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  Partner: UNT Libraries
 Degree Discipline: Biochemistry
 Collection: UNT Theses and Dissertations
NSAID effect on prostanoids in fishes: Prostaglandin E2 levels in bluntnose minnows (Pimephales notatus) exposed to ibuprofen.

NSAID effect on prostanoids in fishes: Prostaglandin E2 levels in bluntnose minnows (Pimephales notatus) exposed to ibuprofen.

Date: August 2009
Creator: Bhandari, Khageshor
Description: Prostanoids are oxygenated derivatives of arachidonic acid with a wide range of physiological effects in vertebrates including modulation of inflammation and innate immune responses. Nonsteroidal anti-inflammatory drugs (NSAIDs) act through inhibition of cyclooxygenase (COX) conversion of arachidonic acid to prostanoids. In order to better understand the potential of environmental NSAIDS for interruption of normal levels COX products in fishes, we developed an LC/MS/MS-based approach for tissue analysis of 7 prostanoids. Initial studies examining muscle, gut and gill demonstrated that prostaglandin E2 (PGE2) was the most abundant of the measured prostanoids in all tissues and that gill tissue had the highest and most consistent concentrations of PGE2. After short-term 48-h laboratory exposures to concentrations of 5, 25, 50 and 100 ppb ibuprofen, 50.0ppb and 100.0 ppb exposure concentrations resulted in significant reduction of gill tissue PGE2 concentration by approximately 30% and 80% respectively. The lower exposures did not result in significant reductions when compared to unexposed controls. Measured tissue concentrations of ibuprofen indicated that this NSAID had little potential for bioaccumulation (BCF 1.3) and the IC50 of ibuprofen for inhibition of PGE2 production in gill tissue was calculated to be 0.4 µM. Short-term laboratory exposure to ibuprofen did not result in ...
Contributing Partner: UNT Libraries
Identifying genetic interactions of the spindle checkpoint in Caenorhabditis elegans.

Identifying genetic interactions of the spindle checkpoint in Caenorhabditis elegans.

Date: May 2009
Creator: Stewart, Neil
Description: Faithful segregation of chromosomes is ensured by the spindle checkpoint. If a kinetochore does not correctly attach to a microtubule the spindle checkpoint stops cell cycle progression until all chromosomes are attached to microtubules or tension is experienced while pulling the chromosomes. The C. elegans gene, san-1, is required for spindle checkpoint function and anoxia survival. To further understand the role of san-1 in the spindle checkpoint, an RNAi screen was conducted to identify genetic interactions with san-1. The kinetochore gene hcp-1 identified in this screen, was known to have a genetic interaction with hcp-2. Interestingly, san-1(ok1580);hcp-2(ok1757) had embryonic and larval lethal phenotypes, but the phenotypes observed are less severe compared to the phenotypes of san-1(ok1580);hcp-1(RNAi) animals. Both san-1(ok1580);hcp-1(RNAi) and san-1(ok1580);hcp-2(RNAi) produce eggs that may hatch; but san-1(ok1580):hcp-1(RNAi) larvae do not survive to adulthood due to defects caused by aberrant chromosome segregations during development. Y54G9A.6 encodes the C. elegans homolog of bub-3, and has spindle checkpoint function. In C.elegans, bub-3 has genetic interactions with san-1 and mdf-2. An RNAi screen for genetic interactions with bub-3 identified that F31F6.3 may potentially have a genetic interaction with bub-3. This work provided genetic evidence that hcp-1, hcp-2 and F31F6.2 interact with spindle checkpoint ...
Contributing Partner: UNT Libraries
Stretching the Flexible Myosin II Subfragment Using the Novel Gravitational Force Spectroscope, and the Uncoiling of S2

Stretching the Flexible Myosin II Subfragment Using the Novel Gravitational Force Spectroscope, and the Uncoiling of S2

