Peptide hormones are ubiquitous in nature, and perform cellular functions that are critical to the survival of the organism. Approximately 50 percent of all known peptide hormones are post-translationally modified at the C-terminus by conversion of glycine-extended precursors to the peptide-amide. The enzyme that performs this modification is peptidylglycine alpha-amidating mono-oxygenase (PAM). Inhibition of PAM in various cell types in culture can be accomplished by the addition of specific PAM inhibitors, such as 4-phenyl-3-butenoic acid, resulting in the accumulation of glycine-extended hormone precursors in the conditioned medium. The medium will be fractionated by chromatography techniques and the individual fractions assayed by sequential enzyme reactions consisting of PAM --> glyoxylate dehydrogenase or PAM > glyoxal oxidase --> horseradish peroxidase, to yield a color reaction. In Phase I, we will optimize the enzyme reactions using model glycine-extended substrates, and demonstrate feasibility by the isolation of known peptide hormones from CA-77 cells using this technology. During Phase II, the optimized technology will be used to detect novel amidated peptide hormones in neuro-endocrine cells, and produce the novel peptides recombinantly to obtain material for a series of physico-chemical and functional characterizations. The success of this project will eventually lead to the development of novel therapeutic peptide drugs.
| Carpenter, Sarah E; Merkler, David J (2003) An enzyme-coupled assay for glyoxylic acid. Anal Biochem 323:242-6 |
