Life Sciences Commons^™

Structural And Mechanistic Investigations Of Phosphothreonine Lyase Class Of Enzymes, Alok Gopalkrishna Shenoy

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Phosphorylation and dephosphorylation are a highly pervasive mechanism in biology that is used by the cell to modulate enzymes and proteins. The presence of a phosphate group can activate or deactivate an enzyme. The phosphate group is linked to a protein by a phosphoester bond that is known to be highly stable in cytoplasmic pH range. Thus the breaking and formation of these bonds need to be effected by enzymes.

Recent discovery of the activity carried out by certain virulence related proteins (OspF released by Shigella and SpvC released by Salmonella) have resulted in a necessity to create a new …

Biochemical Characterization Of Sac9, A Putative Phosphoinositide Phosphatase In Arabidopsis Thaliana, And Its Role In Cellular Abnormalities, Almut H. Vollmer

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Since the early colonization of land by fungi and plants some 700 million years ago, plants have been continuously faced with changes in their environment. Unlike animals, plants are not free to move about, and can therefore not evade many stress factors. How plants sense and respond to their environment has been of interest not only to scientific research but also in more practical applications such as agriculture.

Signals (such as light or salinity) from the outside of plant cells trigger a flow of information to the inside of the cell. The final target for most of the information is …

Studies Of The Distinguishing Features Of Nadph:2-Ketopropyl-Coenzyme M Oxidoreductase/Carboxylase, An Atypical Member Of The Disulfide/ Oxidoreductase Family Of Enzymes, Melissa A. Beighley-Kofoed

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

The metabolism of propylene in Xanthobacter autotrophicus occurs via epoxypropane formation and subsequent metabolism by a three-step, four-enzyme pathway, utilizing the atypical cofactor Coenzyme M (CoM) to form acetoacetate. The last step in the epoxide carboxylase pathway is catalyzed by a distinctive member of the disulfide oxidoreductase (DSOR) family of enzymes, NADPH:2-ketopropyl CoM oxidoreductase/carboxylase (2-KPCC). 2-KPCC catalyzes the unorthodox cleavage of a thioether bond and successive carboxylation of the substrate. The focus of the research presented in this dissertation aims to elucidate the details of 2-KPCC that allow it to perform chemistry unconventional for typical DSOR members. Sitedirected mutagenesis was …

Kinetic, Mechanistic, And Structural Investigation Of Features Controlling Stereoselectivity Of (R)- And (S)-Hydroxypropyl Com Dehydrogenases From Xanthobacter Autrophicus Strain Py2, Dariusz Adam Sliwa

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Enantiopure alcohols are valuable intermediates in fine organic synthesis, in particular for preparation of biologically active compounds. The necessity of preparing single enantiomer drugs in an optically pure form has triggered much research, especially in the pharmaceutical industry. The biocatalytical production of chiral alcohols by alcohol dehydrogenase enzymes is characterized by the asymmetric reduction of the corresponding ketones, usually with high degree of stereoselectivity. The commercial value of the enzymes as stereoselective biocatalysts has been a significant driving force in understanding features that control their mechanism of catalysis and stereoselectivity. This work focuses on two enantiocomplementary dehydrogenase enzymes ((R …

Physiological And Molecular Function Of Hap3b In Flowering Time Regulation And Cold Stress Response, Mingxiang Liang

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Heme-activated proteins (HAPs) are transcription factors that have multiple roles in plant growth and development, such as embryogenesis, flowering time control, and drought tolerance.

In the present study I found that HAP3b was also involved in controlling response to cold stress. Transcript profiling and gene expression analyses indicated that HAP3b repressed the CBF3 regulon under normal growth conditions. As a result, plants with HAP3b-overexpressed showed decreased survival rates while plants homozygous for the null allele hap3b showed an improved freezing tolerance compared to wild-type plants.

To understand the mechanism of HAP3b in Arabidopsis, i.e. whether it also acts …

Host Signaling Response To Adhesion Of Bifidobacterium Infantis, Reed N. Gann

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Investigations of the molecular binding partners of the probiotic bacterium Bifidobacterium longum subspecies infantis (B. infantis) and the pathogen Salmonella enterica subspecies enterica serovar Typhimurium LT2 (Salmonella ser. Typhimurium) found that these two very different bacteria bind gangliosides. However, these organisms lead to completely different host health outcomes when present in the gut. B. infantis is the founding microbial population in the intestinal tract of breast-fed infants. S. typhimurium is the most important food-borne pathogen that results in humans. This study used an in vitro gut epithelial cell model to examine the host cellular response to adhesion …

