Browsing by Subject "Glutamine synthetase"
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Item Cloning of higher plant glutamate synthase genes(Texas Tech University, 1993-08) Nalbantoglu, BarbarosIn all plants, glutamate synthase (GOGAT) is a key enzyme involved in the assimilation of ammonia. GOGAT catalyzes the transamidation of the amido nitrogen from glutamine to 2-oxoglutarate to form two molecules of glutamate. GOGAT exists in higher plants in two forms, each of which requires a specific electron donor for the reaction; one utilizes ferredoxin as a reductant and the other utilizes NAD(P)H. Fd-dependent GOGAT, rather than the NAD(P)H form, is the major component in green tissues and is localized in the chloroplast. Fddependent GOGAT from green leaves is a monomeric protein with a molecular mass ranging from 160 to 140 kDa and contains an iron-sulfur cluster and flavins (FAD and FMN). In this study, I have focused on the cDNA cloning of spinach Fddependent GOGAT in order to elucidate the complete primary structure of the protein and obtain a nucleic acid probe for further genetic studies. The 3.8 kb cDNA insert was isolated from a cDNA library. An additional 1.2 kb coding cDNA fragment was produced by PCR. The total cDNA is 4948 bp in length, and it includes 98% of the coding sequence of the 1483 amino acids of the mature protein. The deduced amino acid sequences reveal a high similarity with Fd-dependent GOGAT from maize and with NADPH-dependent GOGAT from A. brasilense and £. coli. Binding domains for flavin cofactors and ferredoxin, a domain for glutamine binding and activation, and cysteine clusters for iron-sulfur centers formation were tentatively identified on the basis of sequence comparison with flavoproteins, ferredoxin-dependent enzymes, aminotransferases, and iron-sulfur proteins. The cDNA hybridizes to an RNA band of about 5.5 kb. Analysis of genomic DNA indicates the presence of a single-copy gene for Fd-dependent GOGAT in spinach.Item Cytoplasmic foci at the crossroads of artifactual science and biological function(2016-05) Zhao, Alice; Marcotte, Edward M.; Ellington, Andrew D; Zhang, Yan J; Appling, Dean R; Iyer, Vishwanath RDeciphering protein interaction and compartmentalization is crucial to understanding the molecular mechanisms that drive biological processes. Using various high throughput approaches, we have managed to score subcellular dynamic protein re-organization into supramolecular structures and map physical association networks to discover protein complexes on a proteome-wide level. However, the case by case studies of some of these novel structures and interactions reveal difficulties in interpreting their biological basis. This study offers insights into limits inherent in the molecular techniques used to investigate subcellular structures and protein interactions, describing a set of cautionary tales and critical analysis for deciphering cases of confounding data from orthogonal approaches. This study also offers a new experimental technique for high-throughput imaging assays with mammalian cell lines.