Thesis (Ph. D.)--University of Wisconsin--Madison, 1965. / Vita. Typescript. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph. D.)--West Virginia University, 1999. / Title from document title page. Document formatted into pages; contains xii, 96 p. : ill. Includes abstract. Includes bibliographical references.

M.Sc. / Please refer to full text to view abstract

Human glucosephosphate isomerase was subjected to a series of chemical modifications aimed at identifying residues essential for catalytic activity. Specific lysyl, arginyl, tryptophanyl and histidyl residues were found to react stoichiometrically with pyridoxal-5'-phosphate-NaBH4, 2,3-butadione, N-bromosuccinimide and N-bromoacetylethanolamine phosphate, respectively.

Glucose phosphate isomerase (GPI) was purified from human placenta utilizing cross-linked spherical particle phosphocellulose. In three steps, GPI could be purified approximately 5500 fold with greater than 50% recovery. The purified enzyme exhibited four bands upon non-denaturing PAGE and isoelectric focusing (IEF) when stained with GPI specific activity stain. The four isozymes were isolated by preparative IEF. The isoelectric points of the isozymes were determined. Sodium dodecyl sulfate (SDS) gel electrophoresis showed two types of subunits with different molecular weights. Structural analyses showed both types of subunits had blocked amino termini. Other properties of the isozymes and subunits, including immunological reactivity, pH stability, peptide mapping and amino acid composition, were also established.

