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Full-Text Articles in Biochemistry, Biophysics, and Structural Biology

Structural And Functional Studies On Snares-Mediated Membrane Fusion , Yong Chen Jan 2006

Structural And Functional Studies On Snares-Mediated Membrane Fusion , Yong Chen

Retrospective Theses and Dissertations

Membrane fusion is an essential biological process for vesicle trafficking in eukaryotic cells. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are a family of proteins that have been recognized as key components of protein complexes that drive membrane fusion. The SNARE proteins localized in opposite membranes form a SNARE complex, which bridges the two membranes into close proximity and promotes fusion. Despite considerable sequence divergence among SNARE proteins, this fusion machinery is conserved structurally, mechanistically and evolutionally from yeast to human;Site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) are well established techniques to study the structures of membrane ...


Genetic And Molecular Analysis Of Starch Synthases Functions In Maize And Arabidopsis , Xiaoli Zhang Jan 2006

Genetic And Molecular Analysis Of Starch Synthases Functions In Maize And Arabidopsis , Xiaoli Zhang

Retrospective Theses and Dissertations

Understanding the specific functions played by individual starch synthase isoforms in maize and Arabidopsis will provide important evidence for how highly organized starch structure is made. Starch synthases (SS) catalyze the transfer of the glucosyl moiety from ADP-Glc to the terminus of a growing alpha-(1, 4)-linked glucan linear chain. At least five classes of SSs are identified in higher species, referred to as GBSS, SSI, SSII, SSIII, and SSIVN. They have high similarity in the catalytic and starch-binding domains of the C-termini but differ at their N-termini. All of these enzymes are highly conserved in plant kingdom, which ...


Molecular And Genetic Characterization Of A Biotin Biosynthetic Gene In Arabidopsis Encoding Both 7,8-Diaminopelargonic Acid Aminotransferase And Dethiobiotin Synthetase , Elve Chen Jan 2006

Molecular And Genetic Characterization Of A Biotin Biosynthetic Gene In Arabidopsis Encoding Both 7,8-Diaminopelargonic Acid Aminotransferase And Dethiobiotin Synthetase , Elve Chen

Retrospective Theses and Dissertations

Biotin is an essential enzyme cofactor required for different metabolic processes. Plants are a major source of biotin, however, the genes and the protein products of biotin biosynthetic pathway are not fully characterized. We have molecularly characterized the genetically defined Arabidopsis bio1 locus as encoding 7,8-diaminopelargonic acid (DAPA) aminotransferase. Molecular and genetic analysis revealed that the full length BIO1 cDNA is capable of producing a fusion protein that catalyzes not only DAPA aminotransferase, but also dethiobiotin synthetase (BIO3) which is another enzymatic step in the biotin biosynthetic pathway. We refer to this fusion-gene as BIO3/BIO1. This bifunctional enzyme ...


Structural Characterization Of Ligand Binding In Hexacoordinate Hemoglobins , Julie Anne Hoy Jan 2006

Structural Characterization Of Ligand Binding In Hexacoordinate Hemoglobins , Julie Anne Hoy

Retrospective Theses and Dissertations

Hemoglobins are ancient proteins that predate the divergence of prokaryotes and eukaryotes. Although the most thoroughly studied hemoglobins are mammalian oxygen transporters, it has become clear that this function is a recent evolutionary advancement coinciding with the "oxygen revolution" and the development of multicellular organisms. Hemoglobins capable of oxygen transport are highly specialized structurally to maintain pentacoordination of the heme and appropriate ligand rate and equilibrium constants, while preventing autooxidation due to bound oxygen. Their predecessors however, adopt a more energetically favorable hexacoordinate conformation that precludes oxygen transport;Despite the fact that hexacoordinate hemoglobins are ubiquitous in biological organisms, little ...