Date: May 2010
Creator: Dunn, James W.
Description: Familial Hypertrophic cardiomyopathy (HCM) causes ventricle walls to thicken and often leads to sudden death especially in adults. Mutations in the subfragment 2 (S2) of β-cardiac myosin are implicated in the genetic disorder. This S2 region is a coiled-coil rod region resulting from the dimeric form of myosin II. It has been proposed that an elastic quality allows normal S2 to absorb force during the powerstroke according to the sliding filament model. To test the flexibility of single molecules of S2 against levels of physiological force, the Gravitational Force Spectrometer (GFS) is being developed. This novel system employs a standard microscope on an equatorial mount that allows the spectrometer to be rotated freely in space. Stationary glass beads are attached to a microscope slide where the molecule is tethered between the stationary bead and a smaller mobile bead. The GFS is oriented so that the force of gravity can act on the mobile bead and so impart a small force to the tethered subfragment. Additionally, a video system in conjunction with ImageJ software makes a distance measurement of the molecule possible with a resolution of around 11 nm. The S2 can be stretched parallel or perpendicular to the coiled coil ...
Contributing Partner: UNT Libraries
Metabolic Engineering of Raffinose-Family Oligosaccharides in the Phloem Reveals Alterations in Patterns of Carbon Partitioning and Enhances Resistance to Green Peach Aphid

Metabolic Engineering of Raffinose-Family Oligosaccharides in the Phloem Reveals Alterations in Patterns of Carbon Partitioning and Enhances Resistance to Green Peach Aphid

Date: August 2010
Creator: Cao, Te
Description: Phloem transport is along hydrostatic pressure gradients generated by differences in solute concentration between source and sink tissues. Numerous species accumulate raffinose-family oligosaccharides (RFOs) in the phloem of mature leaves to accentuate the pressure gradient between source and sinks. In this study, metabolic engineering was used to generate RFOs at the inception of the translocation stream of Arabidopsis thaliana, which transports predominantly sucrose. To do this, three genes, GALACTINOL SYNTHASE, RAFFINOSE SYNTHASE and STACHYOSE SYNTHASE, were expressed from promoters specific to the companion cells of minor veins. Two transgenic lines homozygous for all three genes (GRS63 and GRS47) were selected for further analysis. Sugars were extracted and quantified by high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD), and 21-day old plants of both lines had levels of galactinol, raffinose, and stachyose approaching 50% of total soluble sugar. All three exotic sugars were also identified in phloem exudates from excised leaves of transgenic plants whereas levels were negligible in exudates from wild type leaves. Differences in starch accumulation or degradation between wild type and GRS63 and GRS47 lines were not observed. Similarly, there were no differences in vegetative growth between wild type and engineered plants, but engineered plants flowered ...
Contributing Partner: UNT Libraries
Interactions of N-Acylethanolamine Metabolism and Abscisic Acid Signaling in Arabidopsis Thaliana Seedlings

Interactions of N-Acylethanolamine Metabolism and Abscisic Acid Signaling in Arabidopsis Thaliana Seedlings

Date: August 2010
Creator: Cotter, Matthew Q.
Description: N-Acylethanolamines (NAEs) are endogenous plant lipids hydrolyzed by fatty acid amide hydrolase (FAAH). When wildtype Arabidopsis thaliana seeds were germinated and grown in exogenous NAE 12:0 (35 µM and above), growth was severely reduced in a concentration dependent manner. Wildtype A. thaliana seeds sown on exogenous abscisic acid (ABA) exhibited similar growth reduction to that seen with NAE treatment. AtFAAH knockouts grew and developed similarly to WT, but AtFAAH overexpressor lines show markedly enhanced sensitivity to ABA. When low levels of NAE and ABA, which have very little effect on growth alone, were combined, there was a dramatic reduction in seedling growth in all three genotypes, indicating a synergistic interaction between ABA and NAE. Notably, this synergistic arrest of seedling growth was partially reversed in the ABA insensitive (abi) mutant abi3-1, indicating that a functional ABA signaling pathway is required for the full synergistic effect. This synergistic growth arrest results in an increased accumulation of NAEs, but no concomitant increase in ABA levels. The combined NAE and ABA treatment induced a dose-dependent increase in ABI3 transcript levels, which was inversely related to growth. The ABA responsive genes AtHVA22B and RD29B also had increased expression in both NAE and ABA treatment. ...
Contributing Partner: UNT Libraries
Functional Characterization of Plant Fatty Acid Amide Hydrolases

