Glycerol trinitrate (GTN) reductase, which enables Agrobacterium radiobacter to utilize GTN and related explosives as sources of nitrogen for growth, was purified and characterized, and its gene was cloned and sequenced. The enzyme was a 39-kDa monomeric protein which catalyzed the NADH-dependent reductive scission of GTN (K m ؍ 23 M) to glycerol dinitrates (mainly the 1,3-isomer) with a pH optimum of 6.5, a temperature optimum of 35°C, and no dependence on metal ions for activity. It was also active on pentaerythritol tetranitrate (PETN), on isosorbide dinitrate, and, very weakly, on ethyleneglycol dinitrate, but it was inactive on isopropyl nitrate, hexahydro-1,3,5-trinitro-1,3,5-triazine, 2,4,6-trinitrotoluene, ammonium ions, nitrate, or nitrite. The amino acid sequence deduced from the DNA sequence was homologous (42 to 51% identity and 61 to 69% similarity) to those of PETN reductase from Enterobacter cloacae, N-ethylmaleimide reductase from Escherichia coli, morphinone reductase from Pseudomonas putida, and old yellow enzyme from Saccharomyces cerevisiae, placing the GTN reductase in the ␣/ barrel flavoprotein group of proteins. GTN reductase and PETN reductase were very similar in many respects except in their distinct preferences for NADH and NADPH cofactors, respectively.Nitrate esters such as glycerol trinitrate (GTN) and pentaerythritol tetranitrate (PETN) are widely used both as explosives and as vasodilators in the treatment of angina. During their long history of exploitation in these applications (24, 34), there has been ample opportunity during production, storage, and use for significant contamination of land sites and water courses to occur, so that site remediation of explosive residues is now an urgent issue worldwide. Moreover, the current acceleration in demilitarization of ordnance and rocket formulations will produce further waste material, raising new environmental concerns. The environmental issues, together with the rarity of naturally occurring analogs (14), make the discovery of microorganisms which can degrade such compounds and thus influence their environmental fate of particular interest. Earlier interest in the elimination of nitrate ester explosives in wastewater treatment plants (35,36) and in their metabolism in fungal organisms (6,7,29,30) led to the first report (21) of denitration of GTN in pure bacterial cultures of Bacillus thuringiensis plus B. cereus and Enterobacter agglomerans, apparently occurring via a hydrolytic pathway, although formation of nitrate was not demonstrated. In contrast, White et al. showed unequivocally that assimilation of nitrogen from GTN in pure cultures of a Pseudomonas sp. (37) and Agrobacterium radiobacter (38) occurred via nitrite (not nitrate) with the concomitant formation of mainly glycerol 1,3-dinitrate (1,3-GDN) and small amounts of the corresponding 1,2-isomer. Cells were able to denitrate both of the dinitrates to mononitrates but not beyond, and they also converted PETN to its tri-and dinitrates. The enzyme responsible was identif...
We describe the cloning, DNA sequence analysis and mRNA expression analysis of human Cctg (HsCctg), a gene that encodes the gamma-subunit of the eukaryotic cytosolic 'chaperonin-containing TCP-1' (CCT). Partial clones representing the 3' region of HsCctg cDNA were isolated from a human kidney cDNA library, and the missing 5' region was amplified directly from human kidney cDNA. The Cctg mRNA transcript is expressed in numerous human and mouse tissues and, like Tcp-1/Ccta, Cctg mRNA is expressed at higher levels in mouse testis when compared with kidney and brain. Southern-blot analysis has also revealed the Cctg gene to be highly conserved in mouse, rat, sheep and frog. The 1901 bp HsCctg cDNA has a coding region of 1635 bp and encodes a predicted 60 kDa protein (544 amino acids). The predicted HsCCT gamma amino acid sequence shares a high degree of sequence similarity with gamma-subunits from the mouse Mus musculus (98% similarity), the yeast Saccharomyces cerevisiae (75% similarity) and the protozoan Tetrahymena pyriformis (76% similarity) as well as with other members of the TF55/TCP-1 family, such as human TCP-1/CCT alpha (55% similarity) and TCP-20/CCT zeta (54% similarity). HsCCT gamma also shares conserved domains previously identified in the TF55/TCP-1 family of chaperonins and more distantly related chaperonins such as GroEL and Hsp60.
We have developed a novel test system for the quantitative assessment of gene transcription. The procedure involves the use of chemiluminescent-labeled oligonucleotide probes in a hybridization protection assay (HPA) format. We have used this technology to measure changes in vitellogenin mRNA to demonstrate the impact of estrogen exposure in the juvenile fathead minnow (Pimephales promelas). Marked changes in mRNA expression were observed in response to intraperitoneal injection of 17beta-estradiol demonstrating the utility of this technique for the identification and monitoring of toxic responses to xenobiotics.
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