Studies Of Intermediates In Snare-Induced Membrane Fusion , Fan Zhang Jan 2006

Studies Of Intermediates In Snare-Induced Membrane Fusion , Fan Zhang

Retrospective Theses and Dissertations

The purpose of this course of study is to understand the intermediates during SNAREs assembly and the membrane fusion mediated by SNAREs. SNAREs have been proposed to be the minimal machinery for the membrane fusion. According to the location distribution, SNAREs can be classified as either v-(vesicles) or t-(target) SNAREs. The assembly of three SNARE components from the opposing membranes into the ternary complex is believed to provide the ultimate driving force for merging the separate bilayers into a continuous entity. Before this step the t-SNAREs associate as the binary complex on the target membrane to serve as ...


Release Of Human Brain Hexokinase From The Mitochondrial Membrane , David Andrew Skaff Jan 2006

Release Of Human Brain Hexokinase From The Mitochondrial Membrane , David Andrew Skaff

Retrospective Theses and Dissertations

Type I hexokinase (HKI) is the pacemaker of glycolysis in the brain and red blood cell. Recent work has tied hexokinase association with the mitochondrial membrane to the prevention of apoptosis. With increased hexokinase activity and expression in tumor cells, the importance in understanding the release of HKI from the membrane has become extremely important. Glucose 6-phosphate and nucleotides have been shown to effectively release HKI from the membrane; however, the mechanisms by which these molecules can induce release have never been studied;Expressed recombinant HKI protein was lacking the N-terminus that is required for binding to the mitochondrial membrane ...


Signaling Capabilities Of A Novel H-Ras Mutant From The Golgi Apparatus , Daniel R. Zamzow Jan 2006

Signaling Capabilities Of A Novel H-Ras Mutant From The Golgi Apparatus , Daniel R. Zamzow

Retrospective Theses and Dissertations

The paradigm that H-Ras needs to be attached to the plasma membrane to signal has been put into question in the last decade. New insights into H-Ras signaling point to a biological requirement for signaling from endomembranes.;This thesis will demonstrate the abilities of a unique H-Ras mutant that accumulates on the Golgi.


An Examination Of Hexacoordinate Hemoglobins Using The Techniques Of Biochemistry, Biophysics And Molecular Biology , James Thomas Trent Iii Jan 2006

An Examination Of Hexacoordinate Hemoglobins Using The Techniques Of Biochemistry, Biophysics And Molecular Biology , James Thomas Trent Iii

Retrospective Theses and Dissertations

Hexacoordinate hemoglobins are found in a truly diverse array of organisms, ranging from cyanobacteria to humans. The prevalence of these proteins in nature coupled with very high sequence identity between species homologues suggests they have a role vital to life. The high sequence identity also implies that hxHb structural features, including the hexacoordination phenomenon, are critical aspects of the physiological function. A detailed understanding of the ligand binding behavior of these proteins will not only facilitate efforts towards discovering their physiological role(s), but will also help explain how hxHbs perform their function. This dissertation consists of four published papers ...


Development Of Aptamer Based Targeted Reversibly Attenuated Probes , Xiangyu Cong Jan 2006

Development Of Aptamer Based Targeted Reversibly Attenuated Probes , Xiangyu Cong

Retrospective Theses and Dissertations

The targeted reversely attenuated probe (TRAP) is an aptamer-based biosensor in which the aptamer activity can be regulated by a specific nucleic acid sequence such as an mRNA. The TRAP has the potential of being developed for imaging gene expression in vivo. The central portion of the TRAP, between the aptamer and the attenuator, is complementary to a target nucleic acid, such as an mRNA, which is referred to as a regulatory nucleic acid (regDNA);I developed 8att-cmRas20 and 9att-cmRas15 ATP DNA TRAPs with regDNA-dependent aptamer activity. The results suggested that, as well as inhibiting the aptamer, the attenuator also ...