Functional Characterization of Plant Fatty Acid Amide Hydrolases

Date: December 2010
Creator: Kim, Sang-Chul
Description: Fatty acid amide hydrolase (FAAH) terminates the endocannabinoid signaling pathway that regulates numerous neurobehavioral processes in animals by hydrolyzing a class of lipid mediators, N-acylethanolamines (NAEs). Recent identification of an Arabidopsis FAAH homologue (AtFAAH) and several studies, especially those using AtFAAH overexpressing and knock-out lines suggest that a FAAH-mediated pathway exists in plants for the metabolism of endogenous NAEs. Here, I provide evidence to support this concept by identifying candidate FAAH cDNA sequences in diverse plant species. NAE amidohydrolase assays confirmed that several of the proteins encoded by these cDNAs indeed catalyzed the hydrolysis of NAEs in vitro. Kinetic parameters, inhibition properties, and substrate specificities of the plant FAAH enzymes were very similar to those of mammalian FAAH. Five amino acid residues determined to be important for catalysis by rat FAAH were absolutely conserved within the plant FAAH sequences. Site-directed mutation of each of the five putative catalytic residues in AtFAAH abolished its hydrolytic activity when expressed in Escherichia coli. Contrary to overexpression of native AtFAAH in Arabidopsis that results in enhanced seedling growth, and in seedlings that were insensitive to exogenous NAE, overexpression of the inactive AtFAAH mutants showed no growth enhancement and no NAE tolerance. However, both active ...
Contributing Partner: UNT Libraries
The Relationship of Force on Myosin Subfragment 2 Region to the Coiled-Coiled Region of the Myosin Dimer

The Relationship of Force on Myosin Subfragment 2 Region to the Coiled-Coiled Region of the Myosin Dimer

Date: December 2011
Creator: Hall, Nakiuda M.
Description: The stability of myosin subfragment 2 was analyzed using gravitational force spectroscopy. The region was found to destabilize under physiological force loads, indicating the possibility that subfragment 2 may uncoil to facilitate actin binding during muscle contraction. As a control, synthetic cofilaments were produced to discover if the observations in the single molecule assay were due to the lack of the stability provided by the thick filament. Statistically, there was no difference between the single molecule assay data and the synthetic cofilament assay data. Thus, the instability of the region is due to intrinsic properties within subfragment 2.
Contributing Partner: UNT Libraries
Identification of Three Symbiosome Targeting Domains in the MtENOD8 Protein and Cell-to-cell MtENOD8 mRNA Movement in Nodules

Identification of Three Symbiosome Targeting Domains in the MtENOD8 Protein and Cell-to-cell MtENOD8 mRNA Movement in Nodules

Access: Use of this item is restricted to the UNT Community.
Date: May 2012
Creator: Meckfessel, Matthew Harold
Description: The model legume, Medicago truncatula, is able to enter into a symbiotic relationship with soil bacteria, known as rhizobia. This relationship involves a carbon for nitrogen exchange in which the plant provides reduced carbon from photosynthesis in exchange for reduced, or “fixed” atmospheric nitrogen, which allows the plant to thrive in nitrogen depleted soils. Rhizobia infect and enter plant root organs, known as nodules, where they reside inside the plant cell in a novel organelle, known as the symbiosome where nitrogen fixation occurs. the symbiosome is enriched in plant proteins, however, little is known about the mechanisms that direct plant proteins to the symbiosome. Using the M. truncatula ENOD8 (MtENOD8) protein as a model to explore symbiosome protein targeting, 3-cis domains were identified within MtENOD8 capable of directing green fluorescent protein (GFP) to the symbiosome, including its N-terminal signal peptide (SP). the SP delivered GFP to the vacuole in the absence of nodules suggesting that symbiosome proteins share a common targeting pathway with vacuolar proteins. a time course analysis during nodulation indicated that there is a nodule specific redirection of MtENOD8-SP from the vacuole to the symbiosome in a MtNIP/LATD dependent manner. GFP expression by the MtENOD8 promoter revealed spatial ...
Contributing Partner: UNT Libraries
Proteomic Responses in the Gill of Zebrafish Following Exposure to Ibuprofen and Naproxen

Proteomic Responses in the Gill of Zebrafish Following Exposure to Ibuprofen and Naproxen