Shen Dong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 97-104). / Abstracts in English and Chinese. / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- High fructose corn syrup (HFCS) --- p.2 / Chapter 1.1.1 --- Status quo and prospect of HFCS --- p.2 / Chapter 1.1.2 --- Industrial process for HFCS production --- p.3 / Chapter 1.1.3 --- Glucose (xylose) isomerase in industrial application --- p.5 / Chapter 1.2 --- Glucose (xylose) isomerase --- p.9 / Chapter 1.2.1 --- Source organisms --- p.9 / Chapter 1.2.2 --- Functions of glucose (xylose) isomerase --- p.12 / Chapter 1.2.3 --- Structure of glucose isomerase --- p.13 / Chapter 1.2.4 --- Catalytic mechanism of glucose isomerase --- p.17 / Chapter 1.2.5 --- Biochemical properties of glucose isomerase --- p.18 / Chapter 1.2.6 --- Immobilization studies --- p.22 / Chapter 1.3 --- Aims of my study --- p.25 / Chapter Chapter 2 --- Materials and Methods --- p.26 / Chapter 2.1 --- Cloning of parental glucose isomerase gene --- p.27 / Chapter 2.1.1 --- Materials --- p.27 / Chapter 2.1.1.1 --- Bacterial strain --- p.27 / Chapter 2.1.1.2 --- Growth media --- p.29 / Chapter 2.1.1.3 --- Antibiotics --- p.29 / Chapter 2.1.1.4 --- Reagents for isolation of chromosomal DNA --- p.30 / Chapter 2.1.1.5 --- Reagents for PCR reaction --- p.30 / Chapter 2.1.1.6 --- Reagents for agarose gel electrophoresis --- p.30 / Chapter 2.1.1.7 --- Reagents for DNA recovery from agarose gel --- p.31 / Chapter 2.1.1.8 --- Vector and enzyme for ligation --- p.31 / Chapter 2.1.1.9 --- Reagents for preparation of competent cells --- p.32 / Chapter 2.1.1.10 --- Reagents for extraction of plasmid DNA --- p.32 / Chapter 2.1.1.11 --- Reagents for DNA sequencing --- p.32 / Chapter 2.1.2 --- Methods --- p.32 / Chapter 2.1.2.1 --- Isolation of chromosomal DNA --- p.32 / Chapter 2.1.2.2 --- Preparation of primers --- p.33 / Chapter 2.1.2.3 --- Amplification of parental glucose isomerase gene --- p.33 / Chapter 2.1.2.4 --- Agarose gel electrophoresis of DNA --- p.35 / Chapter 2.1.2.5 --- DNA recovery from agarose gel --- p.35 / Chapter 2.1.2.6 --- Ligation of purified DNA fragment into vector --- p.36 / Chapter 2.1.2.7 --- Making competent cells --- p.37 / Chapter 2.1.2.8 --- Transformation / Chapter 2.1.2.9 --- Plasmid DNA preparation --- p.38 / Chapter 2.1.2.10 --- DNA sequencing --- p.39 / Chapter 2.2 --- Mutagenesis of glucose isomerase --- p.40 / Chapter 2.2.1 --- Materials --- p.40 / Chapter 2.2.2 --- Methods --- p.40 / Chapter 2.2.2.1 --- Preparation of primers --- p.40 / Chapter 2.2.2.2 --- Introduction of point mutations --- p.42 / Chapter 2.2.2.3 --- Assembly of DNA fragments --- p.44 / Chapter 2.2.2.4 --- Amplification of full-length genes --- p.45 / Chapter 2.2.2.5 --- Agarose gel electrophoresis of DNA --- p.46 / Chapter 2.2.2.6 --- DNA recovery from agarose gel --- p.46 / Chapter 2.2.2.7 --- Ligation of purified DNA fragment into vector --- p.46 / Chapter 2.2.2.8 --- Transformation --- p.46 / Chapter 2.2.2.9 --- Plasmid DNA preparation --- p.46 / Chapter 2.2.2.10 --- DNA sequencing --- p.46 / Chapter 2.3 --- Expression and purification of glucose isomerase --- p.47 / Chapter 2.3.1 --- Materials --- p.47 / Chapter 2.3.1.1 --- Phosphate buffer preparation --- p.47 / Chapter 2.3.1.2 --- Reagents for SDS-PAGE --- p.48 / Chapter 2.3.2 --- Methods --- p.48 / Chapter 2.3.2.1 --- Incubation of bacteria --- p.48 / Chapter 2.3.2.2 --- Extraction of crude protein --- p.49 / Chapter 2.3.2.3 --- Partial purification of glucose isomerase --- p.49 / Chapter 2.3.2.4 --- Further purification of glucose isomerase --- p.50 / Chapter 2.3.2.5 --- SDS-PAGE --- p.51 / Chapter 2.4 --- Enzyme assays --- p.52 / Chapter 2.4.1 --- Materials --- p.52 / Chapter 2.4.1.1 --- Substrate for activity assay --- p.52 / Chapter 2.4.1.2 --- Buffer and bivalent metal cations --- p.52 / Chapter 2.4.1.3 --- Reagents for protein concentration determination --- p.53 / Chapter 2.4.1.4 --- Reagents for activity determination --- p.54 / Chapter 2.4.2 --- Methods --- p.54 / Chapter 2.4.2.1 --- Protein concentration determination --- p.54 / Chapter 2.4.2.2 --- Specific activity assay --- p.55 / Chapter 2.4.2.3 --- Thermostability assay --- p.57 / Chapter 2.4.2.4 --- Temperature curve of activity --- p.57 / Chapter 2.4.2.5 --- pH effects --- p.57 / Chapter 2.4.2.6 --- pH stability assay --- p.58 / Chapter 2.4.2.7 --- Bivalent metal cations --- p.58 / Chapter 2.4.2.8 --- Conversion rate of isomerization --- p.59 / Chapter Chapter 3 --- Results --- p.61 / Chapter 3.1 --- Cloning of parental glucose isomerase gene --- p.62 / Chapter 3.2 --- Mutagenesis of glucose isomerase --- p.64 / Chapter 3.3 --- Expression and purification of glucose isomerase --- p.65 / Chapter 3.4 --- Enzyme assays of glucose isomerase --- p.70 / Chapter 3.4.1 --- Specific activity --- p.70 / Chapter 3.4.2 --- Thermostability --- p.72 / Chapter 3.4.3 --- Activity at different temperatures --- p.76 / Chapter 3.4.4 --- pH effects --- p.77 / Chapter 3.4.5 --- pH stability --- p.78 / Chapter 3.4.6 --- Bivalent metal cations --- p.79 / Chapter 3.4.7 --- Conversion rate of isomerization --- p.84 / Chapter Chapter 4 --- Discussions --- p.87 / Chapter 4.1 --- Different glucose isomerase mutants --- p.88 / Chapter 4.2 --- Enzymatic physicochemical and catalytic properties --- p.94 / Chapter 4.3 --- Future work --- p.95 / References --- p.97