Cloning, Expression, And Characterization Of A Recombinant Bifunctional Protein -- Porcine Air Carboxylase And Saicar Synthetase , Xin Gao Jan 2006

Cloning, Expression, And Characterization Of A Recombinant Bifunctional Protein -- Porcine Air Carboxylase And Saicar Synthetase , Xin Gao

Retrospective Theses and Dissertations

The conversion of aminoimidazole ribonucleotide (AIR) to carboxyaminoimidazole ribonucleotide (CAIR) by phosphoribosylaminoimidazole carboxylase (AIR carboxylase) and the conversion of conversion of ATP, L-aspartate and CAIR to 5-aminoimidazole-4-(N-succinylcarboxamide) ribonucleotide (SAICAR), ADP and phosphate by phosphoribosylaminoimidazolesuccinocarboxamide synthetase (SAICAR synthetase) represent the 7th and 8th steps respectively of de novo purine nucleotide biosynthesis. For the vertebrate enzyme system, AIR carboxylase and SAICAR synthetase are combined in a bifunctional protein encoded by a single gene. Here, a porcine gene encoding AIR carboxylase/SAICAR synthetase was cloned and expressed in Escherichia coli. The native molecular weight of the purified recombinant protein infers an octameric ...


Analysis Of A Nuclear Complex Of Proteins That Form A Spindle Matrix During Mitosis In Drosophila , Uttama Rath Jan 2005

Analysis Of A Nuclear Complex Of Proteins That Form A Spindle Matrix During Mitosis In Drosophila , Uttama Rath

Retrospective Theses and Dissertations

A spindle matrix is a macromolecular structure hypothesized to provide a stationary support or strut for the anchorage of motor proteins during force generation, microtubule sliding, and chromosome segregation in mitosis. Previous work in Drosophila has shown Skeletor, as the first putative molecular candidate of spindle matrix structure since it possesses many features consistent with the proposed spindle matrix structure.;We have identified two more putative spindle matrix candidate proteins, Chromator and Megator, using yeast two-hybrid interaction assay, cross-immunoprecipitation and immunocytochemistry. Chromator is a novel and essential chromodomain containing protein that interacts directly with Skeletor and extensively colocalizes with it ...


Molecular And Chemical Characterization Of Genes Involved In Maize Cuticular Wax Biosynthesis , M. Ann D. N. Perera Jan 2005

Molecular And Chemical Characterization Of Genes Involved In Maize Cuticular Wax Biosynthesis , M. Ann D. N. Perera

Retrospective Theses and Dissertations

The primary surfaces of the arial parts of plants, and the surfaces of reproductive organs are covered by an extra cellular layer called the cuticle. The cuticle provides the foremost protection to the plant in order to adapt to the terrestrial environment. The cuticle composed of a biopolymer (cutin), which is embedded in a coating of cuticular waxes. Understanding the biochemical, genetic and physiological mechanisms for the biosynthesis of these cuticular waxes is relatively poor. This body of work has established methods for the chemical analysis of the cuticular waxes of maize plants. I have used these methods to chemically ...


Understanding 16s Ribosomal Rna Conformational Changes During 30s Subunit Assembly , Kristi L. Holmes Jan 2005

Understanding 16s Ribosomal Rna Conformational Changes During 30s Subunit Assembly , Kristi L. Holmes

Retrospective Theses and Dissertations

The purpose of this course of study is to understand the conformational changes that occur in 16S rRNA during ribosomal assembly. The prokaryotic ribosome comprises two asymmetric subunits, the large (50S) and small (30S) subunits. The 30S subunit contains 16S rRNA and the small ribosomal proteins (S1--S21). Functional 30S ribosomal subunits can be reconstituted in vitro from purified components. Reconstitution performed at low temperatures results in a stall in 30S subunit assembly. This stall produces a R&barbelow;econstitution I&barbelow;ntermediate (RI) which sediments at 21S and contains 16S rRNA and a subset of small subunit proteins. In order for RI ...