Date: August 2012
Creator: Adhikari, Prem R.
Description: Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most abundant environmental pharmaceutical contaminants. In this study, a proteomic analysis was conducted to identify proteins differentially expressed in gill tissue of zebrafish (Danio rerio) after a 14-day exposure to the NSAIDs ibuprofen or naproxen. A total of 104 proteins with altered expression as indicated by 2-dimensional electrophoresis were analyzed by liquid chromatography with ion trap mass spectrometry (MS/MS). A total of 14 proteins fulfilled our requirements for identification which included consistency among replicate gels as well as successful MS/MS ion searches with the MASCOT database. The most prominent feature of the differential protein expression observed after NSAID exposure was an up-regulation of proteins belonging to the globin family which are involved in the transport of oxygen from gills and availability of heme molecules required for synthesis of cyclooxygenase. Differential expression was observed at exposure concentrations as low as 1-10 µg/L indicating that altered gene expression may occur in fish subjected to environmentally realistic levels of NSAID exposure.
Contributing Partner: UNT Libraries
O-Acetylserine Sulhydralase-A from Salmonella typhimurium LT-2: Thermodynamic Properties and SPectral Identification of Intermediates

O-Acetylserine Sulhydralase-A from Salmonella typhimurium LT-2: Thermodynamic Properties and SPectral Identification of Intermediates

Date: August 1993
Creator: Simmons, James Walter
Description: O-Acetylserine Sulfhydrylase (OASS) is a pyridoxal phosphate enzyme that catalyzes the reaction of O-acetyl-Lserine with sulfide to give L-cysteine. OASS is present as two isoforms, designated -A and -B. The kinetic mechanism of OASS-A is well known and there is also much known concerning the acid-base chemistry of the enzyme. However, little is known concerning the location of the rate determining steps, the sequencing of chemical steps that occur at the active site, or the nature of the rate determining transition states. The studies performed to help elucidate these aspects of the OASS-A mechanism included determination of the thermodynamics of both half reactions, along with studies utilizing substrate analogs of OAS halting the reaction at specific points along the reaction pathway allowing the identification of reaction intermediates. The free energy change of the first half reaction was shown to be -5.7 Kcal/mole while the second half reaction was shown to be, for all intents and purposes, irreversible. Intermediates along the reaction pathway that have been previously identified include the internal Schiff base and the a-aminoacrylate. The external Schiff base was identified using the analogs cysteine, alanine, and glycine while the geminal diamine was identified using the analog serine. Formation of ...
Contributing Partner: UNT Libraries
Application of Synthetic Peptides as Substrates for Reversible Phosphorylation

Application of Synthetic Peptides as Substrates for Reversible Phosphorylation

Date: August 1992
Creator: Abukhalaf, Imad Kazem
Description: Two highly homologous synthetic peptides MLC(3-13) (K-R-A-K-A-K-T-TK-K-R-G) and MLC(5-13) (A-K-A-K-T-T-K-K-R-G) corresponding to the amino terminal amino acid sequence of smooth muscle myosin light chain were utilized as substrates for protein kinase C purified from murine lymphosarcoma tumors to determine the role of the primary amino acid sequence of protein kinase C substrates in defining the lipid (phosphatidyl serine and diacylglycerol) requirements for the activation of the enzyme. Removal of the basic residues lysine and arginine from the amino terminus of MLC(3-13) did not have a significant effect on the Ka value of diacylglycerol. The binding of effector to calcium-protein kinase C appears to be random since binding of one effector did not block the binding of the other.
Contributing Partner: UNT Libraries
Identification of Endogenous Substrates for ADP-Ribosylation in Rat Liver

Identification of Endogenous Substrates for ADP-Ribosylation in Rat Liver

Date: May 1992
Creator: Loflin, Paul T. (Paul Tracey)
Description: Bacterial toxins have been shown to modify animal cell proteins in vivo with ADPR. Animal cells also contain endogenous enzymes that can modify proteins. Indirect evidence for the existence in vivo of rat liver proteins modified by ADPR on arginine residues has been reported previously. Presented here is direct evidence for the existence of ADP-ribosylarginine in rat liver proteins. Proteins were subjected to exhaustive protease digestion and ADP-ribosyl amino acids were isolated by boronate chromatography.
Contributing Partner: UNT Libraries
Characterization of a Human 28S Ribosomal RNA Retropseudogene and Other Repetitive DNA Sequence Elements Isolated from a Human X Chromosome-Specific Library

Characterization of a Human 28S Ribosomal RNA Retropseudogene and Other Repetitive DNA Sequence Elements Isolated from a Human X Chromosome-Specific Library

Date: May 1994
Creator: Wang, Suyue
Description: Three genomic clones encompassing human DNA segments (designated LhX-3, LhX-4, and LhX5) were isolated from an X chromosome-specific library and subjected to analysis by physical mapping and DNA sequencing. It was found that these three clones are very rich in repetitive DNA sequence elements and retropseudogenes.
Contributing Partner: UNT Libraries
Kinetic and Chemical Mechanism of 6-phosphogluconate Dehydrogenase from Candida Utilis