The distribution of isozymes of triosephosphate isomerase was normal in all human tissues examined. This finding argues against the existence of tissue-specific isozymes. Normal distributions of isozymes were also found in patients with cri-du-chat syndrome. Thus it is unlikely that a gene for triosephosphate isomerase is located on the short arm of chromosome five in man. When triosephosphate isomerases from a wide range of species were examined by starch gel electrophoresis, definite evolutionary patterns were found. Kinetic studies were conducted on human triosephosphate isomerase under conditions simulating the intracellular environment of the erythrocyte. Calculations using the kinetic parameters obtained indicate that even in triosephosphate isomerase deficiency disease, enough enzyme activity remains that the rate of glycolysis should not become inhibited.

The discovery of the methylerythritol phosphate pathway (the MEP pathway) as an alternate pathway for isoprenoid biosynthesis in some organisms including most bacteria, malarial parasites and plants, but not in animals, has stimulated extensive studies in this area. Research has revealed the potential of finding novel antibacterials, antimalarial drugs, and herbicides from enzyme inhibitors of this pathway. The natural products fosmidomycin and FR900098 appear to be very promising antibacterial and antimalarial compounds. Both compounds have inhibition activities against the second enzyme in the MEP pathway, deoxyxylulose- 5-phosphate reductoisomerase (DXR), which mediates the conversion of deoxyxylulose-5-phosphate (DXP) into methylerythritol-4-phosphate (MEP). This thesis presents one aspect of the MEP pathway studies. Six different analogs of DXP were designed based on the structural features of DXP to understand the requirements of the DXR-substrate binding. Compounds with the trivial names 1-Me-DXP (containing an ethyl ketone moiety), DX-phosphonate (DXP having a phosphonate group rather than a phosphate group), 4-epi-DXP (possessing the opposite stereochemistry at the C��� position compared to DXP), 4-deoxy-DXP (lacking the hydroxyl group at the C��� position), 3-deoxy-DXP (lacking the hydroxyl group at the C��� position), and DXP carboxamide (having a primary amide group rather than the methyl ketone) were synthesized and tested as alternate substrates and enzyme inhibitors against DXR. The compound DX-phosphonate was the only alternate substrate among the synthesized compounds. The remaining analogs of DXP acted as weak competitive inhibitors against DXR. Kinetic studies of these compounds provided an overall picture of how the substrate DXP binds to DXR. Further studies of the compound 1-Me-DXP, using the published X-ray crystal structures of DXR and DXR mutagenesis demonstrated more detail of the DXR active site. The results present useful information for designing better enzyme inhibitors. The mechanism for the rearrangement of DXP to MEP by DXR was also studied. Two possible mechanisms for this rearrangement have been proposed, the ��-ketol rearrangement and the retroaldol/aldol rearrangement. Several approaches including the use of the potential alternate substrates, 4-deoxy-DXP and 3-deoxy-DXP were tried. Unfortunately none of the results obtained can definitively rule out either of the mechanisms. Further studies are needed to completely understand this mechanism and establish additional strategies for inhibition of DXR. Syntheses of an intermediate from the DXR reaction, methylerythrose-4-phosphate, were also attempted in order to better understand the chemistry mediated by DXR. Even though the target compound was not successfully obtained, several synthetic approaches to this compound were useful for the syntheses of the different DXP analogs mentioned above. / Graduation date: 2004

Conjugated linoleic acid (CLA) has gained much attention recently due to its beneficial health and biological effects on animals and humans. However, the CLA-forming enzyme system has not been studied in details. Six strains of Lactobacillus acidophilus L11, L12, L14, L15, Lactobacillus fermentum and Lactobacillus reuteri were used to study the growth conditions and the production of CLA-forming enzyme in MRS media containing linoleic acid concentrations at 37°C. The purification and characterization of a CLA-forming enzyme were reported for the first time. The results showed that this enzyme has a molecular mass of 72 kDa, and is composed of two subunits. The optimal pH and temperature were 7.0 and 37°C, respectively. Kinetic study indicated that the enzyme has a high affinity for linoleic acid having a Km value of 1.49 x 10 -5 M and the Vmax was 17.1 muM/mg/min. The enzyme activity was inhibited by the metal chelators. (Abstract shortened by UMI.)

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