PubMed · 13600089
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S ROCCHIETTA. [Carbonic anhydrase].. https://pubmed.ncbi.nlm.nih.gov/13600089/
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Rice carbonic anhydrase (CA) was successfully expressed as a glutathione-S-transferase (GST) fusion protein in an Escherichia coli expression system. The optimal induction concentration of IPTG and growth temperature was found to be 1.0mM and 28 degrees C. To obtain milligram amounts of homogeneous active recombinant proteins, 150mM NaCl and Mg-ATP solution were used during the purification procedures. After improving the conditions of expression and the purification procedures, final yield of recombinant proteins was 1.3mg/g wet cell weight after enzymatic cleavage of the GST tag, and the molecular weight was about 29kDa. The purified protein had CO(2) hydration activity, and had no detectable esterase activity in vitro. Addition of zinc improved the CO(2) hydration activity of the rice CA produced by E. coli. The effects of acetazolamide (AZ) and the anions N3-, NO3-, I(-), Br(-), and Cl(-) on CO(2) hydration activity of CA were studied. AZ and N3- were found to be strong inhibitors of rice CA. The inhibitory activity of AZ and ions was in the order AZ>N3->NO3->I(-)>Br(-)>Cl(-).
A two-step reaction cascade is applied to the sequence-specific detection of DNA by enzymatic amplification of the molecular into an optical signal.
In the present study, we investigated the role of the extracellular signal-regulated protein kinase (ERK) in regulation of the hypoxia marker, carbonic anhydrase IX (CAIX). U0126, a specific inhibitor of MEK1/2, downregulated CAIX expression induced by true hypoxia and cell density. CA9 promoter activity was similarly affected. Mapping of the U0126 effect revealed that both critical elements within the CA9 promoter, the hypoxia response element and the juxtaposed SP1-binding PR1, were inhibited. This confirmed that ERK signaling modulates CA9 promoter activity via its effects on hypoxia inducible factor-1 (HIF-1) and SP1. Further analysis of the U0126 effect on HIF-1-dependent transcription in MCF-7 cells identified p300, a transcriptional co-activator of HIF-1, as the target of ERK. Constitutively increased ERK activity in isogenic fibrosarcoma cell lines did not cause increased cell density-dependent CAIX expression/CA9 promoter activity. In HeLa cells, an inverse correlation between cell density-induced CAIX expression and ERK activation was observed: sparse cultures did not express CAIX and displayed high ERK activation, whereas CAIX expression in dense cultures was associated with low ERK activation. Collectively, our data do not support any quantitative relationship between ERK activation and CAIX expression. Thus, although ERK signaling is required for optimal CAIX expression, our data are consistent with a model in which constitutive basal ERK activity plays an auxiliary role in CA9 promoter transactivation by modulating activity of the transcription factor SP1 and the transcriptional co-activator p300.