Includes bibliographical references. / 2 v. : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Describes the purification and characterization of human polyamine oxidases found in rectoplacental serum. / Thesis (Ph.D.)--University of Adelaide, Dept. of Paediatrics and Women's and Children's Hospital Dept. of Immunopathology, 1998

Thesis (Ph. D.)--University of Adelaide, Dept. of Paediatrics and Women's and Children's Hospital Dept. of Ummunopathology, 1998. / Includes bibliographical references.

The major findings of this investigation are that polyamine biosynthetic enzymes and polyamine levels are regulated by specific cellular growth factors. A serum-free defined medium was developed for the Chinese hamster ovary cell line to examine the regulation of ornithine decarboxylase (ODCase) (EC.4.1.1.17), S-adenosylmethionine decarboxylase (SAMDCase) (EC.4.1.1.50), as well as polyamine catabolism. The activity of ODCase is dependent primarily on the presence of insulin, and appears to be modulated by transferrin and ferrous sulfate, indicating that iron transport may be important in the expression of ODCase activity. The enzyme activity can also be increased by depriving the substrate ornithine, which probably acts through a putrescine mediated event. This substrate limitation leads to an intracellular decrease in putrescine and spermidine, but not spermine. The activity of SAMDCase is not influenced by alterations in the growth factors or by ornithine deprivation. Since the spermidine levels are lower as compared to cells growing in medium with serum, it appears that SAMDCase activity is not generally regulated in a negative manner by spermidine. The polyamine interconversion enzymes, such as spermidine/spermine N¹-acetyltransferase and polyamine oxidase, appear to be regulated by growth factors other than insulin, transferrin, and ferrous sulfate. Cells maintained in defined medium are much more tightly attached to the surface of the dishes in which they are growing, which may be related to the growth factors present or a lack of cellular polyamines. Vinculin, a cell surface protein associated with focal adhesion plaques, moves away from the cell surface and into the nuclear area in defined medium cells as evidenced by fluorescent antibody staining. The major conclusions of this work are that ODCase synthesis is regulated by growth factors, that enzyme activity is also regulated post-transcriptionally by substrate and end-product, and that general polyamine metabolism is dependant on complex growth factors, other than insulin, which regulate the metabolism is dependant on complex growth factors, other than insulin, which regulate the metabolism and interconversion of polyamines.

Thesis (Ph. D.)--University of Texas at Austin, 2000. / Vita. Includes bibliographical references (leaves 163-176). Available also in a digital version from Dissertation Abstracts.

The speB gene of E. coli encodes agmatine ureohydrolase (AUH). AUH catalyses the hydrolysis of agmatine to urea and putrescine in a polyamine biosynthetic pathway. The plasmid pKA5, derived from an E. coli genomic library, was the source of a 2.97 kb restriction fragment containing the speB gene. Sequencing of this fragment revealed three intact open reading frames, ORF1 and ORF2 on one strand and ORF3 on the opposite strand, as well as a truncated open reading frame, ORF4, which terminated 92 kb upstream from ORF3. ORF2 and ORF3 were convergent, and overlapped by 85% of their sequence. ORF1 and ORF3 were separated by a sequence of two imperfect repeats containing four palindromes, three of which were overlapping. ORF3 represented the coding sequence of the speB gene. Two transcripts were detected from the speB gene: a shorter transcript, initiated 101 bp upstream from ORF3, and a polycistronic message, coding for ORF3 and ORF4. The short transcript was abundantly expressed when ORF4 sequences were deleted, but when ORF4 and its upstream sequences were present, the polycistronic message predominated and the amount of the monocistronic message was drastically reduced. The promoter producing the shorter transcript required only a -12 TATACT sequence for activity. Deletion of a 460 bp fragment comprising the 5'-region of ORF1 from a plasmid containing ORF1, ORF2 and speB reduced the activity of AUH by 83%. This fragment contained two divergently oriented promoters. The presence of ORF1 did not stimulate ß-galactosidase encoded by the speB promoter fused to lacΖ. Agmatine induced transcription from speB but not from the ORF4 nor the ORF1 promoters. cAMP caused an 88% reduction in the AUH activity of wild type E. coli K-12 but had no effect on the activity of plasmid encoded AUH. The activity of neither the speB nor the ORF4 promoters fused to lacΖ or phoA were influenced by cAMP; in contrast, the lacZ promoter fused to lacZ or phoA was stimulated by cAMP. Thus, the role of cAMP and CRP on speB expression is indirect and limited to a single copy state. / Ph. D.

Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.

The polyamines spermine and spermidine play important roles in many cellular processes, and unusual levels of these polyamines have been associated with numerous human diseases. Spermidine/spermine N¹-acetyltransferase (SSAT) is an enzyme involved in polyamine regulation, where acetylation of polyamines by SSAT ultimately leads to their degradation or export from the cell. In this dissertation, x-ray crystallography and nuclear magnetic resonance (NMR) are used to provide insights into the structure and function of this important enzyme. X-ray crystallography provided two distinct views of SSAT: one of the enzyme in complex with coenzyme A (CoA), and another of the enzyme in complex with CoA and the polyamine spermine. Together, the two structures reveal structural plasticity in the active site of the enzyme. The complex with spermine provides a direct view of polyamine binding by SSAT, and shows that the enzyme relies heavily on associated water molecules to bind spermine; these water molecules also appear to form a "proton relay" between the primary amine of spermine and the side-chain of a conserved glutamate residue. Guided by the structural results, NMR methods were used to test hypotheses regarding the enzyme mechanism of SSAT. The activity of the enzyme over a range of solution conditions, and towards different polyamine substrates, was determined; the effects of mutating single amino acids in the enzyme were also evaluated. The enzyme appeared to be most active between pH 8.5 and 9.5, and mutation of the aforementioned glutamate significantly altered this behavior. This suggests the glutamate is directly involved in the acetyltransfer reaction, where it likely functions as a catalytic base though the proton relay in the enzyme active site. These studies advance our general understanding of how polyamines are regulated in mammalian cells, and have the potential to assist in developing new therapeutic options for human diseases involving polyamines. / text