Isolation and compositional analysis of secretion granules and their membrane subfraction from the rat parotid gland.
A secretory granule fraction has been isolated from rat parotid by discontinuous gradient centrifugation using hyperosmotic sucrose-Ficoll solutions of low ionic strength. The secretion granule fraction comprises 25% of the total tissue alpha-amylase activity and is judged to be of high purity, both morphologically and by its low level of contamination by enzyme activities associated with other organelles. Secretion granules were lysed by capitalizing on their lability in KCl-containing media, and the low density granule membranes were separated from residual organelle and soluble contaminants by flotation in a sucrose gradient. Residual, poorly extractable secretory contaminants of the granule membrane subfraction were selectively removed by a saponin- (10 micrograms/ml) Na2SO4 (0.3 M) wash, apparently with negligible disruption of granule membrane structure. Based on detailed consideration of the extent of contamination by residual mitochondria and incompletely removed secretory polypeptides, it is possible to estimate that approximately 95% of the protein associated with the purified secretion granule membrane is bona fide granule membrane protein. Further analyses indicate that gamma-glutamyltransferase constitutes a marker enzymatic activity shared by granule membranes and the apical domain of the plasma membrane. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoretograms of radio-iodinated granule membrane polypeptides are characterized by 20-25 radioactive bands of which 5-6 are suggested to be glycoproteins by virtue of their binding of concanavalin A. The limited polypeptide composition of the secretion granule membrane (in comparison to membranes of other cellular compartments) and the high phospholipid-protein ratio (4.4 mg/mg) may reflect the functional specialization of this storage container for secretory proteins.
D000818:Animals; D002458:Cell Fractionation; D003594:Cytoplasmic Granules / Q000032:analysis* / Q000201:enzymology / Q000648:ultrastructure; D005089:Exocytosis; D006023:Glycoproteins / Q000302:isolation & purification; D007425:Intracellular Membranes / Q000032:analysis*; D008565:Membrane Proteins / Q000302:isolation & purification; D010306:Parotid Gland / Q000032:analysis* / Q000378:metabolism; D010743:Phospholipids / Q000032:analysis; D051381:Rats
D006023:Glycoproteins; D008565:Membrane Proteins; D010743:Phospholipids