Role Of Protein Kinase C Isotypes In 1,25-Dihydroxyvitamin D3 Mediated Signal Transduction Through The 1,25d3 Membrane Associated, Rapid Response Steroid Binding Receptors In Chick Intestinal Cells, Sakara Tunsophon

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

It is now accepted that 1,25(OH)2D3 mediates its rapid actions on the control of phosphate and calcium homeostasis through its membrane receptor termed the 1,25D3-MARRS (membrane associated rapid response steroid binding) protein. I determined the various PKC isotypes involved in the rapid regulation of phosphate uptake and calcium extrusion in chick intestinal cells. 1,25(OH)2D3-mediated phosphate uptake was stimulated within 1 min after addition of the hormone. Western blot analyses on isolated intestinal cells treated with steroid hormone resulted in dose-dependent increases in PKC alpha and PKC beta in postnuclear centrifugation fractions, but not in the low speed centrifugation fractions. The …

A Synthetic Biological Engineering Approach To Secretion- Based Recovery Of Polyhydroxyalkanoates And Other Cellular Products, Elisabeth Linton

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

The costs associated with cellular product recovery commonly account for as much as 80% of the total production expense. As a specific example, significant recovery costs limit commercial use of polyhydroxyalkanoates (PHA), which comprise a class of microbially-accumulated polyesters. PHAs are biodegradable compounds that are of interest as a sustainable alternative to petrochemically-derived plastics. Secretion-based recovery of PHAs was studied to decrease PHA production costs. Type I and II secretory pathways are commonly used for the translocation of recombinant proteins out of the cytoplasm of E. coli. Proteins were targeted for translocation using four signal peptides (HlyA, TorA, GeneIII, …

Development Of Novel Methods And Their Utilization In The Analysis Of The Effect Of The N-Terminus Of Human Protein Arginine Methyltransferase 1 Variant 1 On Enzymatic Activity, Protein-Protein Interactions, And Substrate Specificity, Brenda Bienka Suh-Lailam

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Protein arginine methyltransferases (PRMTs) are enzymes that catalyze the methylation of protein arginine residues, resulting in the formation of monomethylarginine, and/or asymmetric or symmetric dimethylarginines. Although understanding of the PRMTs has grown rapidly over the last few years, several challenges still remain in the PRMT field. Here, we describe the development of two techniques that will be very useful in investigating PRMT regulation, small molecule inhibition, oligomerization, protein-protein interaction, and substrate specificity, which will ultimately lead to the advancement of the PRMT field. Studies have shown that having an N-terminal tag can influence enzyme activity and substrate specificity. The first …

Transesterification And Recovery Of Intracellular Lipids Using A Single Step Reactive Extraction, Daniel R. Nelson

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

A single-step, extractive reaction for extraction of lipids such as biodiesel components, omega-3 fatty acids, or other triglycerides from microbial cells was examined. Conventional methods for lipid extraction use toxic solvents, and require multiple steps and long processing times. When the goal is to produce fatty acid methyl esters or FAMEs, the extracted lipids are subjected to a separate transesterification reaction with simple alcohols in the presence of an acid or base catalyst. A simplified, single-step reactive extraction method can be applied that combines the sequential extraction followed by transesterification using acidified alcohols - a process known as in situ …

A Comparative Study Of The Structural Features And Kinetic Properties Of The Mofe And Vfe Proteins From Azotobacter Vinelandii, Miguel Alejandro Pabon Sanclemente

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Biological nitrogen fixation is accomplished in the bacterium Azotobacter vinelandii by means of three metalloenzymes: The molybdenum, vanadium, and iron-only nitrogenase. The knowledge regarding biological nitrogen fixation has come from studies on the Mo-dependent reaction. However, the V- and Fe-only-dependent reduction of nitrogen remains largely unknown.

By using homology modeling techniques, the protein folds that contain the metal cluster active sites for the V- and Fe-only nitrogenases were constructed. The models uncovered similarities and differences existing among the nitrogenases regarding the identity of the amino acid residues lining pivotal structural features for the correct functioning of the proteins. These differences, …

Characterization Of The Substrate Specificity And Mechanism Of Protein Arginine Methyltransferase 1, Whitney Lyn Wooderchak

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Protein arginine methyltransferases (PRMTs) posttranslationally modify protein arginine residues. Type I PRMTs catalyze the formation of monomethylarginine (MMA) and asymmetric dimethylarginine (ADMA) via methyl group transfer from S-adenosyl methionine onto protein arginine residues. Type II PRMTs generate MMA and symmetric dimethylarginine. PRMT-methylation affects many biological processes. Although PRMTs are vital to normal development and function, PRMT-methylation is also linked to cardiovascular disease, stroke, multiple sclerosis, and cancer.

