Project 1 Introduction: Fluorescently labeled neuropeptides can be used to localize neuropeptide receptors in cells and tissue sections, examine trafficking of neuropeptide receptors in live cells by fluorescence microscopy, and to sort receptor bearing cells by flow cytometry. Methods: Neuropeptides will be labeled with the fluorophores cyanine-3, cyanine-5, and rhodamine and fluorescein. Fluorescently labeled peptides will be separated from unlabeled peptides by high pressure liquid chromatography. Mass spectrometry will be used to identify labeled residues and to determine how many fluorescent groups are linked to the neuropeptide. Results: We have previously used the Mass Spectrometry Facility to characterize fluorescently labeled substance P, neuokinin A and gastrin releasing peptide. We identified that the lysine residues were labeled with the fluorophore cyanine-3, and determined the identity of multiple fluorescent products. These fluorophores were used to localize neuropeptide receptors in both transfected cells and primary cultures of neurons, and to sort receptor bearing cells by flow cytometry. Discussion: We have demonstrated that fluorescent neuropeptides can be used for localizing neuropeptide receptors with very high resolution by confocal microscopy and for enriching populations of receptor bearing cells by flow cytometry. We intend to extend these studies to develop highly fluorescent neuropeptides in which multiple groups are labeled with fluorophores withfull retention of biological activity. Project 2 Introduction: The biological activities of neuropeptides are terminated by enzymatic degradation. We have isolated a number of peptidases that degrade neuropeptides and will use massspectrometry to identify cleavage sites. Methods: Neuropeptides will be incubated with highly purified proteases and the products of degradation will be separated by high pressure liquid chromatography. The cleavage products willbe identified by amino acid composition analysis and also by mass spectrometry. Therefore, we will determine the cleavage sites for the proteases. Results: We have previously used amino acid composition analysis and mass spectrometry to identify cleavage sites in substance P, gastrin releasing peptide, somatostatin and enkephalins by the enzyme neutral endopeptidase. Discussion: We intend to continue purification of proteases from neural tissue and will enzymatically characterize them as described. For these studies it will be important to determine the sites of hydrolysis in a variety of neuropeptides by amino acid composition analysis and mass spectrometry.
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