Graduation Year

2006

Document Type

Dissertation

Degree

Ph.D.

Degree Granting Department

Chemistry

Major Professor

David J. Merkler, Ph.D.

Keywords

Glyoxylate, Platform technololgy, Mouse joining peptide, Glycolate oxidase, Chemi-luminescence, Calcitonin gene related peptide, High performance liquid chromotography

Abstract

Peptide hormones are responsible for cellular functions critical to the survival of an organism. Approximately 50% of all known peptide hormones are post-translationally modified at the C-terminus. Enzymatic oxidative conversion of C-terminal glycine extended peptide precursors results in an a-amidated peptide and glyoxylate. Peptidylglycine a-amidating monooxygenase (PAM) is the single known enzyme responsible for catalyzing this reaction. PAM is an O2, Cu(II), and Zn(II) dependent bifunctional enzyme. Initially, PAM hydroxylates the glycyl a-carbon followed by dealkylation of the hydroxylated intermediate to an a-amidated product and glyoxylate. PAM is also responsible for the conversion of glycine extended fatty acids to fatty acid amides and glyoxylate. PAM catalyzes the activation of all glycine-extended prohormones including biomolecules ranging from neuro to physio-homeostatic hormones.

Identification of a-amidated hormones from a biological source has been severely hindered by the lack of a specific assay for this distinctive class of biological hormones, indicating that numerous a-amidated hormones remain undiscovered. Based on the selective in situ chemistry of PAM, a novel and specific assay was developed for the discovery of a-amidated hormones. The identification of novel a-amidated hormones will lead to an increased understanding of post-translational modifications and will pioneer a new understanding of a-amidated hormone biosynthesis, regulation, and bioactivity. Discovery of novel a-amidated biomolecules could also lead to their use as pharmaceuticals as there are several currently marketed a-amidated peptide based pharmaceuticals.Inhibition of PAM in cell culture leads to the accumulation of glycine-extended hormones in the conditioned medium. The medium was fractionated by chromatographic techniques and each specific fraction was then assayed by the newly developed platform technology for the presence of a-amidated hormones.

For every a-amidated hormone synthesized by PAM, glyoxylate is also formed. Based on this 1:1 molar ratio, several novel spectrophotometric, fluorescent, and chemi-luminescent enzyme linked assays for glyoxylate were developed, which when utilized on cell culture fractions proved positive for the identification of a-amidated hormones. Each novel spectroscopic assay was independently verified by a variety of known methodologies. Moreover the assay was utilized to identify two known a-amidated hormones accumulated from cell culture, which were further verified by Mass Spectral analysis.

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