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  Partner: UNT Libraries
 Degree Discipline: Biochemistry
 Collection: UNT Theses and Dissertations
Hindrance of the Myosin Power Stroke Posed by the Proximity to the Troponin Complex Identified Using a Novel LRET Fluorescent Nanocircuit

Hindrance of the Myosin Power Stroke Posed by the Proximity to the Troponin Complex Identified Using a Novel LRET Fluorescent Nanocircuit

Date: May 2007
Creator: Coffee Castro-Zena, Pilar G.
Description: A novel luminescence resonance energy transfer (LRET) nanocircuit assay involving a donor and two acceptors in tandem was developed to study the dynamic interaction of skeletal muscle contraction proteins. The donor transmits energy relayed to the acceptors distinguishing myosin subfragment-1 (S1) lever arm orientations. The last acceptor allows the detection of S1's bound near or in between troponin complexes on the thin filament. Additionally, calcium related changes between troponin T and myosin were detected. Based on this data, the troponin complex situated every 7 actin monomers, hinders adjacently bound myosins to complete their power stroke; whereas myosins bound in between troponin complexes undergo complete power strokes.
Contributing Partner: UNT Libraries
Function of the ENOD8 gene in nodules of Medicago truncatula.

Function of the ENOD8 gene in nodules of Medicago truncatula.

Date: December 2006
Creator: Coque, Laurent
Description: To elaborate on the function(s) of the ENOD8 gene in the nodules of M. truncatula, several different experimental approaches were used. A census of the ENOD8 genes was first completed indicating that only ENOD8.1 (nt10554-12564 of GenBank AF463407) is highly expressed in nodule tissues. A maltose binding protein-ENOD8 fusion protein was made with an E. coli recombinant system. A variety of biochemical assays were undertaken with the MBP-ENOD8 recombinant protein expressed in E. coli, which did not yield the esterase activity observed for ENOD8 protein nodule fractions purified from M. sativa, tested on general esterase substrates, α-naphthyl acetate, and p-nitrophenylacetate. Attempts were also made to express ENOD8 in a Pichia pastoris system; no ENOD8 protein could be detected from Pichia pastoris strains which were transformed with the ENOD8 expression cassette. Additionally, it was shown that the ENOD8 protein can be recombinantly synthesized by Nicotiana benthamiana in a soluble form, which could be tested for activity toward esterase substrates, bearing resemblance to nodule compounds, such as the Nod factor. Transcription localization studies using an ENOD8 promoter gusA fusion indicated that ENOD8 is expressed in the bacteroid-invaded zone of the nodule. The ENOD8 protein was also detected in that same zone by ...
Contributing Partner: UNT Libraries
N-Acylethanolamine (NAE) Profiles Change During Arabidopsis Thaliana Seed Germination and Seedling Growth

N-Acylethanolamine (NAE) Profiles Change During Arabidopsis Thaliana Seed Germination and Seedling Growth

Date: August 2006
Creator: Wiant, William C.
Description: An understanding of the potential roles as lipid mediators of a family of bioactive metabolites called N-acylethanolamines (NAEs) depends on their accurate identification and quantification. The levels of 18C unsaturated NAEs (e.g. NAE18:2, NAE 18:3, etc.) in wild-type seeds (about 2000 ng/g fw) generally decreased by about 80% during germination and post-germinative growth. In addition, results suggest NAE-degradative fatty acid amide hydrolase (FAAH) expression does not play a major role in normal NAE metabolism as previously thought. Seedlings germinated and grown in the presence of abscisic acid (ABA), an endogenous plant hormone, exhibited growth arrest and secondary dormancy, similar to the treatment of seedlings with exogenous N­lauroylethanolamine (NAE12:0). ABA-mediated growth arrest was associated with higher levels of unsaturated NAEs. Overall, these results are consistent with the concept that NAE metabolism is activated during seed germination and suggest that the reduction in unsaturated NAE levels is under strict temporal control and may be a requirement for normal seed germination and post-germinative growth.
Contributing Partner: UNT Libraries
Genetic Modification of Fatty Acid Profiles in Cotton