Molecular And Functional Analysis Of Drosophila Hillarin And Its Leech Homolog , Yun Ji Jan 2004

Molecular And Functional Analysis Of Drosophila Hillarin And Its Leech Homolog , Yun Ji

Retrospective Theses and Dissertations

This thesis describes the molecular and functional analysis of the leech Hillarin and its Drosophila homolog D-hil. Leech Hillarin was identified by screening a leech expression library with the mAb Lan3-15, which specifically labels the axon hillock region of leech neurons. Database searches revealed that leech Hillarin has a number of potential homologs in a variety of organisms such as yeast, cyanobacterium, fly, nematode, mouse and human and that these proteins share two novel protein domains, the W180 domain and the H domain. The W180 domain is enriched with tryptophans whereas the H domain shares high number of invariant residues ...


Site-Directed Mutagenesis In Photosystem I Complex: Functional Analysis Of P700 , Maria Pantelidou Jan 2004

Site-Directed Mutagenesis In Photosystem I Complex: Functional Analysis Of P700 , Maria Pantelidou

Retrospective Theses and Dissertations

Photosynthesis is the process by which plants and cyanobacteria convert light to chemical energy. Photosystem I and photosystem II are the two photosynthetic reaction centers that perform light-driven electron transport across thylakoid membranes. Using photon energy, photosystem I oxidizes plastocyanin, transfers the electron through a series of cofactors, and finally reduces ferredoxin. Photosystem I, consists of 12 or more proteins, 96 chlorophyll alpha molecules, 22 beta-carotenes, two phylloquinones, two lipid molecules, and three iron-sulfur clusters. PsaA and PsaB, the largest subunits, surround the electron transfer chain cofactors with a two-fold symmetry. These cofactors are: P700, the primary electron donor chlorophylls ...


Assembly Of The 30s Ribosomal Subunit: S15 As The Probe , Indumathi Jagannathan Jan 2004

Assembly Of The 30s Ribosomal Subunit: S15 As The Probe , Indumathi Jagannathan

Retrospective Theses and Dissertations

The aim of this study is to dissect molecular events that occur during the assembly of 30S subunit of the E. coli ribosome. The 30S ribosomal subunit is made up of 16S ribosomal RNA (rRNA) and 21 proteins (S1--S21). Crystal structure of the 30S subunit has answered longstanding questions on the three-dimensional organization of its constituents. However, this view does not expose the nature of interesting cooperative movements and conformational changes that occur during the formation of this macromolecular complex. Hence, biochemical and genetic efforts are required to understand these rearrangements.;In this study, we have employed directed hydroxyl radical ...


Insights Into The Working Of P-Rex2: An Inositol Polyphosphate 4-Phosphatase Domain Containing Guanine Nucleotide Exchange Factor , Raji Elizabeth Joseph Jan 2004

Insights Into The Working Of P-Rex2: An Inositol Polyphosphate 4-Phosphatase Domain Containing Guanine Nucleotide Exchange Factor , Raji Elizabeth Joseph

Retrospective Theses and Dissertations

Inositol polyphosphate 4-phosphatases (IP4Ps) are enzymes that catalyze the hydrolysis of the 4-position phosphate of phosphatidylinositol 3,4-bisphosphate to form phosphatidylinositol 3-phosphate. Database searches for proteins similar to IP4Ps led to the identification of two novel proteins: P-Rex1 and P-Rex2. These proteins, in addition to sharing similarity with IP4Ps, had a Dbl homology (DH) domain followed by a pleckstrin homology (PH) domain, a characteristic feature of the Dbl family of guanine nucleotide exchange factors (GEFs). P-Rex1 was recently isolated from neutrophils, where it plays a key role in stimulating the neutrophil oxidative burst by activating Rac, a component of the ...


