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

Computational Studies Of Glucoamylase Selectivity , Pedro Maldonado Coutinho Jan 1996

Computational Studies Of Glucoamylase Selectivity , Pedro Maldonado Coutinho

Retrospective Theses and Dissertations

Glucoamylase (GA) is an industrial enzyme involved in the production of glucose and fructose syrups from starch. Exhibiting a large spectrum of selectivities, GAs are able to cleave glucose from the nonreducing ends of [alpha]-(1,4) glycosidic bonds of maltooligosaccharide chains and the [alpha]-(1,6) bonds initiating their branches. The high temperatures and glucose concentrations occurring in saccharification lead to the formation of condensation products, reducing the overall glucose yield. Modified versions of Aspergillus GA that better satisfy industrial requirements are desirable. To achieve this, a structure-based multisequence alignment of the primary sequences of the catalytic domains, linkers ...


Molecular Analysis Of A Shaker-Related K+ Channel In Leech , Jamie Lynn Weiss Jan 1996

Molecular Analysis Of A Shaker-Related K+ Channel In Leech , Jamie Lynn Weiss

Retrospective Theses and Dissertations

Voltage-gated K+ channels play a critical role in regulating the excitability of neurons and muscle. In the nervous system of many species voltage-gated K+ channels are primarily represented by four subfamilies, Shaker, Shal, Shab, and Shaw. We have cloned a K+ channel in the leech Haemopis marmorata, called LK-1, using a PCR based screen that was designed to isolate any of the four defined subfamilies of voltage-gated K+ channels. LK-1 primary amino acid sequence reveals that it is a Shaker-related channel sharing the highest amino acid identity within the transmembrane domains (48-58%) to classified Shaker homologs in species both higher ...


Purification, Characterization And Molecular Cloning Of Muscle Paranemin , Philip Mark Hemken Jan 1996

Purification, Characterization And Molecular Cloning Of Muscle Paranemin , Philip Mark Hemken

Retrospective Theses and Dissertations

Paranemin is an incompletely characterized ~280 kilodalton protein previously identified and immunolocalized in embryonic chick skeletal muscle. Paranemin has been purified from the same tissue source, has the same molecular weight by SDS-PAGE, and has the same antibody localization at the Z-lines of adult avian cardiac muscle. The method developed for preparation of purified paranemin from embryonic (chick) skeletal muscle includes homogenization, centrifugation and gel filtration, hydroxyapatite, and DEAE-cellulose chromatography. By using this method, ~2 mg of purified paranemin was routinely obtained. Amino acid analysis revealed that paranemin has a high acidic to basic amino acid ratio, which agrees with ...


Molecular Marker Analysis Of Iron Efficiency In Soybean (Glycine Max (L.) Merr.) , Shun-Fu Lin Jan 1996

Molecular Marker Analysis Of Iron Efficiency In Soybean (Glycine Max (L.) Merr.) , Shun-Fu Lin

Retrospective Theses and Dissertations

The objectives of the studies were to map genes affecting iron deficiency chlorosis in soybean, to test the hypothesis that two genetic mechanisms control iron deficiency chlorosis in soybean, and to determine the effectiveness of nutrient solution evaluation for iron deficiency chlorosis in soybean. Chlorosis symptoms of one hundred and twenty F2:4 lines from a Pride B216 x A15 population, and 92 F2:4 lines from an Anoka x A7 population grown in a field of calcareous soil in 1993 and 1994 were evaluated by visual scores and spectrometric chlorophyll determinations. Each population also was evaluated separately with two ...


Integration Specificity Of The Retrovirus-Like Transposable Element Ty5 Of Saccharomyces , Sige Zou Jan 1996

Integration Specificity Of The Retrovirus-Like Transposable Element Ty5 Of Saccharomyces , Sige Zou

Retrospective Theses and Dissertations

The retrotransposons and retroviruses display a strong preference in selecting integration sites. The molecular mechanisms underlying this process, however, are largely unknown. I have characterized a new retrotransposon, called Ty5, in diverse strains of Saccharomyces cerevisiae and related species. A functional element, Ty5-6p, was identified from S. paradoxus. To investigate Ty5 target preference, the locations of 13 native insertions were determined in S. cerevisiae and S. paradoxus. Twelve were found near the telomeres and the mating locus HMR. To determine if this distribution is a consequence of targeted integration, a Ty5 transposition assay was developed in S. cerevisiae using Ty5-6p ...


