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Abstract
Grant Number: 1R43DK063812-01 PI Name: CONSALVO, ANGELO P. PI Email: consalvo@frontiernet.net PI Title: Project Title: Method for Identification of alpha-Amidated Peptides Abstract: DESCRIPTION (provided by applicant): 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.
Thesaurus Terms:
amidation /deamidation, enzyme activity, glycine, method development, oxygenase, peptide hormone
biological signal transduction, biotherapeutic agent, enzyme inhibitor, posttranslational modification, short chain fatty acid
high performance liquid chromatography, mass spectrometry, polymerase chain reaction, tissue /cell culture
Institution: UNIGENE LABORATORIES, INC. 110 LITTLE FALLS RD FAIRFIELD, NJ 07004 Fiscal Year: 2003 Department: Project Start: 01-APR-2003 Project End: 31-MAR-2004 ICD: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES IRG: ZRG1