Functional Interactions Among Starch Biosynthetic Enzymes , Seungtaek Kim Jan 2004

Functional Interactions Among Starch Biosynthetic Enzymes , Seungtaek Kim

Retrospective Theses and Dissertations

Understanding the starch biosynthetic mechanism is often hampered by the existence of many isoforms of the involved enzymes as well as numerous pleiotropic effects of mutations affecting these enzymes. This dissertation investigated interactions among starch biosynthetic enzymes, focusing on both physical association of the proteins and functional interactions in a transgenic in vivo system. Specific protein-protein interactions among the starch biosynthetic enzymes that were indicated by yeast two-hybrid experiments were confirmed directly by affinity chromatography. The functional significance of such interactions, specifically between starch synthase and starch branching enzyme, was studied using heterologous expression in the yeast Saccharomyces cerevisiae. A ...


Structural And Molecular Analysis Of Leech Neuronal Proteins, Calsensin And Filamin , Deepa V. Venkitaramani Jan 2004

Structural And Molecular Analysis Of Leech Neuronal Proteins, Calsensin And Filamin , Deepa V. Venkitaramani

Retrospective Theses and Dissertations

This thesis describes the structural and molecular analysis of two leech neuronal proteins, Calsensin and Filamin. Calsensin is an EF-hand calcium-binding protein expressed by a subset of peripheral sensory neurons which fasciculate into a single tract in the leech central nervous system. Calsensin is a 9 kD protein with two EF-hand calcium-binding motifs. Calsensin can multimerize via disulfide bridge formation involving the two cysteine residues in vitro. Furthermore, Calsensin shows differential gel migration depending on the presence or absence of calcium suggesting a conformational change upon calcium binding. Using multidimensional NMR spectroscopy we have determined the solution structure and backbone ...


The Essential Jil-1 Kinase Regulates Histone H3 Phosphorylation And Maintains Chromatin Structure In Drosophila , Weiguo Zhang Jan 2004

The Essential Jil-1 Kinase Regulates Histone H3 Phosphorylation And Maintains Chromatin Structure In Drosophila , Weiguo Zhang

Retrospective Theses and Dissertations

JIL-1 encodes a chromatin-associated tandem serine/threonine kinase in Drosophila melanogaster. JIL-1 predominantly associates with interbands on polytene chromosomes. It appears at higher levels on the X chromosome than on autosomes in males, and distributes about equally on all chromosomes in females. In addition, in males JIL-1 overlaps with the MSL (for male specific lethal) complex that specifically associates with the male X chromosome on which expression of most genes are upregulated.;We isolated JIL-1 mutants by inducing imprecise excision of an EP element inserted in JIL-1. We found that JIL-1 is required for viability of both females and males ...


Structural And Biochemical Insight Into The Inositol Polyphosphate 4-Phosphatase Family Of Proteins , Colin Tucker Shearn Jan 2004

Structural And Biochemical Insight Into The Inositol Polyphosphate 4-Phosphatase Family Of Proteins , Colin Tucker Shearn

Retrospective Theses and Dissertations

Inositol polyphosphate 4-phosphatases (IP4Ps) are lipid phosphatases that remove the 4-position phosphate from the lipid second messenger phosphatidylinositol 3,4-bis-phosphate converting it to phosphatidylinositol 3-phosphate. There are 2 families of IP4P's that are 37% homologous to each other denoted type I and type II. Previously, several different isoforms of both type I and type II IP4P had been identified. These include the beta-spliceoforms of both type I and II that contained a hydrophobic tail instead of a hydrophilic tail and a novel deletion spliceoform found in brain tissue that is approximately 1kDa smaller than type Ialpha called type Ialpha2 ...


Escherichia Coli 30s Ribosomal Subunit Assembly: A Novel Role For The Dnak Chaperone System , Jennifer Anne Maki Jan 2004

Escherichia Coli 30s Ribosomal Subunit Assembly: A Novel Role For The Dnak Chaperone System , Jennifer Anne Maki

Retrospective Theses and Dissertations

The prokaryotic ribosome is a 2.5 MDa particle comprised of two asymmetric subunits, the large (50S) and small (30S) subunits. The large subunit contains two RNAs (5S and 23S) in addition to thirty-four proteins. The small subunit consists of one RNA (16S rRNA) and twenty-one proteins. Although the crystal structure has been solved, much remains to be revealed concerning the assembly of this macromolecular structure. Our laboratory is focused on the assembly of the small subunit. In vitro assembly of this structure was achieved in the late 1960's and early 1970's. At low temperature when 16S rRNA ...


