Purification and characterization of the apical plasma membrane of the rat pancreatic acinar cell.
A method is described for the rapid purification of the apical plasma membrane from the rat pancreatic acinar cell. It makes use of wheat germ agglutinin affinity chromatography to selectively bind vesicles with N-acetyl glucosamine present at their surface. Particular conditions (150 mM NaCl) had then to be used to keep membrane vesicles in the coveted orientation, i.e. as right-side-out vesicles. Due to its specific apical location in many epithelial cells, gamma-glutamyltranspeptidase was chosen to monitor the purification procedure. The final fraction was enriched in gamma-glutamyltranspeptidase by a factor of 75 relative to the homogenate. Na,K-ATPase, a strict basolateral membrane marker, was not detectable in the fraction. No membranes originating from other compartments, more particularly expected from zymogen granules, or from other cell types, did contaminate the preparation. As expected for an epithelial cell apical plasmalemma, lipid composition showed a very high ratio of glycolipids (37.5%). The absence of membrane-bound GP-2, and the exceptionally high specific activity of gamma-glutamyltranspeptidase suggest that the apical membrane would not be made up by the exocytosis of secretory granule, but instead by the fusion of specialized secretory vesicles very likely originating from the constitutive secretory pathway. In conclusion, this report describes a method of obtaining a fraction highly enriched in the secretory apex of the pancreatic exocrine cell that would be directly involved in exocytosis with zymogen granules and also in local anion transport.
Paul
E
E
Département de Biologie, Faculté des Sciences, Université de Sherbrooke, Québec, Canada.
Hurtubise
Y
Y
LeBel
D
D
D000818:Animals; D002458:Cell Fractionation / Q000379:methods*; D002462:Cell Membrane / Q000737:chemistry; D002498:Centrifugation; D002846:Chromatography, Affinity; D008297:Male; D010179:Pancreas / Q000737:chemistry* / Q000648:ultrastructure; D051381:Rats; D011919:Rats, Inbred Strains