| Budget Amount *help |
¥7,700,000 (Direct Cost: ¥7,700,000)
Fiscal Year 1985: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1984: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1983: ¥5,200,000 (Direct Cost: ¥5,200,000)
|
|---|
| Research Abstract |
We attempted to develop sensitive methods of analysing peptides in the nervous tissue using high performance liquid chromatography (HPLC), mass spectrometry and sequence analysis.
As many bioactive neuropeptides contain amidated carboxyl-termianls, procedures for determination of peptides containing amidated carboxylterminals were studied. After the peptide sample was digested with an endopeptidase and was dansylated, dansylated amino acid amides were selectively extracted with ethyl acetate under alkaline condition and separated by HPLC with Develosil C8-5 using a linear gradient of methanol in Tris buffer. By analysis of acid-acetone extracts of bovine spinal cord, several amino acid amides, such as Pro-amide,Met-amide,Leu-amide and Ser-amide, were detected. In the subfractions of extracts by Biogel P2, Leu-amide was detected in fraction B (Kav=0.2) and Met-amide in fraction C (Kav=0.3). This HPLC system was sensitive enough to detect up to pmol level of dansylated amino acid amides and was useful for analysis of neuropeptides.
In order to develop analytical procedures of hydrophobic peptides, we solubilized a hydrophobic glycoprotein, PO protein, and digested it with trypsin. The tryptic peptides were separated into 12 peaks by HPLC with a macromolecular polystyrene resin (Hitachi 3013-0). Amino acid sequence of isolated peptides were determined by gas phase sequence analyser and gasmass spectrometry. For gas-mass analysis, the peptides were digested by acid, acylated, permethylated and analysed using Chrom pack Sil-5 column. By these procedures, amino acid sequence of the amino terminal peptide, the glycopeptide and the fatty acid containing peptide were determined. These sensitive methods were also applicable for analysis of neuropeptides.
|