Identification And Functional Analysis Of Thylakoid Membrane Proteome , Yingchun Wang Jan 2003

Identification And Functional Analysis Of Thylakoid Membrane Proteome , Yingchun Wang

Retrospective Theses and Dissertations

Membrane proteins play crucial roles in many biological functions. Identities and functions of most membrane proteins remain to be revealed. New technological breakthroughs in proteomics together with the availability of genomic sequence information make it possible to study functions of membrane proteins on a genome-wide scale. We used a multidisciplinary approach combining biochemistry, genetics, proteomics and bioinformatics to study the functions of the thylakoid proteome of Synechocystis sp. PCC6803. The thylakoid membrane proteins were separated into peripheral and integral fractions and resolved into 2-D gels with different pH ranges. The protein spots in the 2-D gels were subjected to peptide ...


Roles Of The Mitogen-Induced Proteins, Mitogen Regulated Protein/Proliferin And 24p3/Uterocalin, During Mouse Development , Pierig Jean-Marie Lepont Jan 2002

Roles Of The Mitogen-Induced Proteins, Mitogen Regulated Protein/Proliferin And 24p3/Uterocalin, During Mouse Development , Pierig Jean-Marie Lepont

Retrospective Theses and Dissertations

Mitogens, serum and growth factors, induce the secretion of several proteins in cell cultures. Mitogen regulated protein/proliferin (MRP/PLF) is one such protein secreted from 3T3 cells 20 hours after the addition of fibroblast growth factor (FGF). 24p3/uterocalin is another protein induced 10 hours after FGF addition. When combined with cycloheximide, 24p3/uterocalin secretion is increased by 5 fold.;MRP/PLFs are glycoproteins, belonging to the prolactin-growth hormone family, expressed at high concentration during mid-gestation in the mouse placenta. MRP/PLFs are thought to stimulate angiogenesis in the placenta and the uterus, and stimulate uterine cell growth during ...


Proline Cis/Trans Isomerization Regulates A T Cell Specific Tyrosine Kinase , Kristine Nicole Brazin Jan 2002

Proline Cis/Trans Isomerization Regulates A T Cell Specific Tyrosine Kinase , Kristine Nicole Brazin

Retrospective Theses and Dissertations

This dissertation examines the role of protein-protein interactions that regulate the conformation and function of the Tec family kinase, interleukin-2 tyrosine kinase (Itk). Tec family kinases are expressed in hematopoetic cells and modulate intracellular signaling cascades in response to external stimuli. The domain structure of Tec family members contains the conserved SH3, SH2, and catalytic domains common to many kinase families, yet they are distinguishable by the presence of a unique N-terminal sequence. The mechanism by which Itk is regulated is not well understood. Both nuclear magnetic resonance spectroscopy and functional assays were used to elucidate a novel regulatory mechanism ...


Conformational Variability In Adenylosuccinate Synthetase As Revealed By Crystal Structures Of Mouse-Muscle And Escherichia Coli Enzymes , Cristina Valeria Iancu Jan 2002

Conformational Variability In Adenylosuccinate Synthetase As Revealed By Crystal Structures Of Mouse-Muscle And Escherichia Coli Enzymes , Cristina Valeria Iancu

Retrospective Theses and Dissertations

Adenylosuccinate synthetase governs the committed step of AMP biosynthesis from IMP: the generation of 6-phosphoryl-IMP from GTP and IMP, followed by the formation of adenylosuccinate from 6-phosphoryl-IMP and L-aspartate. Prokaryotes such as Escherichia coli (E. coli) have a single form of adenylosuccinate synthetase but vertebrates have two isozymes of the synthetase. The basic isozyme, which predominates in muscle, participates putatively in the purine nucleotide cycle, has a higher Km for IMP and exhibits substrate inhibition by this nucleotide. Compared to E. coli structures, there are conformational variations in the IMP pocket both in the ligand-free and IMP-ligated structures of the ...