The Structure And Function Of Human Brain Hexokinase , Chenbo Zeng Jan 1996

The Structure And Function Of Human Brain Hexokinase , Chenbo Zeng

Retrospective Theses and Dissertations

An ATP binding site model for human brain hexokinase was established based on the similarity of the ATP-binding domains among actin, heat shock protein, glycerol kinase and brain hexokinase. Site directed mutagenesis was performed on the residues in the putative ATP-binding site in order to substantiate or refute the proposed model. The k cat values decreased 1000- and 200-fold for the Asp532Lys and Asp532Glu mutants, 114- and 12-fold for the Arg539Ile and Arg539Lys mutants, 2000- and 2-fold for the Thr680Val and Thr680Ser mutants, respectively. It is suggested that these residues are important for catalysis by either keeping the [gamma]-phosphoryl ...


Molecular Characterization Of The Chloroplastic Acetyl-Coa Carboxylase Of Arabidopsis Thaliana , Joong-Kook Choi Jan 1996

Molecular Characterization Of The Chloroplastic Acetyl-Coa Carboxylase Of Arabidopsis Thaliana , Joong-Kook Choi

Retrospective Theses and Dissertations

Acetyl-CoA carboxylase (ACCase) is a biotin-containing enzyme that catalyzes the formation of malonyl-CoA from acetyl-CoA. Dicot plants such as Arabidopsis contain two structurally distinct types of ACCases that are located in different subcellular compartments. The ACCase located in the cytosol of plant cells generates malonyl-CoA for the synthesis of plant secondary metabolites, including a variety of polyketides and derivatives, and malonylated phytochemicals. The ACCase that is located in chloroplasts generates malonyl-CoA for de novo fatty acid biosynthesis. This enzyme has a heteromeric structure consisting of at least four subunits: the biotin-carrier subunit, coded by the CAC1 gene, the biotin carboxylase ...


Identification Of Mouse Sip24/24p3 As A New Acute Phase Protein And Study Of Its Expression , Quansheng Liu Jan 1996

Identification Of Mouse Sip24/24p3 As A New Acute Phase Protein And Study Of Its Expression , Quansheng Liu

Retrospective Theses and Dissertations

Mouse SIP24 was originally discovered as a 24 k Da inducible protein secreted from quiescent Balb/c 3T3 fibroblast cells. Based on the analysis of peptide sequencing results and Western blot studies, SIP24 was identified as identical to the mouse protein encoded by 24P3 and is thus referred to as SIP24/24P3 in this thesis. SIP24/24P3 is a member of the lipocalin protein family, and its human homolog NGAL (neutrophil gelatinase associated lipocalin) has been reported to bind the chemotactic factor, fMLP (N-formylmethionyl-leucyl-phenylalanine);Studies were carried out to test the hypothesis that SIP24/24P3 is an acute phase protein ...


The Role Of Rat Brain Ptp-1 Surface Basic Residues In Substrate Reactivity , Deborah Sue Baedke Jan 1996

The Role Of Rat Brain Ptp-1 Surface Basic Residues In Substrate Reactivity , Deborah Sue Baedke

Retrospective Theses and Dissertations

Previous work by others has demonstrated that rat brain protein tyrosine phosphatase (rbPTP-1) exhibited increased reactivity toward substrates containing acidic residues N-terminal to phosphotyrosine. We show that substrates containing a single acidic residue C-terminal to the phosphotyrosine also enhanced reactivity with rbPTP-1. A substrate aspartate residue at the P+2 position exhibited the greatest enhancement of reactivity;Surface basic residues of rbPTP-1 were investigated for their role in the increased reactivity due to substrate acidic residues. Mutation of R47 or K120 to alanine decreased the enhancement of reactivity due to acidic residues N-terminal to phosphotyrosine by 78% and 73% respectively ...


Single Cell Analysis By Capillary Electrophoresis With Laser-Induced Native Fluorescence Detection , Sheri Joanne Lillard Jan 1996

Single Cell Analysis By Capillary Electrophoresis With Laser-Induced Native Fluorescence Detection , Sheri Joanne Lillard

Retrospective Theses and Dissertations

Individual mammalian cells were analyzed by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). This technique was chosen due to its high separation efficiencies, small sample volumes and sensitive detection. Native fluorescence was used, in which the analyte was not tagged with a fluorophore. 275-nm excitation gave attomole (amol = 10-18 mol) detection limits for the intracellular species of interest. Two projects are described in which hemoglobin (Hb) variants were determined in single red blood cells. In the third experiment, individual mast cells were degranulated on-column, and exocytosis and serotonin release were monitored temporally;First, single red blood cells, in which Hb ...


