On the mechanism of hormone action. IX. Stimulation of RNA polymerase activity of rat-liver nuclei by cortisol in vitro

Special care to prevent proteolysis during yeast RNA polymerase B purification leads to the appearance of two forms of enzymes, BI and BII, with different molecular weight (465000 and 435000, respectively). The two forms of enzyme can be separated by ion‐exchange chromatography or polyacrylamide gel electrophoresis. Their subunit structures were compared by sodium dodecylsulfate gel electrophoresis, the only observed difference between the two enzymes is in the molecular weight of the heaviest subunit which is 220000 for enzyme BI and 180000 for enzyme BII. Otherwise, the two enzymes have seven common subunits of molecular weights 150000, 45000, 26000, 22500, 14500, 12500 and 9000. Two additional polypeptide chains of 32000 and 16500 Mr are dissociated from the enzyme upon polyacrylamide gel electrophoresis or DEAF Sephadex chromatography. The largest subunit of enzyme BI (Mr 220000) can be specifically cleaved in vitro by a yeast protease extract, generating a polypeptide chain indistinguishable from the largest subunit of enzyme BII. This proteolytic cleavage of enzyme BI in vitro is inhibited by phenylmethylsulfonyl fluoride and does not significantly change the activity of the enzyme with single‐stranded or double‐stranded DNA as template. The precursor‐product relationship of the different forms of class B RNA polymerises in eukaryotic cells is discussed.

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“…Assays were incubated at 30 "C for 30 min. Acidinsoluble radioactivity was determined as described by Lukacs and Sekeris [25].…”

Section: Assay Of R N a Polymerase Bmentioning

confidence: 99%

Two Forms of RNA Polymerase B in Yeast

Dezélée¹,

Wyers²,

Sentenac³

et al. 1976

European Journal of Biochemistry

104

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“…Amino acid analysis was then performed by standard procedures with a Beckman Unichrom amino acid analyser. The assay for RNA polymerase activity was essentially the same as described by Lukacs & Sekeris (1967). Under these conditions both amanitinsensitive and amanitin-insensitive DNA-dependent RNA polymerases are assayed (W. Schmid & C. E. Sekeris, unpublished work).…”

Section: Methodsmentioning

confidence: 99%

Effect of cortisol on the thiol content of rat liver nuclear proteins

Doenecke

Beato

Congote

et al. 1972

Biochemical Journal

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Administration of cortisol to normal or adrenalectomized rats leads within 15-30min to an increased thiol content of nuclear proteins, measured by the incorporation of iodo[(3)H]-acetate or N-[(14)C]ethylmaleimide or by colorimetric methods. The same effect is observed after incubation of isolated rat liver nuclei with corticosteroids. The increased thiol content of the nuclear proteins shows the same time-dependence as the stimulation of RNA synthesis by corticosteroids observed in vivo and in vitro. Amino acid analysis of the carboxymethylated proteins reveals that in the experiments in vivo most of the label is present as carboxymethylcysteine with small amounts of carboxymethyl-lysine and carboxymethylhistidine, whereas in the experiments in vitro more carboxymethyl-lysine and carboxymethylhistidine than carboxymethylcysteine are found. The increase in the content of thiol groups is due to cleavage of the disulphide bridges between the nuclear proteins. Polyacrylamide-gel electrophoresis of the acid-soluble fraction reveals that most of the iodo[(3)H]acetate label is incorporated into a non-histone fraction with a molecular weight of approx. 45000 whereas in the acid-insoluble fractions many protein bands are labelled.

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“…The reaction was stopped by pipetting aliquots of 120 ~1 on filter papers, which were immediately immersed in ice-cold 5% trichloroacetic acid (TCA). The filters were then washed 3 times in TCA, 3 times in ethanol (96%), 2 times in ethanol/ether (1: 1) and ether, dried and counted as described,:earlier [9] . …”

Section: Test For Rnase H Activitymentioning

confidence: 99%