Kinetic and Chemical Mechanism of 6-phosphogluconate Dehydrogenase from Candida Utilis

Date: May 1993
Creator: Berdis, Anthony J. (Anthony Joseph)
Description: A complete initial velocity study of the 6-phosphogluconate dehydrogenase from Candida utilis in both reaction directions suggests a rapid equilibrium random kinetic mechanism with dead-end E:NADP:(ribulose 5-phosphate) and E:NADPH:(6- phosphogluconate) complexes. Initial velocity studies obtained as a function of pH and using NAD as the dinucleotide substrate for the reaction suggest that the 2'-phosphate is critical for productive binding of the dinucleotide substrate. Primary deuterium isotope effects using 3-<i-6-phosphogluconate were obtained for the 6-phosphogluconate dehydrogenase reaction using NADP and various alternative inucleotide substrates.
Contributing Partner: UNT Libraries
Dependence of the Kinetic Mechanism of Adenosine 3',5'-Monophosphate Dependent Protein Kinase Catalytic Subunit in the Direction of Magnesium Adenosine 5'-Diphosphate Phosphorylation on pH and the Concentration of Free Magnesium Ions

Dependence of the Kinetic Mechanism of Adenosine 3',5'-Monophosphate Dependent Protein Kinase Catalytic Subunit in the Direction of Magnesium Adenosine 5'-Diphosphate Phosphorylation on pH and the Concentration of Free Magnesium Ions

Date: December 1992
Creator: Qamar, Raheel
Description: To define the overall kinetic and chemical mechanism of adenosine 3',5'-monophosphate dependent protein kinase catalytic subunit, the mechanism in the direction of MgADP phosphorylation was determined, using studies of initial velocity in the absence and presence of dead-end inhibitors. The kinetic mechanism was determined as a function of uncomplexed Mg^2+ (Mg_f) at pH 7.2 and as a function of pH at low (0.5 mM) Mg_f. At pH 7.2 data are consistent with a random kinetic mechanism in the direction of MgADP phosphorylation with both pathways allowed: the pathway in which MgADP binds to enzyme prior to phosphorylated peptide (PSP) and that in which PSP binds before MgADP. One or the other pathway predominates, depending on Mg_f concentration. At 0.5 mM Mg_f, the mechanism is steady-state ordered with the pathway where PSP binds first preferred; at 10 mM Mg_f, the mechanism is equilibrium ordered, and the pathway in which MgADP binds first preferred. This change in mechanism to equilibrium ordered is due to an increase in affinity of enzyme for MgADP and a decrease in affinity for PSP. There is also a pH-dependent change in mechanism at 0.5 mM Mg_f. At pH 6 the mechanism is equilibrium ordered with the pathway ...
Contributing Partner: UNT Libraries
Cottonseed Microsomal N-Acylphosphatidylethanolamine Synthase: Identification, Purification and Biochemical Characterization of a Unique Acyltransferase

Cottonseed Microsomal N-Acylphosphatidylethanolamine Synthase: Identification, Purification and Biochemical Characterization of a Unique Acyltransferase

Date: December 1998
Creator: McAndrew, Rosemary S. (Rosemary Smith)
Description: N-Acylphosphatidylethanoiamine (NAPE) is synthesized in the microsomes of cotton seedlings by a mechanism that is possibly unique to plants, the ATP-, Ca2+-, and CoA-independent acylation ofphosphatidylethanolamine (PE) with unesterified free fatty acids (FFAs), catalyzed by NAPE synthase. A photoreactive free fatty acid analogue, 12-[(4- azidosalicyl)amino]dodecanoic acid (ASD), and its 125I-labeled derivative acted as substrates for the NAPE synthase enzyme.
Contributing Partner: UNT Libraries
N-Acylethanolamines and Plant Phospholipase D

