The research project, The Enzymatic Synthesis of CoA Derivatives Using a New ATP Regeneration System, describes the multiple lab trials conducted to develop an ATP regeneration system using various concentrations of specific substrates and the two enzymes MatB and PrpE. Reactions were combined using different concentrations of ADP, the addition or removal of ADK (adenylate kinase), and the substitution of either MatB or PrpE. Further reactions were combined using trans-crotonyl, trifluoroacetic acid, butyric acid, acetic acid, and creatine phosphokinase. This report also includes the methods used and analysis of the different chromatographs of each sample tested. / text

Staphylococcus aureus protease V8 (SPV8), also known as Endoproteinase Glu-C (EC 3.4.21.19), is an enzyme isolated from the bacteria Staphylococcus aureus. This unusual enzyme has been found to cleave specifically at glutamyl and aspartyl peptide bonds and has been used as a tool in the preparation of protein substrates for amino acid sequence analysis. SPV8 has been reported to show some stability toward various denaturants (Drapeau, G.R. (1977) Methods in Enzymology_, 47:189-191). In order to more adequately assess the denaturant stability of SPV8, the effect of guanidine hydrochloride (HC1), a common protein denaturant, on the proteolytic action of SPV8 was studied. The extent of cleavage of the glutamyl peptide bond in adrenocorticotropic hormone 1-10 (ACTH 1-10) was found to decrease with increasing concentrations of guanidine HC1.At 22°C in the presence of 3.0 M guanidine HCl, only 25% of SPV8's proteolytic activity was retained. In the presence of 4.0, 5.0, or 6.0 M guanidine HC1, virtually all proteolytic activity toward the glutamyl bond of ACTH 1-10 was lost, presumably due to the inactivation of the protease by denaturation or increased autolysis mediated by the guanidine HC1. At temperatures above 22°C, SPV8 was more susceptible to inactivation by guanidine HC1. Thus SPV8 appears to retain some proteolytic activity in the presence of guanidine HC1, but only at concentrations less than 4.0 M. There was no difference in the proteolytic activity of SPV8 toward the glutamyl peptide bond of ACTH 1-10 when incubation was carried out in ammonium bicarbonate buffer (pH 7.80), phosphate buffer (pH 7.80), or Tris-HC1 buffer (pH 7.80). The presence of 1 mM calcium chloride in the 3.0 M guanidine HC1/phosphate buffer solution enhanced the enzymatic action of SPV8. The presence of 1 mM calcium chloride in Tris-HC1 buffer (pH 7.80) does not effect the proteolytic activity of SPV8 at 22°C. However, there was slight reduction in SPV8's enzymatic action toward ACTH 1-10 when the 1 mM calcium chloride was present in the 3.0 M guanidine HC1/ammonium bicarbonate buffer (pH 7.80) solution. / Department of Chemistry

Polyphenol oxidase (PPO) enzymatic extracts were recovered from apple fruit and potato tubers and enriched by an acetone precipitation. The enriched PPO extracts were immobilized by adsorption onto a wide range of inorganic supports, including chitin, alumina oxide, glass beads, Celite, Dowex and Silica gel using selected media, including water, sodium phosphate buffer and a ternary micellar system. The highest immobilization efficiencies and specific activities were obtained when the PPO extracts were suspended in sodium phosphate buffer and adsorbed onto alumina oxide. Biocatalysis of the free and immobilized PPO extracts was investigated in selected organic solvent media, including hexane, heptane, toluene and dichloromethane, using chlorogenic acid, catechin, and the endogenous phenolic compounds from apple fruit and potato tubers as substrate models. In the organic solvent media, the free PPO extracts from apple and potato demonstrated optimal enzymatic activities at 28°C and between 25 to 35°C, respectively, whereas the immobilized extracts both showed optimal enzymatic activities at 30°C. The free and immobilized extracts from apple and potato also showed similar pH values for optimal enzymatic activity in the range of 6.0 to 6.5. The immobilized apple and potato PPO extracts demonstrated a 1.5 to 1.8 and 2.1 to 3.2-fold increases in PPO activity, respectively, compared to those observed with their free counterparts, and the lowest Km values were obtained with chlorogenic acid followed by catechin and the endogenous phenolic compounds. The immobilized and free PPOs from apple and potato also showed higher Vmax values in the hexane medium followed the heptane, toluene and dichloromethane media. The end products of PPO biocatalysis were purified by size-exclusion chromatography and detected at 280 nm for the residual catechin and endogenous phenolic compounds, and at 320 nm for the PPO-catalyzed end products. Spectroscopic scanning

Thesis (M.S.)--Rochester Institute of Technology, 1991. / Spine title: Active site configuration of GDH. Typescript. References: leaves 73-75.