Genetic Modification of Fatty Acid Profiles in Cotton

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Date: August 2005
Creator: Rommel, Amy A.
Description: The industrial uses of cottonseed oil are limited by its fatty acid composition. Genetic modification of cotton lipid profiles using seed-specific promoters could allow cotton growers to produce valuable new oils in the seed without adverse effects on fiber quality and yield, therefore making this crop more commercially profitable. Transgenic cotton callus harboring a diverged fatty acid desaturase gene (FADX) from Momordica charantia was characterized for production of alpha-eleostearic acid (conjugated double bonds: 18:3 D9 cis, 11 trans, 13 trans), not normally found in cotton. Gas chromatography (GC) in conjunction with mass spectrometry (MS) confirmed production of alpha-eleostearic acid in the transgenic cotton tissues. A second series of transformation experiments introduced the cotton fatty acid thioesterase B (FATB) cDNA, fused to the seed-specific oleosin promoter into cotton to promote the over-expression of FATB, to generate cotton with increased palmitate in the cottonseed. PCR amplification, as well as fatty acid analysis by gas chromatography, confirmed introduction of the FATB cDNA in transgenic tissues. Collectively, these results demonstrate the feasibility of manipulating the fatty acid composition in cotton via transgenic approaches and form the basis for continued efforts to create novel oils in cottonseed.
Contributing Partner: UNT Libraries
Molecular and biochemical characterization of phospholipase D in cotton (Gossypium hirsutum L) seedlings.

Molecular and biochemical characterization of phospholipase D in cotton (Gossypium hirsutum L) seedlings.

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Date: May 2005
Creator: McHugh, John
Description: N-Acylethanolamines (NAEs) are enriched in seed-derived tissues and are believed to be formed from the membrane phospholipid, N-acylphosphatidylethanolamine (NAPE) via the action of phospholipase D (PLD). In an effort to identify a functional NAPE-PLD in cotton seeds and seedlings, we have screened a cotton seedling cDNA (cotyledon mRNA from 48 h dark grown seedlings) library with a 1.2 kb tobacco partial cDNA fragment encoding the middle third of a putative PLDβ/γ (genbank accession, AF195614) isoform. Six plaques were isolated from the Uni-ZAP lambda library, excised as pBluescript SK(-) phagemids and subjected to nucleotide sequence analysis. Alignment of derived sequences with Arabidopsis PLD family members indicated that the cDNAs represent six different PLD gene products -three putative PLD β isoforms and three putative PLD δ isoforms. The PLD β isoforms, designated Ghpldβ1a, GHpldβ1b and a truncated Ghpldβ1b isoform. Both the full-length PLD β proteins contained characteristic HKxxxxD catalytic domains, a PC-binding domain, a PIP2-binding domain and a C2 domain. In addition both cotton PLD β isoforms had a N-terminal "SPQY" rich domain which appeared to be unique to these PLDs. The three PLD δ isoforms, designated Ghpldδ1a, Ghpldδ1b and Ghpldδ1b-2 encode full-length PLDδ proteins, and like the above PLDs, contained the ...
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The structure and function of troponin T upon metal ion binding and the detection of nucleic acid sequence variations.

The structure and function of troponin T upon metal ion binding and the detection of nucleic acid sequence variations.

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Date: May 2005
Creator: Zhang, Zhiling
Description: Numerous troponin T (TnT) isoforms are generated by alternative RNA splicing primarily in its NH2-terminal hypervariable region, but the functions of these isoforms are not completely understood. In this dissertation work, calcium and terbium binding behavior of several forms of TnT were investigated by spectroscopic and radioactive techniques. Chicken breast muscle TnT binds calcium and terbium through its NH2-terminal Tx motif (HEEAH)n with high affinity (10-6 mM) and fast on-rate (106 - 107 M-1 s-1). Chicken leg muscle TnT and a human cardiac TnT NH2-terminal fragment, which both lack the Tx motif on their NH2-terminal regions, do not have affinities for calcium in the physiological range. Computational predictions on TnT N47 suggest that the TnT NH2-terminal region might fold into an elongated structure with at least one high affinity metal ion binding pocket comprised primarily of the Tx motif sequence and several lower affinity binding sites. In addition, calcium binding to TnT N47 might alter its conformation and flexibility. Luminescence resonance energy transfer measurements and other experimental observations are consistent with the computational predictions suggesting the computational simulated atomic model is reasonable. TnT mutations are responsible for 15% of familiar hypertrophic cardiomyopathy (FHC) cases with a phenotype of relatively mild ...
Contributing Partner: UNT Libraries
N-Acylethanolamine Metabolism During Seed Germination: Molecular Identification of a Functional N-Acylethanolamine Amidohydrolase

N-Acylethanolamine Metabolism During Seed Germination: Molecular Identification of a Functional N-Acylethanolamine Amidohydrolase

