Isolation and compositional analysis of secretion granules and their membrane subfraction from the rat parotid gland

Richard S. Cameron, J. David Castle

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

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 α-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 μg/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 ∼95% of the protein associated with the purified secretion granule membrane is bona fide granule membrane protein. Further analyses indicate that γ-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.

Original languageEnglish (US)
Pages (from-to)127-144
Number of pages18
JournalThe Journal of Membrane Biology
Volume79
Issue number2
DOIs
StatePublished - Jun 1 1984
Externally publishedYes

Fingerprint

Parotid Gland
Membranes
Secretory Vesicles
Organelles
Peptides
Sucrose
Ficoll
Proteins
Saponins
Amylases
Concanavalin A
Radio
Centrifugation
Sodium Dodecyl Sulfate
Osmolar Concentration
Phospholipids
Glycoproteins
Mitochondria
Membrane Proteins
Cell Membrane

Keywords

  • exocytosis
  • parotid
  • secretion
  • secretory granule membranes

ASJC Scopus subject areas

  • Biophysics
  • Physiology
  • Cell Biology

Cite this

Isolation and compositional analysis of secretion granules and their membrane subfraction from the rat parotid gland. / Cameron, Richard S.; Castle, J. David.

In: The Journal of Membrane Biology, Vol. 79, No. 2, 01.06.1984, p. 127-144.

Research output: Contribution to journalArticle

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