of such a system necessitates the purification of several subcellular components. Two 5α-dihydrotestosterone–receptor complexes are located in the prostatic soluble supernatant fraction, separable by selective ammonium sulphate fractionation, and one complex can be isolated from the nuclear fraction. In the presence of all these complexes, stimulation of RNA polymerase in intact nuclei and nucleoli was observed. The complexes also increased the activity of the enzyme solubilized from whole nuclei. Greater stimulation of this system was noted in the presence of prostatic chromatin as template, as compared with that observed with calf thymus DNA or liver chromatin as template. The effects of the complexes on subnuclear forms of RNA polymerase, of nucleolar and extranucleolar origin, are also described. RNA polymerase solubilized from nucleoli is more susceptible to stimulation by the 5α-dihydrotestosterone–receptor complexes than is the ‘nucleoplasmic’ enzyme. Stimulation occurs less readily in the presence of Mn2+and at high ionic strength than in the presence of Mg2+and at low ionic strength. Preliminary experiments show that prostatic nucleolar RNA polymerase transcribes prostatic chromatin poorly as compared with the nucleoplasmic enzyme. The observations reported indicate an involvement of non-histone proteins associated with DNA in the process by which stimulation of enzyme activity by the 5α-dihydrotestosterone–receptor complexes is achieved. The implications of these findings in the mechanism of steroid hormone action is considered.

FURTHER STUDIES ON THE STIMULATION OF PROSTATIC RIBONUCLEIC ACID POLYMERASE BY 5α-DIHYDROTESTOSTERONE—RECEPTOR COMPLEXES

1974 ◽  
Vol 62 (2) ◽  
pp. 385-400 ◽  
Author(s):  
PETER DAVIES ◽  
KEITH GRIFFITHS
Keyword(s):  
Rna Polymerase ◽  
Active Form ◽  
Polymerase Activity ◽  
Selective Inhibitor ◽  
High Ionic Strength ◽  
Receptor Complexes ◽  
Associated Proteins ◽  
Rna Polymerase Activity ◽  
Stimulation Of

SUMMARY The stimulation in vitro of prostatic RNA polymerase activity by prostatic 5α-dihydrotestosterone—receptor complexes has been previously reported. Further investigations into the nature of the stimulation have now been carried out. By use of the selective inhibitor, α-amanitin, and by varying the concentration of ammonium sulphate in the assay media, both the nucleolar and extranucleolar forms of RNA polymerase could be stimulated, depending upon the ionic conditions employed. High ionic strength inhibited stimulation, either by interference with the association between steroid—receptor complexes and chromatin components, or by blocking the conversion of cytoplasmic complexes to a more 'active' form of the complex. 5α-Dihydrotestosterone—receptor complexes appeared to affect the template availability of prostatic chromatin, possibly in a way similar to that of the chromatin-associated proteins.

scholarly journals Differential inhibition of mammalian ribonucleic acid polymerases by an exotoxin from Bacillus thuringiensis. The direct observation of nucleoplasmic ribonucleic acid polymerase activity in intact nuclei

1972 ◽  
Vol 127 (4) ◽  
pp. 619-624 ◽  
Author(s):  
T. Beebee ◽  
A. Korner ◽  
R. P. M. Bond
Keyword(s):  
Ionic Strength ◽  
Bacillus Thuringiensis ◽  
Rna Polymerase ◽  
Ribonucleic Acid ◽  
Polymerase Activity ◽  
High Ionic Strength ◽  
Rna Polymerases ◽  
Differential Inhibition ◽  
Rna Polymerase Activity ◽  
Soluble Enzymes

The effects of the exotoxin from Bacillus thuringiensis on DNA-dependent RNA polymerases from rat liver were examined. The exotoxin inhibits all RNA polymerase activity at both low and high ionic strength in intact nuclei, and soluble enzymes are similarly affected. This inhibition is relieved by ATP. Dephosphorylated exotoxin did not inhibit the soluble enzymes. Nucleolar and nucleoplasmic RNA polymerases respond to different concentration ranges of exotoxin, and the compound can be used in intact nuclei to isolate the nucleoplasmic activity.

scholarly journals Studies on the pathogenesis of liver necrosis by α-amanitin. Effect of α-amanitin on ribonucleic acid synthesis and on ribonucleic acid polymerase in mouse liver nuclei

1967 ◽  
Vol 105 (2) ◽  
pp. 779-782 ◽  
Author(s):  
F. Stirpe ◽  
L. Fiume
Keyword(s):  
Rna Polymerase ◽  
Ammonium Sulphate ◽  
Ribonucleic Acid ◽  
Mouse Liver ◽  
Orotic Acid ◽  
Acid Synthesis ◽  
Liver Necrosis ◽  
Liver Nuclei