Studies On The Structure-Function Relationships And Regulation Mechanisms Of Escherichia Coli Adenylosuccinate Synthetase , Wenyan Wang Jan 1996

Studies On The Structure-Function Relationships And Regulation Mechanisms Of Escherichia Coli Adenylosuccinate Synthetase , Wenyan Wang

Retrospective Theses and Dissertations

Molecular biological, biochemical, and biophysical techniques were utilized to explore structure-function relationship of Escherichia coli adenylosuccinate synthetase. For the first time, residues Arg303, Arg304 and Arg305 were implicated as the determinants in the recognition and binding of substrate L-aspartate. Replacing Arg305 with leucine resulted in over a 100-fold decrease in kcat, suggesting a possible role of Arg305 in catalysis. pH dependent kinetic studies indicated that the side chain of Gln224 is not only involved in recognition of IMP, but also in stabiling the intermediate 6-phosphoryl-IMP. Substitution of the side chain amide with a carboxylate group resulted in loss of catalytic ...


Regulation Of Translation By The 3' Untranslated Region Of Barley Yellow Dwarf Virus-Pav Rna , Shanping Wang Jan 1996

Regulation Of Translation By The 3' Untranslated Region Of Barley Yellow Dwarf Virus-Pav Rna , Shanping Wang

Retrospective Theses and Dissertations

Eukaryotic translation initiation is an important step for gene expression. This process is stimulated by the interaction of 5' cap and 3' poly(A) tail mediated by trans-acting factors. Positive sense viral RNA can differ from the cellular mRNA by lacking either a 5' cap, or 3' poly(A) tail or both. Therefore, various strategies have evolved by the RNA viruses to achieve efficient translation. Barley yellow dwarf virus-PAV serotype (BYDV-PAV) lacks both a 5' cap and 3' poly(A) tail. I found that a sequence (called 3'TE for 3' translation enhancer) at the 3'UTR of barley yellow ...


Applications Of Partial Filling Micellar Electrokinetic Chromatography: From Electrospray Mass Spectrometry To Fluorescence Polarization Immunodetection , Wendy Marie Nelson Jan 1996

Applications Of Partial Filling Micellar Electrokinetic Chromatography: From Electrospray Mass Spectrometry To Fluorescence Polarization Immunodetection , Wendy Marie Nelson

Retrospective Theses and Dissertations

Mechanistic studies of partial filling micellar electrokinetic chromatography PF-MEKC) and a theoretical model for the prediction of separation behavior in PF-MEKC was developed in this work. Along with the theoretical development of PF-MEKC, two different detection modes were applied to PF-MEKC, in contrast to the traditional UV detection used in micellar electrokinetic chromatography (MEKC). The first detection mode coupled PF-MEKC with eletrospray ionization mass spectrometry. The second detection mode developed in this work is PF-MEKC coupled with fluorescence polarization immunodetection;First, in the mechanistic studies of PF-MEKC a theoretical model was proposed for predicting the separation behavior of triazine herbicides ...


X-Ray Crystallographic Studies Of The Nucleotide Binding Sites On Adenylosuccinate Synthetase From Esherichia Coli , Bradley Wayne Poland Jan 1996

X-Ray Crystallographic Studies Of The Nucleotide Binding Sites On Adenylosuccinate Synthetase From Esherichia Coli , Bradley Wayne Poland

Retrospective Theses and Dissertations

The crystal structures of unligated adenylosuccinate synthetase from Esherichia coli in the space groups P21 and P212121 have been refined to R-factors of 0.199 and 0.206 against 2.0 and 2.5A data, respectively. The dominant structural motif of the synthetase monomer is a central 10 stranded [beta]-sheet composed of 9 parallel strands and a 10th antiparallel strand. Four subdomains extend off of this [beta]-sheet and contribute loops of residues which are disordered. The dimer interface involves two nearly-independent regions. A pair of helicea (H4 & H5) interacting with their symmetry equivalent mates defines the first region. The second region involves structural elements which connect strands of the central [beta]-sheet. Residues in the putative IMP binding site are also located at or near the dimer interface;The P212121 crystal form allows gradual infusion of guanosine triphosphate ribonucleotides ...


Cloning Of Bovine Intercellular Adhesion Molecules And Characterization Of Expression Of Neutrophil Adhesion Molecules In Periparturient Cows And Calves , Eunkyung Lee Jan 1996

Cloning Of Bovine Intercellular Adhesion Molecules And Characterization Of Expression Of Neutrophil Adhesion Molecules In Periparturient Cows And Calves , Eunkyung Lee

Retrospective Theses and Dissertations

The interactions of circulating leukocytes with the endothelium represent a key point in the effector functions of the immune system by regulating the specificity and strength of cell-cell interactions. The recruitment of neutrophils to sites of inflammation is initiated by chemoattractants. The initial contact of neutrophils on vascular endothelial cells is a rolling interaction mediated by members of the selectin family. These include E-selectin and P-selectin on the surface of activated endothelial cells, and L-selectin on neutrophils. The subsequent activation of leukocyte [beta]2 integrins, which bind to the glycoprotein intercellular adhesion molecule-1 (ICAM-1) on endothelial cells, is essential for ...