Thus far, nine human PRMT isoforms have been identified with orthologues present in yeast, plants, and fish. PRMT1 predominates, performing an estimated 85% of all protein arginine methylation in vivo. Yet, the substrate …

Expression Analysis Of Plant Defense Responses During The Establishment Of Biotrophy And Role Of Abiotic Stress In The Infection Of Dyer’S Woad (Isatis Tinctoria) By Puccinia Thlaspeos, Elizabeth Thomas

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

The kinetics and amplitude of the salicylic acid-responsive pathogenesis-related (PR) genes and the cytochrome P450 gene ItCYP79B2 in the compatible interaction between Puccinia thlaspeos and dyer’s woad (Isatis tinctoria) during the first 72 hours of inoculation were examined. Immediately following penetration of the host by the rust pathogen, there was a modest up-regulation of PR genes but a significant down-regulation of ItCYP79B2 expression. During haustoria formation, a significant pathogen-mediated suppression of PR genes was observed with a corresponding up-regulation of ItCYP79B2. This potentially facilitates haustoria formation by P. thlaspeos. After haustoria formation, a more …

Role Of Iron In The Mechanism Of Asbestos-Induced Apoptosis In Human Lung And Pleural Target Cells, Aleksander Baldys

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Occupational exposure to asbestos has been associated with increased incidence of pulmonary interstitial fibrosis, mesothelioma of the pleura, and bronchogenic carcinoma. Although the mechanism by which asbestos causes cancer remains unknown, iron associated with asbestos is thought to play a role in the pathogenic effects of fibers.

The aim of this research was to examine and compare the asbestos-induced signaling phenomena in relevant human lung and pleural target cells, and to determine the role of iron from asbestos fibers in these events. Exposure of human airway epithelial (A549) cells, human pleural mesothelial (MET5A) cells, and normal human small airway epithelial …

Studies On The Roles Of Atp In Nitrogenase Catalysis, Wei Wu

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Nitrogenase is the enzyme that catalyzes the reduction of nitrogen to ammonia in a reaction requiring MgATP hydrolysis. Two component proteins of nitrogenase are the iron protein (Fe protein) and the molybdenum-iron protein (MoFe protein).

Nitrogenase contains two nucleotide binding sites. During catalysis, the Fe protein binds two MgATP first. The conformational changes induced upon MgATP binding allow the Fe protein to associate with the MoFe protein. After the formation of the Fe protein-MoFe protein complex, a single electron is transferred from the Fe protein to the MoFe protein, an event that is coupled to MgATP hydrolysis in the Fe …

Phylogenetic Studies Of The United States Bluetongue Viruses And Characterization Of The Viral Vp4 Protein, I-Jen Huang

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Bluetongue virus (BTV) is transmitted by arthropod vectors and causes bluetongue disease with serious economic loss in many regions of the world. The replication mechanism of bluetongue virus is still not clear. To have a better understanding regarding the viral replication, the function of each individual protein has to be identified. This study used molecular biology techniques to investigate the function of the inner core protein VP4.

The M1 genes of United States bluetongue virus serotypes-2, -10, -11, -13, and -17 were cloned and sequenced. The length of each of the five M1 genes is 1981 nucleotides. The coding region …

Characterization Of An Axial Ligand Substitution In Sperm Whale Myoglobin, Michael J. Chen

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Of central importance to the study of heme proteins are the effects imposed by axial ligand(s) on the heme structure and, therefore, on the overall activity of the protein. In this study, we confirm and extend the spectroscopic characterization of a mutated sperm whale myoglobin in which the proximal Histidine is replaced with a Tyrosine residue (MbH93Y). The MbH93Y, as well as wild-type sperm whale myoglobin and horse erythrocyte catalase (HEC), was purified and characterized by optical absorption and x-ray absorption (XAS) spectroscopies. Optical absorption spectra of HEC and the metmyoglobin, cyanometmyoglobin, reduced, oxy, and carbon-monoxy forms of both sperm …

Extended X-Ray Absorption Fine Structure And Redox Potential Studies Of Heme-Substituted Horseradish Peroxidase And Myoglobin, Bing He

