Differential extraction of eosinophil granule proteins.
Eosinophil granules contain several toxic cationic proteins that contribute to the pathophysiology of allergic diseases. These include eosinophil peroxidase, two ribonucleases, and two forms of the major basic protein (MBP). Extraction of eosinophil granules by exposure to acid solution and fractionation on Sephadex G-50 characteristically yields a distinctive profile of three discrete peaks, and these proteins are usually recovered in good quantities, except for the eosinophil major basic protein homolog (MBP2). We investigated the effect of multiple granule extractions by dilute HCl on the recovery of granule proteins. Isolated granules were repetitively extracted, up to 31 times, in 0.01 M HCl, and the extracts fractionated on Sephadex G-50. Whereas initial extracts yielded the characteristic three-peak fractionation pattern, later extracts yielded four discrete peaks. Characterization of the novel fourth peak showed that it contained MBP2. These results indicate that repetitive extraction of eosinophil granules yields an increased amount of all granule proteins, and that MBP2 can now be recovered in good quantities and in a relatively pure form.
Ohnuki
Lyo E
LE
Department of Dermatology, University of Utah, Salt Lake City, 84132, USA.
Wagner
Lori A
LA
Georgelas
Ann
A
Loegering
David A
DA
Checkel
James L
JL
Plager
Douglas A
DA
Gleich
Gerald J
GJ
D001798:Blood Proteins / Q000302:isolation & purification; D002458:Cell Fractionation; D002469:Cell Separation; D002850:Chromatography, Gel; D003594:Cytoplasmic Granules / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D047150:Eosinophil Cationic Protein / Q000302:isolation & purification; D047091:Eosinophil Granule Proteins / Q000302:isolation & purification*; D047093:Eosinophil Major Basic Protein; D047209:Eosinophil Peroxidase / Q000302:isolation & purification; D047208:Eosinophil-Derived Neurotoxin / Q000302:isolation & purification; D004802:Eosinophilia / Q000097:blood; D004804:Eosinophils / Q000737:chemistry* / Q000166:cytology; D005415:Flavins / Q000737:chemistry; D006801:Humans; D007118:Immunoassay; D008163:Luminescent Measurements; D011509:Proteoglycans / Q000302:isolation & purification; D013050:Spectrometry, Fluorescence
D001798:Blood Proteins; D047091:Eosinophil Granule Proteins; D005415:Flavins; D011509:Proteoglycans; C074129:PRG2 protein, human; D047209:Eosinophil Peroxidase; D047208:Eosinophil-Derived Neurotoxin; D047150:Eosinophil Cationic Protein; D047093:Eosinophil Major Basic Protein; C479220:RNASE3 protein, human