Identification And Characterization Of Proteins Expressed In The Nectar Of Ornamental Tobacco , Clay Jeremy Carter Jan 2002

Identification And Characterization Of Proteins Expressed In The Nectar Of Ornamental Tobacco , Clay Jeremy Carter

Retrospective Theses and Dissertations

A small set of proteins, termed Nectarins, are secreted into the nectar of ornamental tobacco (Nicotiana langsdorffii x Nicotiana sanderae). The most highly expressed protein, Nectarin I (29 kD monomer), was originally identified as a germin-like protein (GLP) of unknown function. This remarkably stable protein was later shown to possess manganese dependent superoxide dismutase activity. Hydrogen peroxide, the product of SOD activity, was found to accumulate in nectar at levels up to 4 mM. The promoter (1.2 kb) of the Nectarin I gene was fused to the reporter gene chloramphenicol acetyl transferase (CAT) and shown to be expressed in ...


Irreversible Oxidation Of Protein Cysteine Residues , Michael James Hamann Jan 2002

Irreversible Oxidation Of Protein Cysteine Residues , Michael James Hamann

Retrospective Theses and Dissertations

Cysteine residues have a reduced thiol group that frequently exerts an influence on the function of proteins. Oxidation of the cysteine through S-thiolation, S-nitrosylation, or irreversible oxidation perturbs these functions. Sulfinic and sulfonic acids produced by irreversible oxidation are exceptionally damaging since these modifications are permanent in cells. In order to detect and quantitate both protein sulfinic and sulfonic acids, it was necessary to develop an assay using a novel amino acid analysis procedure. Purified creatine kinase and carbonic anhydrase III were used to establish the method, and the assay produced results that agreed with total cysteine irreversible oxidation determined ...


Characterization Of The De Novo Pyrimidine Biosynthetic Pathway In Arabidopsis Thaliana , Christopher W. Kafer Jan 2002

Characterization Of The De Novo Pyrimidine Biosynthetic Pathway In Arabidopsis Thaliana , Christopher W. Kafer

Retrospective Theses and Dissertations

We have characterized the enzyme cytidine deaminase. The full length cDNA was expressed in E. coli and purified to apparent homogeneity using a Ni-NTA agarose column. The Km and Vmax for cytidine were determined to be 226uM and 39.7 umoles/min respectively. The enzyme was also able to use 2'-deoxycytidine as substrate with an apparent Km of 49 uM and Vmax of 24 umoles/min.;We have also developed a single tube semi-quantitative relative reverse transcription polymerase chain reaction (RT-PCR) assay. We have used this assay to quantitate the relative transcript levels of the UMP synthase gene and ...


Allosteric Regulation Of Fructose-1,6,-Bisphosphatase , Scott William Nelson Jan 2002

Allosteric Regulation Of Fructose-1,6,-Bisphosphatase , Scott William Nelson

Retrospective Theses and Dissertations

Fructose-1,6-bisphosphatase (FBPase) catalyzes the reaction of fructose-1,6-bisphosphate to fructose-6-phosphate and inorganic phosphate. The investigations described here concern the structure and function of FBPase, with special focus on those properties relating to the allosteric regulation of catalysis. It is our hypothesis that a loop spanning residues 52 through 72 plays a crucial role in the mechanism of allosteric regulation. Testing this hypothesis, a fluorescent reporter group was incorporated into loop 52-72, confirming the existence of three states of the loop dependent on the state of ligation: disordered, disengaged, and engaged. Furthermore, restriction of backbone flexibility by replacement of two ...