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Heme-substituted horseradish peroxidases and myoglobins were reconstituted from the apoenzyme using mesoheme and diacetyldeuteroheme. X-ray absorption spectroscopy was used to determine the dimensions of the active sites of these heme-substituted proteins, and were compared with those of the proto-hemeproteins. The change in the active-site structure corresponded with the electron withdrawing and donating effects of the different side chains. The oxidation-reduction potentials of Fe⁴⁺/Fe³⁺ couples of the heme-substituted proteins were measured at pH 7 with K₂IrCl₆. The oxidation-reduction potential sequence for compound I/compound II was diacetyldeutero- > proto- > meso- in horseradish peroxidase. The oxidation-reduction potential …

Mobilization Of Iron Enhances The Iron-Dependent Biochemical Reactivity Of Asbestos In Vitro And Contributes To The Cytotoxicity Of Asbestos In Cultured Cells, Loren Glen Lund

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Asbestos related research began approximately 60 years ago, yet, the mechanism(s) by which asbestos exerts its biological effects is not well understood. The hypothesis upon which this dissertation is based is that mobilization of iron from asbestos enhances the iron-dependent biochemical reactivity of asbestos in vitro and contributes to asbestos-dependent cytotoxicity. The specific aims for this hypothesis were, 1) to determine whether iron was responsible for the biochemical reactivity of asbestos in vitro and asbestos-induced cytotoxicity in cultured cells, and 2) to determine whether mobilization of iron from asbestos enhanced the reactions catalyzed by asbestos in vitro and contributes to …

Energetic Requirements For Bacterial Protein Export, John Andrew Corbett

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Bacterial protein export involves the translocation of precursor proteins across the inner cytoplasmic membrane. Over 100 proteins are exported from Escherichia coli. This study showed that energy in the form of ATP and membrane gradient energy is essential for the export of leucine specific binding protein and β-lactamase precursors. Ionophores or combinations of ionophores (SF6847, valinomycin/nigericin and valinomycin/monensin) which dissipate protonmotive force inhibit protein export. Valinomycin alone also inhibits export, but not as well as reagents which dissipate protonmotive force. Nigericin or monensin alone slightly stimulate protein export. These results suggest that the transmembrane electrical potential (∆Ψ) is the …

A Study Of Denitrosation Of N-Nitroso Compounds By Irradiation With Long-Wavelength Uv Light, Zhenyu J. Wang

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

N-Nitrosamines and N-nitrosamides have been reported to be mutagenic. N-Nitrosamides are direct mutagens that need no activation to be mutagenic, whereas N-nitrosamines need to be enzymatically activated to exert their mutagenicity. Oxidative demethylation of nitrosamines is a commonly accepted activation mechanism. Another pathway of nitroso compounds, denitrosation, has recently been proposed. The mechanism of denitrosation, however, is still unknown.

The purpose of this study was to use a photo -reaction model to explore the possible denitrosation mechanism of N-nitroso compounds. An N-nitrosamine, N-nitrosomorpholi ne (NMOR), and an N-nitrosamide, N-methyl-N'-nitrosoguanidine (MMNG), were irradiated with long-wavelength UV light in the presence of …

Kinetics Of Two-Substrate Reactions Using Integrated Rate Equations, Leonard D. Holmes

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

The usual procedure employed in enzyme kinetic analysis is the method of initial rates. However, it has been appreciated for years that the analysis of enzyme-catalyzed reactions could, in principle, be more efficiently performed by examining the entire time course. There is much more information contained in a progress curve than in a simple initial rate. With the appearance of the computer, the formidable computations necessary for the use of integrated-rate equations are quite possible. The intention of this research was to develop the analytical and statistical methodology for applying an integrated-rate equation to a two-substrate reaction. I have analyzed …

Mechanism Of Iron Transport In Mycelia Sterilia Ep-76, Jonathan P. Adjimani

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

The cyclic trihydroxamic acid, N, N', N' '-triacetylfusarinine C, produced by Mycelia sterilia EP-76, is shown to be a ferric ionophore for this organism. The association constant for ferric-N, N', N' '-triacetylfusarinine C complex was determined to be log K=32.5. Other iron chelating agents, such as rhodotorulic acid, citric acid, or the monomeric subunit of triacetylfusarinine C, N-acetyl- fusarinine, delivered iron to the cells by an indirect mechanism involving iron exchange into triacetylfusarinine C. In vitro ferric ion exchange was found to be rapid with triacetylfusarinine C. Gallium uptake rates comparable to those of iron were observed with the chelating …

An Improved Method For Transferring Nucleic Acids To Nylon Membranes, Bruce D. Parker

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Methods currently in use for the separation and identification of specific segments of nucleic acids involve long transfer periods or elaborate apparatuses and result in the production of a single blot. Contamination by organisms or enzymes is always a factor to be dealt with. An improved method for transferring nucleic acids from acrylamide or agarose gels for use in hybridization has been developed. This method uses NaOH as the blotting medium to improve the rate and efficiency of transfer to nylon membranes. As many as six blots can be obtained within one hour using this method. This method is effective …

