Browsing by Subject "Catalase"
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Item Analysis of transgenic tobacco that express maize catalase3(Texas Tech University, 1999-05) Schake, Sheryl A.Catalases (H2O2: H2O2 oxidoreductase, EC 1.11.1.6; CAT) are hemecontaining tetramers that are important in destroying H2O2 found in different cellular compartments. Maize Cats has been shown to be capable of dismutating H2O2 via either a catalatic or peroxidatic reaction. In addition, increased maize CAT3 transcripts were detected during periods of chilling acclimation. In this study, a maize CatS cDNA was isolated using reverse transcriptase polymerase chain reaction. To better understand the role of maize CAT3 in oxidative stress, we have introduced the transgene that expresses this enzyme into wild type Xanthi NN tobacco (Nicotiana tabacum). Total catalase activities were only slightly higher in transgenic plants as compared to Xanthi NN. While total peroxidatic activity of these transgenic plants was found to be 12-fold higher than in the wild-type tobacco. Thetransgenic Cat3 plants were exposed to various abiotic stresses such as, low temperatures, high temperatures, salinity, chemical treatments, and photooxidation. Increased seedling growth was evident in transgenic seedlings during treatments at low temperatures, high temperatures, and salinity which could implicate increased protection from oxidative damage. No significant protection was evident when transgenic seedlings were treated with methyl viologen or photooxidatlve stress. In addition, lower lipid peroxidation levels in transgenic plants correlated with increased peroxidatic activity in these plants. These data suggests that in wild-type tobacco that express maize CAT3 have increased protection against various forms of oxidative stress.Item Functional Annotation and Mechanistic Characterization of Enzymes with Unknown Functions: Studies on Adenine Deaminase, N-6-Methyladenine Deaminase and the C-P Lyase Pathway(2012-10-19) Kamat, SiddheshAdenine deaminase (ADE) catalyzes the conversion of adenine to hypoxanthine. Mechanistic characterization of ADE from Escherichia coli was performed along with biophysical studies. The structure of ADE was solved from A. tumefaciens. The structure, along with the biochemical and biophysical characterization, enabled the elucidation of the mechanism of the deaminase reaction of ADE. Elucidation of the origin of the oxygenation reactions within ADE led to the discovery of a promiscuous catalase reaction. The diiron ADE from all tested bacterial species exhibited this unusual reaction, along with the generation of superoxide and hydroxyl radicals, the latter being responsible for the oxygenation of the protein. The residues that were identified to be oxygenated were primarily the metal binding residues implying the origin of this reaction was the binuclear iron center. A group of bacterial enzymes that are co-localized in the same genomic operon as ADE but of unknown function were identified. The enzyme Bh0637 from Bacillus halodurans, a representative member of this group of enzymes was characterized. This enzyme was shown to preferentially catalyze the deamination of epigenetic base, N-6-methyadenine. Lastly, gram-negative bacteria have a highly conserved phn operon composed of 14 genes to break the C-P bond of inert alkylphosphonates. The genes phnGHIJKLM are absolutely critical for this activity. We discovered that methylphosphonate reacts first with MgATP to form alpha-D-ribose-1-methylphosphonate-5-triphosphate (RPnTP) and adenine by the action of PhnI, PhnG, PhnH and PhnL. PhnI by itself was shown to perform a novel nucleosidase reaction converting MgATP to ribose-5-triphosphate and adenine. The triphosphate moiety of RPnTP is then hydrolyzed to pyrophosphate and alpha-D-ribose-1-methylphosphonate-5-phosphate (PRPn) by PhnM. The carbon-phosphorus bond of PRPn is subsequently cleaved via a radical-based reaction to alpha-D-ribose-1,2-cyclic-phosphate-5-phosphate (PRcP) and methane in the presence of S-adenosyl-L-methionine by PhnJ.Item Regulation of catalase gene expression in soybean(Texas Tech University, 1992-08) Isin, Sibel HadiyeCatalase is one of the important enzymes in the cellular active-oxygen scavenging system of aerobic organisms. In plants, catalase is located in peroxisomes where it converts hydrogen peroxide generated during 6-oxidation of fatty acids and photorespiration to oxygen and water. This study attempts to serve as an initial step in understanding the regulation of catalase gene expression in plants. Multiple isoforms of catalase are present in several plant species such as maize, cotton and tobacco. Expression of these isoforms is under developmental and environmental control. In pea and soybean, a single catalase isoform has been detected in all tissues and developmental stages tested. Genomic DNA blot analysis suggested that there is a single catalase gene in pea genome, while soybean appears to have two catalase loci. A pea cDNA that includes the entire coding sequence for a catalase subunit was isolated and characterized. The deduced amino acid sequence proved to be highly similar to several other plant catalase sequences. Using the pea cDNA as probe, a soybean genomic clone that includes catalase coding sequences and 1.8 kb of upstream sequence was isolated. Sequence analysis indicated that the soybean catalase gene contains six introns, and the deduced amino acid sequence has a high percentage of identity with the pea catalase subunit. In soybean seedlings, very low levels of catalase activity were detected in germinated seeds one day after imbibition (DAI), but catalase activity increased rapidly, reaching a maximum by 4 DAI. Catalase mRNA was detectable by 1 DAI and increased gradually to reach maximum levels within 4 DAI. In dark grown seedlings, catalase mRNA was maintained at relatively high levels through 6 DAI . Exposure of seedlings to light after 3 DAI caused a more rapid decline in catalase mRNA levels when compared to dark grown seedlings. This pattern of differential accumulation in light/dark conditions was not observed in the catalase activity levels which were determined during the same developmental period. The upstream region of the soybean catalase gene was fused to the GUS (P-glucuronidase) reporter gene, and a series of 5' deletions were generated using Exonuclease III. Six of these deletion constructs were used to transform tobacco plants. Fluorometric GUS analysis indicated that GUS expression levels were much higher in imbibed seeds than in mature leaves, suggesting that this catalase promoter is seed-specific. Histochemical GUS staining of transgenic plant tissues showed that GUS is expressed at a high level in the cotyledons at early stages of seedling development, but the expression is maintained primarily in the vascular tissues, specifically in the phloem of the mature plants.Item The Relationship of Catalase Activity in Raw Milk to Quality of the Processed Product(Texas Tech University, 1972-12) Smith, Edgar D.Not Available.