1. Injection of α-amanitin to mice causes a decreased incorporation of [6−14C]-orotic acid into liver RNA in vivo. 2. The activity of RNA polymerase activated by Mn2+ and ammonium sulphate is greatly impaired in liver nuclei isolated from mice poisoned with α-amanitin, and is inhibited by the addition of the same toxin in vitro. 3. The activity of the Mg2+-activated RNA polymerase is only slightly affected by α-amanitin either administered to mice or added in vitro.

scholarly journals Inhibition by α-amanitin of ribonucleic acid polymerase solubilized from rat liver nuclei

1970 ◽  
Vol 116 (2) ◽  
pp. 177-180 ◽  
Author(s):  
F. Novello ◽  
L. Fiume ◽  
F. Stirpe
Keyword(s):  
Rna Polymerase ◽  
Ammonium Sulphate ◽  
Rat Liver ◽  
Ribonucleic Acid ◽  
Isolated Rat Liver ◽  
Rat Liver Nuclei ◽  
Liver Nuclei ◽  
Isolated Rat

1. α-Amanitin inhibits in vitro the RNA polymerase solubilized from isolated rat liver nuclei. 2. In contrast with previous observations with whole nuclei, the inhibition occurs approximately to the same extent in the presence and in the absence of ammonium sulphate. 3. Evidence is presented that the toxin acts by interacting with the enzyme itself and not with DNA or other components.

scholarly journals Parthenogenesis in Xenopus eggs requires centrosomal integrity.

1990 ◽  
Vol 110 (2) ◽  
pp. 405-415 ◽  
Author(s):  
C Klotz ◽  
M C Dabauvalle ◽  
M Paintrand ◽  
T Weber ◽  
M Bornens ◽  
...  
Keyword(s):  
Ionic Strength ◽  
Protein Unfolding ◽  
Protein Structures ◽  
High Ionic Strength ◽  
Native State ◽  
Apparent Paradox ◽  
Complete Protein ◽  
Duplication Cycle ◽  
Structure And Activity

Xenopus eggs are laid arrested at second metaphase of meiosis lacking a functional centrosome. Upon fertilization, the sperm provides the active centrosome that is required for cleavage to occur. The injection of purified centrosomes mimics fertilization and leads to tadpole formation (parthenogenesis). In this work we show that the parthenogenetic activity of centrosomes is inactivated by urea concentrations higher than 2 M. The loss of activity is correlated with a progressive destruction of the centriolar cylinder and extraction of proteins. This shows that centrosomes are relatively sensitive to urea since complete protein unfolding and solubilization of proteins normally occurs at urea concentrations as high as 8-10 M. When present, the parthenogenetic activity is always associated with a pelletable fraction showing that it cannot be solubilized by urea. The parthenogenetic activity is progressively inactivated by salt concentrations higher than 2 M (NaCl or KCl). However, only a few proteins are extracted by these treatments and the centrosome ultrastructure is not affected. This shows that both parthenogenetic activity and centrosomal structure are resistant to relatively high ionic strength. Indeed, most protein structures held by electrostatic forces are dissociated by 2 M salt. The loss of parthenogenetic activity produced at higher salt concentrations, while the structure of the centrosome is unaffected, is an apparent paradox. We interpret this result as meaning that the native state of centrosomes is held together by forces that favor functional denaturation by high ionic strength. The respective effects of urea and salts on centrosomal structure and activity suggest that the centrosome is mainly held together by hydrogen and hydrophobic bonds. The in vitro microtubule nucleating activity of centrosomes can be inactivated at salt or urea concentrations that do not affect the parthenogenetic activity. Since egg cleavage requires the formation of microtubule asters, we conclude that the extracted or denatured microtubule nucleating activity of centrosomes can be complemented by components present in the egg cytoplasm. Both parthenogenetic and microtubule nucleating activities are abolished by protease treatments but resist nuclease action. Since we find no RNA in centrosomes treated by RNase, they probably do not contain a protected RNA. Taken together, these results are consistent with the idea that the whole or part of the centrosome structure acts as a seed to start the centrosome duplication cycle in Xenopus eggs.

Divalent cation stimulation of in vitro fibre assembly from epidermal keratin protein

1978 ◽  
Vol 33 (1) ◽  
pp. 255-263
Author(s):  
K. Fukuyama ◽  
T. Murozuka ◽  
R. Caldwell ◽  
W.L. Epstein
Keyword(s):  
Ionic Strength ◽  
Divalent Cation ◽  
Divalent Cations ◽  
Urea Concentration ◽  
Newborn Rats ◽  
Filament Formation ◽  
Stimulation Of ◽  
Millimolar Concentration

Keratin was extracted from purified cornified cells of newborn rats in Tris-HCl-buffered 8 M urea containing beta-mercaptoethanol. Microfilaments were assembled in vitro by reducing the ionic strength of buffer and the urea concentration. One millimolar concentration of KCl and NaCl did not affect filament formation, but the same concentration of divalent cations greatly altered this process. CaCl2 and MgCl2 induced gelation of keratin by formation of bundles of birefringent macrofilaments. ZnCl2, CuSO4 and HgCl2 formed greater numbers of macrofilaments and the protein aggregated.

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