Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.6 (RNA polymerase)
 34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The DNA-dependent RNA polymerase (RPase) from Escherichia coli contained 2 mol of Zn/mol of holoenzyme (alpha 2 beta beta' sigma). An in vitro protocol involving sequential denaturation of RPase in 8 M urea and low pH (2.2), in the presence of 10 mM ethylenediaminetetraacetic acid (EDTA), was developed to completely remove the two intrinsic Zn ions. Subsequent reconstitution of the denatured, Zn-free RPase in the absence and presence of 10(-5) approximately 10(-4) M ZnCl2 yielded respectively the inactive apoenzyme and active (50 +/- 10%) RPase containing one Zn ion (rec-Zn1-RPase). Active rec-Cd1-RPase was similarly obtained when CdCl2 instead of ZnCl2 was used in the reconstitution. The use of 65Zn as a tracer in the two-step reconstitution procedure showed that the metal was incorporated into renatured enzyme only in the last step of reconstitution. The subunit location of the incorporated metal was identified to be in the beta subunit by the use of Affi-Gel Blue column chromatography of rec-Cd1-RPase. The analysis of apo- and rec-Zn1-RPases by sucrose density gradient sedimentation showed that the inactive apo-RPase appeared to be consisted of randomly folded protein species with S20,w values ranging from 5 to 18 S, while rec-Zn1-RPase contained a major, active 13S RPase species and a minor, inactive 7.9S species that could be separated by DNA-cellulose column chromatography. Both 13S and 7.9S RPase contained 1 mol of Zn and the five subunits.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Intrinsic zinc ion is essential for proper conformation of active Escherichia coli RNA polymerase. 639 24

New improved methods were developed for the purification to apparent homogeneity of alpha, beta, beta', and sigma subunits of Escherichia coli RNA polymerase (RNAP) from corresponding overproducing strains. The purified subunits were assembled into enzymatically active RNAP holoenzyme (alpha 2 beta beta' sigma) using the optimal subunit molar ratio (alpha:beta:beta':sigma = 2:8:4:1) at a total protein concentration of 0.5 mg/ml. The presence of sigma subunit and 10 microM ZnCl2 in the reconstitution mixture increased the yield of RNAP approximately 4 times. The assembled RNA polymerase was purified by two successive chromatographic steps using size-exclusion Superose 6 and anion exchange Mono Q FPLC columns, which resulted in the electrophoretically homogeneous holoenzyme with overall yield of 56%. The specific activity of the recombinant RNAP estimated by the standard T4 transcription assay was 6.5 nmol of [3H]UTP incorporated into acid-insoluble RNA product per microgram of RNAP per 1 h.
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PMID:Recombinant Escherichia coli RNA polymerase: purification of individually overexpressed subunits and in vitro assembly. 828 46

Adult pancreatic nonendocrine cells represent a potential alternative source of insulin-producing tissue for the treatment of diabetes. Differentiation of these cells is regulated by various signaling pathways including the phosphoinositide 3-kinase (PI3K) pathway. Therefore, we evaluated the effect of PI3K on this process. Compared with untreated cells the differentiation of human nonendocrine pancreatic cells into insulin-producing elements was increased after treatment with IGF-1, EGF, and Exendin-4, growth factors known to be activators of the PI3K pathway (12.2 +/- 3.2% vs 9.1 +/- 3.2%). Treatment with PI3K pathway inhibitor wortmannin reduced the number of differentiated beta cells from 9.1 +/- 3.2 to 0.7 +/- 0.4%. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis revealed that insulin-like growth factor-1 (IGF-1), epidermal growth factor (EGF), and Exendin-4 significantly increased the expression of the transcription factor neurogenin-3, whereas the expressions of pancreatic and duodenal homeobox 1 (PDX-1), neurogenic differentiation 1 (NeuroD) were increased only among samples treated with ZnCl2 and not significantly affected by treatment with the tested growth factors. Successful differentiation of IGF-1, EGF-, and Exendin-4-treated cells into functional beta cells was confirmed by C-peptide secretion in response to 5 versus 20 mmol glucose stimulation (0.24 vs 0.91 pmol C-peptide/microg DNA). These results showed that activation of the PI3K signaling pathway might be used to stimulate the differentiation of nonendocrine pancreatic cells into insulin-producing elements.
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PMID:An acidic pH and activation of phosphoinositide 3-kinase stimulate differentiation of pancreatic progenitors into insulin-producing cells. 2069 12