Large-scale purification of calf pancreatic zymogen granule membranes.
A protocol for isolating milligram quantities of highly purified zymogen granule membranes from calf pancreas was developed. The method provides a fivefold enriched zymogen granule fraction that is virtually free from major isodense contaminants, such as mitochondria and erythrocytes. Isolated granules are osmotically stable in isosmotic KCl buffers with half-lives between 90 and 120 min. They display specific ion permeabilities that can be demonstrated using ionophore probes to override intrinsic control mechanisms. A Cl- conductance, a Cl-/anion exchanger, and a K+ conductance are found in the zymogen granule membrane, as previously reported for rat pancreatic, rat parotid zymogen granules, and rabbit pepsinogen granules. Lysis of calf pancreatic secretory granules in hypotonic buffers and subsequent isolation of pure zymogen granule membranes yield about 5-10 mg membrane protein from approximately 1000 ml pancreas homogenate. The purified zymogen granule membranes are a putative candidate for the rapid identification and purification of epithelial Cl- channels and regulatory proteins, since they contain fewer proteins than plasma membranes.
Thévenod
F
F
Department of Physiology and Biophysics, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106.
Haase
W
W
Hopfer
U
U
D000818:Animals; D002417:Cattle; D002458:Cell Fractionation / Q000379:methods*; D002712:Chlorides / Q000378:metabolism*; D003594:Cytoplasmic Granules / Q000378:metabolism / Q000648:ultrastructure*; D004792:Enzyme Precursors; D007425:Intracellular Membranes / Q000378:metabolism* / Q000648:ultrastructure; D007473:Ion Channels / Q000378:metabolism; D008854:Microscopy, Electron; D009994:Osmolar Concentration; D010179:Pancreas / Q000378:metabolism / Q000648:ultrastructure*; D011188:Potassium / Q000378:metabolism
D002712:Chlorides; D004792:Enzyme Precursors; D007473:Ion Channels; D011188:Potassium