Secretion granules of the rabbit parotid gland. Isolation, subfractionation, and characterization of the membrane and content subfractions.

Abstract

A fraction of secretion granules has been isolated from rabbit parotid by a procedure which was found to be especially effective in reducing contamination resulting from aggregation and/or cosedimentation of granules with other cell particulates. The fraction, representing 15% (on the average) of the total tissue amylase activity, was homogeneous as judged by electron microscopy and contaminated to exceedingly low levels by other cellular organelles as judged by marker enzymatic and chemical assays.Lysis of the granules was achieved by their gradual exposure to hypotonic NaHCOs, containing 0.5 mM EDTA. The content and the membranes separated by centrifugation of the granule lysate were characterized primarily by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis which indicated that the content was composed of a limited number of molecular weight classes of polypeptides of which three bands (having approximate mol wt 58,000, 33,000, and 12,000) could be considered major components. The gel profile of the membrane subfraction was characterized by 20-30 Coomassie brilliant blue-staining bands of which a single species of tool wt ~40,000 was the conspicuous major polypeptide.Two types of experiments employing gel electrophoretic analysis were carried out for identifying and assessing the extent of residual secretory protein adsorbed to purified granule membranes: (a) examination of staining and radioactivity profiles after mixing of radioactive secretion granule extract with nonradioactively labeled granule membranes and (b) comparison of gel profiles of secretion granule extract and granule membranes with those of unlysed secretion granules and secretory protein discharged from lobules in vitro or collected by cannulation of parotid ducts, the last two samples being considered physiologic secretory standards. The results indicated that the membranes were contaminated to a substantial degree by residual, poorly extractable secretory protein even though assays of membrane fractions for a typical secretory enzyme activity (amylase) indicated quite thorough separation of membranes and content. Hence, detailed examination of membrane subfractions for residual content species by gel electrophoresis points to the general utility and sensitivity of this technique as a means for accurately detecting a defined set of polypeptides occurring as contaminants in cellular fractions or organelle subfractions.The secretion granule in exocrine cells represents the intracellular storage compartment for protein destined for export. Within the exocrine cell the population of accumulated granules, which can occupy as much as half of the cellular volume, is located in the apical cytoplasm between the Golgi region, where granules originate (2,13,28,29,54,55), and the apical cell membrane, where the period of storage is terminated by release of the granule contents into the extracellular space by the process of exocytosis (57).In our previous studies (13) we undertook a characterization by autoradiography of the secre...

Isolated secretion granules from parotid glands of chronically stimulated rats possess an alkaline internal pH and inward-directed H+ pump activity.

“…Granule samples were pelleted by centrifugation (3-5 rain, ,,o3,000 g) after aldehyde fixation and all specimens were then postfixed in OsO4, stained with umnyl acetate, dehydrated, and embedded (in either Epon 812 or Spurr's resin) as described previously (16). Methylene blue-stained 0.5-ttm sections were examined using a Zeiss photomicroscope whereas ,-otO-nm sections (stained with uranyl acetate and lead citrate) were viewed on a Philips 300 electron microscope.…”

Association of a protease (plasminogen activator) with a specific membrane fraction isolated from transformed cells.

“…Cytochrome oxidase was used to monitor subcellular fractions for the presence of mitochondria and was assayed according to the procedure of Cooperstein and Lazarow (17) as modified by Castle et al (11). NADH diaphorase (NADH oxidase E.C.…”

Antipain and leupeptin restrict uterine DNA synthesis and function in mice.

“…Particulate fractions were found on top of the 20% sucrose (A band), the 40% sucrose (B band), and 60% sucrose (C band) phases. The solid fractions were separated and each was tested for cytochrome oxidase (8,9), f3-N-acetylglucosiminidase (10, 11), 5'-nucleotidase (12), and protease (6). Relative concentrations of the enzyme activities in the A, B, and C bands were calculated.…”

“…The females were then sacrificed and the uteri examined for~fetuses anid implantation sites. (12); fl-N-acetylglucosaminidase, ymol of p-nitrophenol released per hr/100 ,g of protein (10,11); cytochrome oxidase, presence (+) or absence (-) of activity determined by decrease in A of reduced cytochrome at 550 nm (8,9). * Particulate material above 20% (band A), 40% (band B), and 60%…”