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves 129-137).

Thesis (Ph. D.)--University of Wisconsin--Madison, 1970. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Kyoto University (京都大学) / 0048 / 新制・課程博士 / 博士(工学) / 甲第17601号 / 工博第3760号 / 新制||工||1573(附属図書館) / 30367 / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 濵地 格, 教授 松田 建児, 教授 秋吉 一成 / 学位規則第4条第1項該当

Preliminary observations of the restricted regionalization of the enzyme 2' ,3'-cyclic nucleotide-3’ -phosphohydrolase led to an investigation of the subcellular regionalization of this enzyme in cerebral white matter. Since bovine corpus callosum contained eighteen times as much enzyme activity as grey matter, an association of the enzyme with myelin was suggested. Subsequent fractionation of bovine cerebral white matter by sucrose density gradient according to the procedure of Autilio, Norton, and Terry for the purification of myelin (1), showed that greater than 60% of the total activity was associated with the myelin rich fractions. In order to fractionate cerebral white matter more thoroughly, a modified De Robertis fractionation procedure was utilized allowing for separation of nuclear, mitochondrial, and microsomal pellets by differential centrffugation (2). Phosphohydrolase activity was distributed in all fractions, and electron microscopy demonstrated the presence of myelin in all of these fractions. Subsequent fractionation of these primary fractions on a discontinuous sucrose density gradient, showed essentially all of the phosphohydrolase activity in the lightest fraction at the top of each gradient. This band was comprised primarily of myelin figures as verified by electron microscopy. These studies indicated that the enzyme was associated with myelin. The foregoing result was further supported by a study of the increase in enzyme activity during myelination in rats. Myelination is known to occur early in the life of the rat, being initiated a few days after birth, entering a rapid phase of onset at about 10 days and being essentially complete after 50 days (3). Cholesterol was shown to increase in a corresponding manner indicating that myelination was indeed proceeding. Further evidence that the enzyme is associated with myelin came from an investigation of mutant mice. Quaking mice have been shown to be deficient in myelin, containing, according to Bauman and co-workers (4), only 62% of the normal galactolipid levels. Since galactolipids are presently accepted markers for myelin, and since adult quaking mice had 50% of the control enzyme activity, in agreement with the published galactolipid values, it was thought not unlikely that the two phenomena were related. This result also inferred an association of the enzyme with myelin. In attempt to further uncover the physiological role of the 2’,3'-cyclic nucleotide-3'-phosphqhydrolase, investigations have been directed towards elucidation of the substrate specificity of the enzyme. Uridine and guanosine-2’,3'-cyclic phosphothioates, kindly donated by Dr. Fritz Eckstein of the Max Planck Institut für Experimentelle Medizin, were hydrolyzed at rates of l.4 and l4.3% that of adenosine-2’,3'-cyclic phosphate. Cyclic inositol phosphate, synthesized from inositol-2-phosphate in the presence of dicyclohexylcarbodi-imide and pyridine, and glucose-l,2-cyclic phosphate, synthesized from (formula omitted)-D-glucose-l-phosphate, in a similar manner, were not hydrolyzed td any measureable extent. Preliminary results also show that ribose cyclic phosphates are not hydrolyzed, indicating a requirement for a purine or pyrimidine ring in the substrate molecule. These results are discussed with respect to their possible physiological significance. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

Pyrimidine salvage pathways are essential to all cells. They provide a balance of RNA synthesis with the biosynthetic pathway in pyrimidine prototrophs and supply all the pyrimidine requirements in auxotrophs. While the pyrimidine biosynthetic pathway is found in almost all organisms and is nearly identical throughout nature, the salvage pathway often differs from species to species, with aspects of salvage seen in every organism. Thus significant taxonomic value may be ascribed to the salvage pathway. The pyrimidine salvage pathways were studied in 55 microorganisms. Nine different salvage motifs, grouped I-IX, were identified in this study based on the presence of different combinations of the following enzymes: cytidine deaminase (Cdd), cytosine deaminase (Cod), uridine phosphorylase (Udp), uracil phosphoribosyltransferase (Upp), uridine hydrolase (Udh), nucleoside hydrolase (Nuh), uridine/cytidine kinase (Udk), 5'-nucleotidase and CMP kinase (Cmk).

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