Bacterial Physiology And Enzymology Of The Membrane-Associated Methane Monooxygenase And Ammonia Oxidation System From Methylococcus Capsulatus Bath, 1992-1996 , James Allan Zahn Jan 1996

Bacterial Physiology And Enzymology Of The Membrane-Associated Methane Monooxygenase And Ammonia Oxidation System From Methylococcus Capsulatus Bath, 1992-1996 , James Allan Zahn

Retrospective Theses and Dissertations

The enzymology and microbial physiology of methane and ammonia oxidation by the membrane-associated methane monooxygenase (pMMO) from the obligate methylotroph, Methylococcus capsulatus Bath were studied. Conditions leading to the stabilization of the pMMO were defined, and an active preparation of the pMMO from M. capsulatus Bath was isolated by ion exchange and hydrophobic interaction chromatography using dodecyl-[beta]-D-maltoside as the detergent. The active preparation consisted of three major polypeptides with molecular masses of 47,000 and 27,000, and 25,000 Da and contained 2.5 iron and 14.5 copper atoms per mole of enzyme. The 27,000 ...


Genetic Construction And Biochemical Analysis Of Thermostability Mutants Of Glucoamylase From Aspergillus Awamori , Yuxing Li Jan 1996

Genetic Construction And Biochemical Analysis Of Thermostability Mutants Of Glucoamylase From Aspergillus Awamori , Yuxing Li

Retrospective Theses and Dissertations

To study the molecular basis of Aspergillus awamori glucoamylase (GA) thermostability, eighteen mutants were constructed by site-directed mutagenesis and expressed in Saccharomyces cerevisiae based on thermostability theories. Four lysine residues, K61, K279, K352 and K404, were replaced with arginine, with all but K404 well exposed to the solvent and far away from the enzyme activity site. Mutations K61F/D65E and H254W/E326Q were made to fill a packing void around the inner set of six [alpha]-helices of GA and to displace water molecules inside the void. Five residues (A27, A393, A435, Ser436 and Ser460) were replaced with proline. Two ...


Characterization Of Membrane-Associated Nuclease Activity In Mycoplasma Pulmonis , Karalee Jean Jarvill-Taylor Jan 1996

Characterization Of Membrane-Associated Nuclease Activity In Mycoplasma Pulmonis , Karalee Jean Jarvill-Taylor

Retrospective Theses and Dissertations

Mycoplasmas are deficient in numerous biosynthetic pathways. Consequently, growth is dependent upon the acquisition of structural and macromolecular precursors from the environment. These precursors include phospholipids, fatty acids and nucleotide bases. DNases and RNases are postulated to be involved in the acquisition of nucleotides. The goals of this study were to determine if nuclease activities are universally associated with mycoplasmas, characterize the nucleases by subcellular distribution and apparent molecular weight, and to identify and analyze the nuclease gene(s) in Mycoplasma pulmonis;Nuclease activity was present in all mycoplasma species tested. The level of activity did not correlate with virulence ...


Characterization Of Biomacromolecules By Nuclear Magnetic Resonance Spectroscopy , Wayne Robert Baker Jan 1996

Characterization Of Biomacromolecules By Nuclear Magnetic Resonance Spectroscopy , Wayne Robert Baker

Retrospective Theses and Dissertations

Two proteins were examined by Nuclear Magnetic Resonance (NMR) spectroscopy. The Emb-1 protein of Daucus carota was found to have few spectral characteristics indicative of a stable tertiary structure. In particular, line-widths were narrow, chemical shift dispersion was poor and nuclear Overhauser effect spectra were sparse. The NMR spectra of Ribonuclease A was examined at a range of pH values and the chemical shifts of a selected number of resonances was plotted as a function of the pH. From these titrations curves pKa values were determined and assigned to specific ionizing groups within the protein based, in part, on physical ...


Biology Of Rpv Barley Yellow Dwarf Virus Satellite Rna , Lada Rasochova Jan 1996

Biology Of Rpv Barley Yellow Dwarf Virus Satellite Rna , Lada Rasochova

Retrospective Theses and Dissertations

Satellite RNAs (satRNAs) depend on their helper viruses for replication, encapsidation and spread. The goal of the research was to determine helper virus specificity and host range of the RPV barley yellow dwarf luteovirus (BYDV-RPV) satRNA (satRPV RNA) and assay satRPV RNA for the ability to affect the accumulation and modulate disease symptoms of its helper viruses in protoplasts and plants. Although similar in structural genes, subgroup I and II luteoviruses have very different polymerases. BYDV-RPV and beet western yellows virus (BWYV), members of subgroup II luteoviruses, supported satRPV RNA replication in monocotyledonous and dicotyledonous hosts, respectively. In contrast, BYDV-PAV ...