Institutionen för medicinsk biokemi och biofysik (MBB) / Department of Medical Biochemistry and Biophysics
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