Isolation and characterization of regulatory peptides and bioactive compounds

Abstract: Isolation of peptides and other bioactive compounds is an important and often necessary step to get the total information about their structures. This is demonstrated by a number of different characterizations in this thesis. Bioactive peptides and small organic molecules can act as signaling substances and messengers in multicellular organisms and are fundamental to higher forms of life. The following bioactive peptides and compounds were studied. 1) Different assays can be used for detection of novel peptides. Chemical assays are simple, but still robust, and have proven successful in several cases. In this thesis, an assay was developed to detect peptides that contain tryptophan. An N-terminal dipeptide Gly-Trp is characteristic for galanin, which was cleaved off, separated and visualized. In this manner a novel form of galanin from chicken was detected and isolated. 2) Antibacterial peptides were first found in insects and later in mammals. Many of these peptides are basic, some are Pro/Arg-rich (e.g. peptide PR-39). Investigation of basic peptides from pig spleen, resulted in the isolation of a new variant form of NK-lysin, previously only detected in a cDNA library from porcine bone marrow. PR-39 was identified by a method suitable for Pro/Arg-rich peptides using ladder sequence analysis with mass spectrometry. 3) Another biological assay, involving cAMP production in cell culture of SK-N-MC cells, was used to screen peptide fractions, and resulted in the isolation of a novel form of the hormone PYY, with a phosphate group attached to the Ser-13 residue. This finding demonstrates the benefit of peptide isolation to find posttranslational modifications not directly obvious from the corresponding DNA sequence. Using nano-electrospray tandem mass spectrometry, the phosphorylated form of porcine PYY was identified and the modification localized to Ser- 13. 4) Specific antibodies raised against peptides are a valuable tool in peptide chemistry. We have isolated PEC-60 from pig and rat brains with a method that combines column purification procedures with the specificity of a radioimmunoassay (RIA) and the sensitivity of mass spectrometry to directly identify the peptide. The results show that PEC-60, like many other peptides, is localized in the gastrointestinal tract and also in the central nervous system. The specific regional brain distribution may imply a specific function. 5) Using RIA and HPLC purification, 4 novel forms of modified galanin were isolated and characterized by mass spectrometry. The modified forms contained beta-aspartic shifts and oxidized tyrosine. 6) Hypoglycemic agents have been described in plants. Gynostemma pentaphyllum (Cucurbitaceae), an East-Asian herb, has been reported to have different activities, such as antitumor, cholesterol-lowering, immunopotentiating, hypoglycemic and antioxidant effects. We have isolated a novel insulin-releasing substance from this extract, determined the structure by NMR and mass spectrometry and characterized the effects on insulin release. We found that it is a novel saponin and we named it phanoside. The results provide us with new knowledge about localization, structure, and processing of hormones and bioactive molecules.