N-Acylethanolamines and Plant Phospholipase D

Date: December 1998
Creator: Brown, Shea Austin
Description: Recently, three distinct isoforms of phospholipase D (PLD) were identified in Arabidopsis thaliana. PLD α represents the well-known form found in plants, while PLD β and γ have been only recently discovered (Pappan et al., 1997b; Qin et al., 1997). These isoforms differ in substrate selectivity and cofactors required for activity. Here, I report that PLD β and γ isoforms were active toward N-acylphosphatidylethanolamine (NAPE), but PLD α was not. The ability of PLD β and γ to hydrolyze NAPE marks a key difference from PLD α. N-acylethanolamines (NAE), the hydrolytic products of NAPE by PLD β and γ, inhibited PLD α from castor bean and cabbage. Inhibition of PLD α by NAE was dose-dependent and inversely proportional to acyl chain length and degree of unsaturation. Enzyme kinetic analysis suggested non-competitive inhibition of PLD α by NAE 14:0. In addition, a 1.2-kb tobacco (Nicotiana tabacum L.) cDNA fragment was isolated that possessed a 74% amino acid identity to Arabidopsis PLD β indicating that this isoform is expressed in tobacco cells. Collectively, these results provide evidence for NAE producing PLD activities and suggest a possible regulatory role for NAE with respect to PLD α.
Contributing Partner: UNT Libraries
A Study of the Intrinsic Fluorescence of O-Acetyl-L-Serine Sulfhydrylase-A from Salmonella typhimurium

A Study of the Intrinsic Fluorescence of O-Acetyl-L-Serine Sulfhydrylase-A from Salmonella typhimurium

Date: May 1993
Creator: McClure, G. David (George David)
Description: O-Acetyl-L-serine sulfhydrylase-A (OASS-A) forms acetate and L-cysteine from O-acetyl-L-serine (OAS) and sulfide. One molecule of the cofactor pyridoxal 5'- phosphate (PLP) is bound in each holoenzyme protomer.
Contributing Partner: UNT Libraries
Autophosphorylation and Autoactivation of an S6/H4 Kinase Isolated From Human Placenta

Autophosphorylation and Autoactivation of an S6/H4 Kinase Isolated From Human Placenta

Date: May 1994
Creator: Dennis, Patrick B. (Patrick Brian)
Description: A number of protein kinases have been shown to undergo autophosphorylation, but few have demonstrated a coordinate increase or decrease in enzymatic activity as a result. Described here is a novel S6 kinase isolated from human placenta which autoactivates through autophosphorylation in vitro. This S6/H4 kinase, purified in an inactive state, was shown to be a protein of Mr of 60,000 as estimated by SDS-PAGE and could catalyze the phosphorylation of the synthetic peptide S6-21, the histone H4, and myelin basic protein. Mild digestion of the inactive S6/H4 kinase with trypsin was necessary, but not sufficient, to activate the kinase fully
Contributing Partner: UNT Libraries
Fumarate Activation and Kinetic Solvent Isotope Effects as Probes of the NAD-Malic Enzyme Reaction

Fumarate Activation and Kinetic Solvent Isotope Effects as Probes of the NAD-Malic Enzyme Reaction

Date: December 1992
Creator: Lai, Chung-Jeng
Description: The kinetic mechanism of activation of the NAD-malic enzyme by fumarate and the transition state structure for the oxidation malate for the NAD-malic enzyme reaction have been studied. Fumarate exerts its activating effect by decreasing the off-rate for malate from the E:Mg:malate and E:Mg:NAD:malate complexes. The activation by fumarate results in a decrease in K_imalate and an increase in V/K_malate by about 2-fold, while the maximum velocity remains constant. A discrimination exists between active and activator sites for the binding of dicarboxylic acids. Activation by fumarate is proposed to have physiologic importance in the parasite. The hydride transfer transition state for the NAD-malic enzyme reaction is concerted with respect to solvent isotope sensitive and hydride transfer steps. Two protons are involved in the solvent isotope sensitive step, one with a normal fractionation factor, another with an inverse fractionation factor. A structure for the transition state for hydride transfer in the NAD-malic enzyme reaction is proposed.
Contributing Partner: UNT Libraries
Desensitized Phosphofructokinase from Ascaris suum: A Study in Noncooperative Allostery

Desensitized Phosphofructokinase from Ascaris suum: A Study in Noncooperative Allostery

Date: May 1993
Creator: Payne, Marvin A.
Description: The studies described in this dissertation examine the effects of F-2,6-P2 and AMP or phosphorylation on the kinetic mechanism of d-PFK. The effect of varied pH on the activation by F-2,6-P2 is also described.
Contributing Partner: UNT Libraries
Kinetic and Chemical Mechanism of O-Acetylserine Sulfhydrylase-B from Salmonella Typhimurium