Date: August 2004
Creator: Shrestha, Rhidaya
Description: N-Acylethanolamines (NAEs) are endogenous lipid metabolites that occur in a variety of dry seeds, and their levels decline rapidly during the first few hours of imbibition (Chapman et al., 1999, Plant Physiol., 120:1157-1164). Biochemical studies supported the existence of an NAE amidohydrolase activity in seeds and seedlings, and efforts were directed toward identification of DNA sequences encoding this enzyme. Mammalian tissues metabolize NAEs via an amidase enzyme designated fatty acid amide hydrolase (FAAH). Based on the characteristic amidase signature sequence in mammalian FAAH, a candidate Arabidopsis cDNA was identified and isolated by reverse transcriptase-PCR. The Arabidopsis cDNA was expressed in E. coli and the recombinant protein indeed hydrolyzed a range of NAEs to free fatty acids and ethanolamine. Kinetic parameters for the recombinant protein were consistent with those properties of the rat FAAH, supporting identification of this Arabidopsis cDNA as a FAAH homologue. Two T-DNA insertional mutant lines with disruptions in the Arabidopsis NAE amidohydrolase gene (At5g64440) were identified. The homozygous mutant seedlings were more sensitive than the wild type to exogenously applied NAE 12:0. Transgenic seedlings overexpressing the NAE amidohydrolase enzyme showed noticeably greater tolerance to NAE 12:0 than wild type seedlings. These results together provide evidence in vitro ...
Contributing Partner: UNT Libraries
Use of luminescence energy transfer probes to detect genetic variants.

Use of luminescence energy transfer probes to detect genetic variants.

Date: August 2004
Creator: Vaccaro, Carlos
Description: The purpose of this research was to study the hybridization of molecular beacons under different conditions and designs. Data collected suggest that the inconsistency found in the emission intensity of several of these probes may be caused by 3 important factors: length of the probe, nucleotide sequence and, the formation of an alternative complex structure such as a dimer. Of all three factors, dimer formation is the most troublesome, since it reduces the emission of the reporter molecules. A new probe design was used to reduce dimer formation. The emission signal of the improved probe was several folds stronger than those probes with the early design. In this research, dimer formation is detected, furthermore a new probe with a different design was tested. If dimer formation can be reduced molecular beacons can be integrated into more complex hybridization systems providing an important tool in research and diagnosis of genetic disorders.
Contributing Partner: UNT Libraries
Identification and quantification of lipid metabolites in cotton fibers: Reconciliation with metabolic pathway predictions from DNA databases.

Identification and quantification of lipid metabolites in cotton fibers: Reconciliation with metabolic pathway predictions from DNA databases.

Date: May 2004
Creator: Wanjie, Sylvia W.
Description: The lipid composition of cotton (Gossypium hirsutum, L) fibers was determined. Fatty acid profiles revealed that linolenate and palmitate were the most abundant fatty acids present in fiber cells. Phosphatidylcholine was the predominant lipid class in fiber cells, while phosphatidylethanolamine, phosphatidylinositol and digalactosyldiacylglycerol were also prevalent. An unusually high amount of phosphatidic acid was observed in frozen cotton fibers. Phospholipase D activity assays revealed that this enzyme readily hydrolyzed radioactive phosphatidylcholine into phosphatidic acid. A profile of expressed sequence tags (ESTs) for genes involved in lipid metabolism in cotton fibers was also obtained. This EST profile along with our lipid metabolite data was used to predict lipid metabolic pathways in cotton fiber cells.
Contributing Partner: UNT Libraries
Studies on actomyosin crossbridge flexibility using a new single molecule assay.

Studies on actomyosin crossbridge flexibility using a new single molecule assay.

Date: May 2004
Creator: Gundapaneni, Deepika
Description: Several key flexure sites exist in the muscle crossbridge including the actomyosin binding site which play important roles in the actomyosin crossbridge cycle. To distinguish between these sources of flexibility, a new single molecule assay was developed to observe the swiveling of rod about a single myosin. Myosins attached through a single crossbridge displayed mostly similar torsional characteristics compared to myosins attached through two crossbridges, which indicates that most of the torsional flexibility resides in the myosin subfragment-2, and thus the hinge between subfragment-2 and light meromyosin should contribute the most to this flexibility. The comparison of torsional characteristics in the absence and presence of ADP demonstrated a small but significant increase in twist rates for the double-headed myosins but no increase for single-headed myosins, which indicates that the ADP-induced increase in flexibility arises due to changes in the myosin head and verifies that most flexibility resides in myosin subfragment-2.
Contributing Partner: UNT Libraries
Tobacco Phospholipase D β1: Molecular Cloning and Biochemical Characterization

