Purification and identification of secernin, a novel cytosolic protein that regulates exocytosis in mast cells.
After permeabilization with the pore-forming toxin streptolysin-O mast cells can be triggered to secrete by addition of both calcium and a GTP analogue. If stimulation is delayed after permeabilization, there is a progressive decrease in the extent of secretion upon stimulation, eventually leading to a complete loss of the secretory response. This loss of secretory response can be retarded by the addition of cytosol from other secretory tissues, demonstrating that the response is dependent on a number of cytosolic proteins. We have used this as the basis of a bioassay to purify Secernin 1, a novel 50-kDa cytosolic protein that appears to be involved in the regulation of exocytosis from peritoneal mast cells. Secernin 1 increases both the extent of secretion and increases the sensitivity of mast cells to stimulation with calcium.
Way
Gemma
G
Department of Biological and Biomedical Sciences, University of Durham, United Kingdom.
Morrice
Nicholas
N
Smythe
Carl
C
O'Sullivan
Antony J
AJ
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002417:Cattle; D002845:Chromatography; D002850:Chromatography, Gel / Q000379:methods*; D003600:Cytosol / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D017886:Durapatite / Q000494:pharmacology; D005089:Exocytosis; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D008297:Male; D013058:Mass Spectrometry; D008407:Mast Cells / Q000378:metabolism*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D010957:Plasmids / Q000378:metabolism; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012685:Sepharose / Q000031:analogs & derivatives* / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors
D009419:Nerve Tissue Proteins; D011506:Proteins; D011993:Recombinant Fusion Proteins; C471696:SCRN1 protein, human; C091883:phenyl-superose; C471695:secernin 1 protein, mouse; D012685:Sepharose; D017886:Durapatite; C049225:Superose 12; D005982:Glutathione Transferase; D002118:Calcium