Kinetic and Chemical Mechanism of O-Acetylserine Sulfhydrylase-B from Salmonella Typhimurium

Date: August 1993
Creator: Tai, Chia-Hui
Description: Initial velocity studies of O-acetylserine sulfhydrylase-B (OASS-B) from Salmonella typhimurium using both natural and alternative substrates suggest a Bi Bi ping pong kinetic mechanism with double substrate competitive inhibition. The ping pong mechanism is corroborated by a qualitative and quantitative analysis of product and dead-end inhibition. Product inhibition by acetate is S-parabolic noncompetitive, indication of a combination of acetate with E followed by OAS. These data suggest some randomness to the OASS-B kinetic mechanism. The pH dependence of kinetic parameters was determined in order to obtain information on the acid-base chemical mechanism for the OASS-B reaction. A mechanism is proposed in which an enzyme general base accepts a proton from α-amine of O-acetylserine, while a second enzyme general base acts by polarizing the acetyl carbonyl assisting in the β-elimination of the acetyl group of O-acetylserine. The ε-amine of the active site lysine acts as a general base to abstract the α-proton in the β-elimination of acetate. At the end of the first half reaction the ε-amine of the active site lysine that formed the internal Schiff base and the general base are protonated. The resulting α-aminoacrylate intermediate undergoes a Michael addition with HS‾ and the active site lysine donates its ...
Contributing Partner: UNT Libraries
Manipulating Sucrose Proton Symporters to Understand Phloem Loading

Manipulating Sucrose Proton Symporters to Understand Phloem Loading

Date: August 2013
Creator: Dasgupta, Kasturi
Description: Phloem vascular tissues transport sugars synthesized by photosynthesis in mature leaves by a process called phloem loading in source tissues and unloading in sink tissues. Phloem loading in source leaves is catalyzed by Suc/H+ symporters (SUTs) which are energized by proton motive force. In Arabidopsis the principal and perhaps exclusive SUT catalyzing phloem loading is AtSUC2. In mutant plants harboring a T-DNA insertion in each of the functional SUT-family members, only Atsuc2 mutants demonstrate overtly debilitated phloem transport. Analysis of a mutant allele (Atsuc2-4) of AtSUC2 with a T-DNA insertion in the second intron showed severely stunted phenotype similar to previously analyzed Atsuc2 null alleles. However unlike previous alleles Atsuc2-4 produced viable seeds. Analysis of phloem specific promoters showed that promoter expression was regulated by Suc concentration. Unlike AtSUC2p, heterologous promoter CoYMVp was not repressed under high Suc conc. Further analysis was conducted using CoYMVp to test the capacity of diverse clades in SUT-gene family for transferring Suc in planta in Atsuc2 - / - mutant background. AtSUC1 and ZmSUT1 from maize complemented Atsuc2 mutant plants to the highest level compared to all other transporters. Over-expression of the above SUTs in phloem showed enhanced Suc loading and transport, but against ...
Contributing Partner: UNT Libraries
Studies of the Mechanism of Plasma Cholesterol Esterification in Aged Rats

Studies of the Mechanism of Plasma Cholesterol Esterification in Aged Rats

Date: December 1989
Creator: Lee, Sun Min
Description: The study was performed to determine factors influencing the esteriflcation of plasma cholesterol in young and aged rats. The distribution of LCAT activity was determined following gel nitration chromatography and ultracentrifugation of whole plasma respectively. When rat plasma was fractionated on a Bio-Gel A-5 Mcolumn, LCAT activity was found to be associated with the HDL fraction. A similar result was observed upon 24 hr density gradient ultracentrifugation of the plasma. However, following prolonged 40 hr preparative ultracentrifugation, the majority of the LCAT activity was displaced into the lipoprotein-free infranatant fraction (d> 1.225 g/ml). The dissociation of LCAT from the HDL fraction occured to a smaller extent in aged rat plasma than in young rat plasma. Plasma incubation (37°C) experiments followed by the isolation of lipoproteins and the subsequent analysis of their cholesterol content revealed that in vitro net esteriflcation of free cholesterol (FC) by LCAT as well as the fractional ufilization of HDL-FC as substrate were lower in the plasma of the aged animal as compared to that of the young animal despite the fact that the total pool of FC was higher in the former. The net transfer of FC from lower density lipoproteins (d<1.07 g/ml) to HDL provided ...
Contributing Partner: UNT Libraries