Tobacco Phospholipase D β1: Molecular Cloning and Biochemical Characterization

Date: December 2002
Creator: Hodson, Jane E.
Description: Transgenic tobacco plants were developed containing a partial PLD clone in antisense orientation. The PLD isoform targeted by the insertion was identified. A PLD clone was isolated from a cDNA library using the partial PLD as a probe: Nt10B1 shares 92% identity with PLDβ1 from tomato but lacks the C2 domain. PCR analysis confirmed insertion of the antisense fragment into the plants: three introns distinguished the endogenous gene from the transgene. PLD activity was assayed in leaf homogenates in PLDβ/g conditions. When phosphatidylcholine was utilized as a substrate, no significant difference in transphosphatidylation activity was observed. However, there was a reduction in NAPE hydrolysis in extracts of two transgenic plants. In one of these, a reduction in elicitor- induced PAL expression was also observed.
Contributing Partner: UNT Libraries
Palmitoyl-acyl Carrier Protein Thioesterase in Cotton (Gossypium hirsutum L.): Biochemical and Molecular Characterization of a Major Mechanism for the Regulation of Palmitic Acid Content

Palmitoyl-acyl Carrier Protein Thioesterase in Cotton (Gossypium hirsutum L.): Biochemical and Molecular Characterization of a Major Mechanism for the Regulation of Palmitic Acid Content

Date: August 2001
Creator: Huynh, Tu T
Description: The relatively high level of palmitic acid (22 mol%) in cottonseeds may be due in part to the activity of a palmitoyl-acyl carrier protein (ACP) thioesterase (PATE). In embryo extracts, PATE activity was highest at the maximum rate of reserve accumulation (oil and protein). The cotton FatB mRNA transcript abundance also peaked during this developmental stage, paralleling the profiles of PATE enzyme activity and seed oil accumulation. A cotton FatB cDNA clone was isolated by screening a cDNA library with a heterologous Arabidopsis FatB probe (Pirtle et al., 1999, Plant and Cell Physiology 40: 155-163). The predicted amino acid sequence of the cotton PATE preprotein had 63% identity to the Arabidopsis FatB thioesterase sequence, suggesting that the cotton cDNA clone probably encoded a FatB-type thioesterase. When acyl-CoA synthetase-minus E. coli mutants expressed the cotton cDNA, an increase in 16:0 free fatty acid content was measured in the culture medium. In addition, acyl-ACP thioesterase activity assays in E. coli lysates revealed that there was a preference for palmitoyl-ACP over oleoyl-ACP in vitro, indicating that the cotton putative FatB cDNA encoded a functional thioesterase with a preference for saturated acyl-ACPs over unsaturated acyl-ACPs (FatA). Overexpression of the FatB cDNA in transgenic cotton ...
Contributing Partner: UNT Libraries
Plastidial carbonic anhydrase in cotton (Gossypium hirsutum L.): characterization, expression, and role in lipid biosynthesis

Plastidial carbonic anhydrase in cotton (Gossypium hirsutum L.): characterization, expression, and role in lipid biosynthesis

Date: August 2001
Creator: Hoang, Chau V.
Description: Recently, plastidial carbonic anhydrase (CA, EC 4.2.1.1) cDNA clones encoding functional CA enzymes were isolated from a nonphotosynthetic cotton tissue. The role of CA in photosynthetic tissues have been well characterized, however there is almost no information for the role of CA in nonphotosynthetic tissues. A survey of relative CA transcript abundance and enzyme activity in different cotton organs revealed that there was substantial CA expression in cotyledons of seedlings and embryos, both nonphotosynthetic tissues. To gain insight into the role(s) of CA, I examined CA expression in cotyledons of seedlings during post-germinative growth at different environmental conditions. CA expression in cotyledons of seedlings increased from 18 h to 72 h after germination in the dark. Seedlings exposed to light had about a 2-fold increase in CA activities when compared with seedlings kept in the dark, whereas relative CA transcript levels were essentially the same. Manipulation of external CO2 environments [zero, ambient (350 ppm), or high (1000 ppm)] modulated coordinately the relative transcript abundance of CA (and rbcS) in cotyledons, but did not affect enzyme activities. On the other hand, regardless of the external CO2 conditions seedlings exposed to light exhibited increase CA activity, concomitant with Rubisco activity and increased ...
Contributing Partner: UNT Libraries
Noncovalent Crosslinking of SH1 and SH2 to Detect Dynamic Flexibility of the SH1 Helix

Noncovalent Crosslinking of SH1 and SH2 to Detect Dynamic Flexibility of the SH1 Helix