Time Course Of Enzyme Catalyzed Reactions: The Stoichiometry A --> P + Q, Tamra Tuttle

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

The feasibility of using an integrated rate equation to analyze the kinetics of a second-order, enzyme-catalyzed reaction has been investigated. The inducible arginine decarboxylase from Escherichia coli B. was chosen for this study because it catalyzes an irreversible reaction with stoichiometry A --> P + Q, the simplest second order case. Values for five of the eight possible kinetic constants were determined from 21 time courses. Of the remaining three, the uncompetitive product inhibition constant for CO₂ was shown to be between 0 and 0.06, while the values of the competitive product inhibition for CO₂ and the uncompetitive …

Photoaffinity Labeling Of The Antimycin Binding Site In Rhodopseudomonas Sphaeroides, Emily Wilson

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

The purpose of this study was to identify the site of interaction of antimycin with the ubiquinone-cytochrome b-c1 oxidoreductase in the photosynthetic bacteria, Rhodopseudomonas sphaeroides. To accomplish this goal, three areas of research were undertaken: the synthesis of a radiolabeled, photoaffinity analog of antimycin, identification of the inhibitory characteristics of this analog, and the photoaffinity labeling of the antimycin binding site. All three areas were accomplished.

The major finding of this study was the identification of an 11,000 dalton polypeptide as the predominantly labeled protein. Although this polypeptide was not exclusively labeled, it was consistently labeled and showed competition …

Hemoglobins Of The Cutthroat Trout Salmo Clarki, Jonathan N. Southard

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Nine hemoglobins have been isolated from the blood of cutthroat trout. All nine hemoglobins bind oxygen cooperatively and appear to be tetramers with molecular weights of -64,000. The oxygen equilibria and subunit structures of the purified hemoglobins were studied. In addition, the red blood cells of cutthroat trout were examined for the presence of ATP and GTP, which are known to be physiological modulators of hemoglobins in fishes.

Five hemoglobins with isoelectric points from 9.1 to 7.0 are classified as cathodal hemoglobins. These five hemoglobins have identical oxygen binding properties by the criteria tested. All have oxygen equilibria which are …

Subunit Interactions In The Inducible Arginine Decarboxylase From Escherichia Coli B, Catalina N. Depusoy

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

The nature of the subunit interactions in the inducible arginine decarboxylase from Escherichia coli B is of considerable interest because of the observed differences in the catalytic activities of the dimer and the decamer; the decamer is active and the dimer is inactive. To study these interactions, inactive dimers were prepared by sodium borohydride reduction of the E-amino--pyridoxal-P Schiff base. Hybrid decamers were then prepared from varying molar ratios of native and reduced dimers. The hybrid decamers were indistinguishable from native decamers as observed in the analytical ultracentrifuge and on acrylamide gel electrophoresis. Kinetic studies indicated that true hybrids were …

A Study Of The Action Of Bovine Cathepsin D On Intramuscular Connective Tissue, Elimosaria Elikalia Maeda

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

Cathepsin D has been postulated to play a role in the tenderization of meat with ageing. Meat toughness has been related to connective tissue content and degree of myofibrillar shortening. This investigation was undertaken to determine the activity of cathepsin D on bovine intramuscular connective tissue.

Highly purified cathepsin D preparations from bovine spleen and bovine sternomandibularis muscle were obtained by ammonium sulfate fractionation followed by affinity chromatography on pepstatin coupled to controlled pore size alkyl amine glass beads. Maximum proteolytic activity using hemoglobin as substrate was obtained at pH 3.5 and 3.8 for bovine spleen and muscle cathepsin D, …

The Isolation And Characterization Of A Novel Iron Protein From Desulfovibrio Gigas, Alan Kay Smith

All Graduate Theses and Dissertations, Spring 1920 to Summer 2023

The isolation, purification, and partial characterization of a novel iron-containing protein from the sulfate-reducing anaerobic bacterium, Desulfovibrio gigas, is described. The highly insoluble protein was isolated from the cell debris following osmotic shock of the bacteria. The insoluble fraction consistently contained about 90% of the cell-associated iron. This protein was treated with acid, chelating agents, detergents and proteases in order to study their effect on the iron solubility. The results of elemental analysis of a crude protein preparation were 5.3% iron, 2.9% sulfur and 11 .9% nitrogen. An independent colorimetric iron analysis showed 6.4% iron. The iron could be …