| Budget Amount *help |
¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 1996: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1995: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Research Abstract |
Screening of new GPI-anchored proteins were carried out in mouse B cell myelome, NS-1 and bovine erythrocytes. At least eight GPI-anchored proteins (170/150kD,96kD,80/76kD,66kD,63kD,59kD,44kD and 27kD) were detected on the outer surface of plasma memberane of mouse myeloma NS-1 cells. From bovine erythrocyte menbrane, we also detected several GPI-anchored proteins, 55kD,42kD,40kD and 30kD,other than acetylcholinesterase. These preteins were confirmrd by detecting myo-inositol moeity in their structures by gas chromatography (GC) -mass spectrometry.
We applied the improved sensitivity and soft Ionization characteristic of electrospray ionization mass/mass spectrometry (ESI-MS/MS) and matrix-assisted laser dissorption/ionization (MALDI) -time of fright (TOF) mass spectrometry (MS)to the analysis of GPI-anchored structure. Heterogeneous peaks of GPI-anchored peptides and their caracteristic fragment ions were detected in positive mode analysis. ESI-MS/MS and MALDI-TOF-MS is very effective and sensitive methods in analyzing small amount of GPI-anchored structure less than 10pmol.
chiro-Inositol, an inositol isomer other than myo-inositol was identified in hydrolytic products from several GPI-anchored proteins. The chiro-inositol contents in several different GPI-anchored proteins varied with hydrolytic conditions of these GPI anchor. Isomerization of 20-60% of myo-inositol occurred on the hydrolysis in 6N HCl solution. In the hydrolytic condition under HCl gas stream in place of solution, however, isomerization was very low (less than 0.1%) . In the hydrolysis of phosphatidylinositol or myo-inositol-1-phosphate, however, significant amount of chiro-inositol was not detected in 6N HCl solution. These facts indicated that isomerization occurred during the hydrolysis of GPI anchor, where myo-inositol is substituted by glucosamine at 6-OH and is substituted by phosphate at 1-OH.
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