Date: August 2000
Creator: Park, Hyunguk
Description: In this experiment, fluorescent N- (1-pyrenyl) iodoacetamide modified the two reactive thiols, SH1 (Cys 707) and SH2 (Cys 697) on myosin to detect SH1-SH2 a -helix melting. The excimer forming property of pyrene is well suited to monitor the dynamics of the SH1 and SH2 helix melting, since the excimer should only form during the melted state. Decreased anisotropy of the excimer relative to the monomeric pyrene fluorescence is consistent with the disordering of the melted SH1-SH2 region in the atomic model. Furthermore, nucleotide analogs induced changes in the anisotropy of the excimer, suggesting that the nucleotide site modulates the flexibility of SH1-SH2 region.
Contributing Partner: UNT Libraries
Conformational Studies of Myosin and Actin with Calibrated Resonance Energy Transfer

Conformational Studies of Myosin and Actin with Calibrated Resonance Energy Transfer

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Date: May 2000
Creator: Xu, Jin
Description: Resonance energy transfer was employed to study the conformational changes of actomyosin during ATP hydrolysis. To calibrate the technique, the parameters for resonance energy transfer were defined. With conformational searching algorithms to predict probe orientation, the distances measured by resonance energy transfer are highly consistent with the atomic models, which verified the accuracy and feasibility of resonance energy transfer for structural studies of proteins and oligonucleotides. To study intramyosin distances, resonance energy transfer probes were attached to skeletal myosin's nucleotide site, subfragment-2, and regulatory light chain to examine nucleotide analog-induced structural transitions. The distances between the three positions were measured in the presence of different nucleotide analogs. No distance change was considered to be statistically significant. The measured distance between the regulatory light chain and nucleotide site was consistent with either the atomic model of skeletal myosin subfragment-1 or an average of the three models claimed for different ATP hydrolysis states, which suggested that the neck region was flexible in solution. To examine the participation of actin in the powerstroke process, resonance energy transfer between different sites on actin and myosin was measured in the presence of nucleotide analogs. The efficiencies of energy transfer between myosin catalytic domain and actin ...
Contributing Partner: UNT Libraries
Nucleotide Inhibition of Glyoxalase II

Nucleotide Inhibition of Glyoxalase II

Date: May 1999
Creator: Gillis, Glen S
Description: The glyoxalase system mediates the conversion of methylglyoxal, a toxic ketoaldehyde, to D-lactic acid. The system is composed of two enzymes, glyoxalase I (Glo-I) and glyoxalase II (Glo-II), and exhibits an absolute requirement for a catalytic quantity of glutathione (GSH). Glo-I catalyzes the isomerization of a hemithioacetal, formed non-enzymatically from methylglyoxal and GSH, to the corresponding a -D-hydroxyacid thioester, s-D-lactoylglutathione (SLG). Glo-II catalyzes the irreversible breakdown of SLG to D-lactate and GSH. We have observed that ATP or GTP significantly inhibits the Glo-II activity of tissue homogenates from various sources. We have developed a rapid, one step chromatography procedure to purify Glo-II such that the purified enzyme remains "sensitive" to inhibition by ATP or GTP (Glo-II-s). Studies indicate that inhibition of Glo-II-s by nucleotides is restricted to ATP, GTP, ADP, and GDP, with ATP appearing most effective. Kinetics studies have shown that ATP acts as a partial non-competitive inhibitor of Glo-II-s activity, and further suggest that two kinetically distinguishable forms of the enzyme exist. The sensitivity of pure Glo-II-s to nucleotide inhibition is slowly lost on storage even at -80° C. This loss is accelerated at higher temperatures or in the presence of ATP. Kinetics studies on the resultant "insensitive" ...
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
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
Regulation of an S6/H4 Kinase in Crude Lymphosarcoma P1798 Preparations

Regulation of an S6/H4 Kinase in Crude Lymphosarcoma P1798 Preparations

Date: December 1998
Creator: Taylor, Allison Antoinette
Description: Purified S6/H4 kinase (Mr 60,000) requires autophosphorylation for activation. A rabbit anti-S6/H4 kinase peptide (SVIDPVPAPVGDSHVDGAAK) antibody recognized both the S6/H4 kinase holoenzyme and catalytic domain. Immunoreactivity with p60 kinase protein, and S6/H4 kinase activity were precisely correlated in fractions obtained from ion exchange chromatography of P1798 lymphosarcoma extracts. An enzyme which catalyzed the MgATP-dependent phosphorylation and activation of S6/H4 kinase coeluted with immunoreactivity from Mono 5, but not Mono Q chromatography. Since S6/H4 kinase is homologous with rac-activated PAK65, the observation that phosphorylation is also required for activation suggests a complex mechanism for in vivo activation of the S6/H4 kinase.
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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
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
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
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
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