No description available.

In this research, a model system for studying the effects of the toxic metabolite, methylglyoxal, was created using Chinese Hamster Ovary (CHO) cells which produce tissue plasminogen activator (t-PA). The human gene for glyoxalase I was subcloned into an inducible mammalian expression vector. This vector was then used to create three stable CHO integrants, two control and one putative glyoxalase I producing cell lines. The CHO clones were characterized for the production of glyoxalase I using both SDS-PAGE gels and glyoxalase activity assays. In addition, the cell lines were evaluated to determine the levels of free methylglyoxal produced. The putative glyoxalase producer showed higher levels of glyoxalase I activity than the parent cell line and produced a unique protein band at the correct molecular weight. They also had a significantly lower level of free methylglyoxal than either of the two control cell lines. These cells can now be used as a tool to determine the specific effect of methylglyoxal on the yield and quality of tissue plasminogen activator produced. / Graduation date: 2000

Thesis (Ph.D.)--University of Minnesota, 1999. / Includes bibliographical references (leaves 98-112). Also available on the World Wide Web as a PDF file.

Thesis (M. Phil.)--University of Hong Kong, 2000. / Includes bibliographical references (leaves.

Thesis (Ph. D.)--University of Queensland, 2001. / Includes bibliographical references.

Thesis (Ph. D.)--University of Texas at Austin, 2001. / Vita. Includes bibliographical references (leaves 76-87). Available also in a digital version from Dissertation Abstracts.

published_or_final_version / Biochemistry / Master / Master of Philosophy

No description available.

No description available.

The carboxyl-terminal sequences of the $ alpha$ and $ beta$ subunits of the secreted yeast K1 toxin have been determined by protein sequencing and amino acid analysis of peptide fragments generated from the purified toxin. It revealed that the $ alpha$ and $ beta$ subunits consist of amino acid residues 45-147 and 236-316 from the preprotoxin, respectively. The preprotoxin configuration can be represented as: prepropeptide-ArgPro-$ alpha$-ArgArg-$ gamma$-LysArg-$ beta$. This structure for the preprotoxin defines a specific processing pathway in yeast involving a dibasic endoprotease, encoded by the KEX2 gene and a carboxypeptidase B like enzyme which is probably encoded by the KEX1 gene. / By using the patch-clamp technique, it is shown both in vivo with sensitive yeast spheroplasts and in vitro with asolectin liposomes that the toxin forms ion permeable channels. The toxin induced ion channels are voltage independent with a unit conductance of 118pS, often appearing in pairs and prefer monovalent cations. / K1 toxin has a much wider killing spectrum at the spheroplast level than at the whole cell level as demonstrated by the fact that the toxin kills spheroplasts from the genera Candida, Kluyveromyces, and Schwanniomyces, whose cells are toxin insensitive. A toxin binding study shows that the wall receptor can define toxin specificity and is necessary but not sufficient for toxin action on intact cells. / Using various mutagenesis techniques, a set of mutations throughout regions encoding the $ alpha$ and $ beta$ subunits that allow secretion of mutant toxins were generated. By analyzing the phenotypes of these mutant toxins, the ion channel forming domain is assigned exclusively to the hydrophobic $ alpha$ subunit and the cell wall receptor binding domain is localized to both the $ alpha$ and $ beta$ subunits.