Composition of Cellular Membranes in the Pancreas of the Guinea Pig

112

In the acinar cells of the rat parotid gland the two membranes participating in exocytosis, i.e., the luminal plasmalemma and the secretory granule membrane, are clearly distinguishable in freeze-fracture because of their different densities in particles. In order to obtain point-specific information about the fusion-fission of these two membranes that occurs during the secretory cycle, glands were studied at various times (5 min to 6 h) after stimulation with isoproterenol. We observed that, in the course of the release of secretion products and shortly afterwards, the enlarged luminal plasmalemma exhibits a mosaic organization consisting of an alternation of membrane patches of high (original plasmalemma) and low (fused granule membrane) particle density. The transition between these two patterns is usually sharp. Later, concomitant with the reformation of acinar canaliculi, the low particle density membrane is found at the cell surface but only bounding vacuolar infoldings, and then it finally disappears.These results suggest that (a) fusion of these membranes does not result in a random intermixing of the molecular components of the participating membranes, which retain their structural identity; and (b) the enlarged luminal plasmalemma reverts to its original size by a progressive, specific removal of the regions of low particle density from the cell surface.In secretory cells the exportable proteins are known to be vectorially transported from the site of synthesis to the extracellular space through a pathway which includes a number of distinct membrane-bounded organelles (8,9,23,24): in sequence, the rough endoplasmic reticulum, the Golgi complex, and the secretion granules. The content of the latter is then discharged by exocytosis, i.e., by fusion of the granule limiting membrane with the plasmalemma followed by an opening at the point of fusion (36). Since the transport of the secretion products between adjacent cell compartments occurs in bulk, by means of membrane-bounded vesicles, a concomitant transfer of membranes must be postulated.The fate of the transferred membrane is still debated. Biochemical studies carried out on various secretory systems have clearly shown that the turnover rate of the molecular components of all the membranes involved is much slower than that of the secretory products (28,29,46,47); thus, it is most likely that these molecular components are reutilized in several secretory cycles (28,29). However, the mechanisms of this reutilization are still unclear, since it is not known whether membrane patches, after fusing with a membrane of a

mentioning

confidence: 75%

Dynamic changes of the luminal plasmalemma in stimulated parotid acinar cells. A freeze-fracture study.

Camilli¹,

Peluchetti²,

Meldolesi³

1976

112

“…In recent years, the exocrine pancreas has been a subject of interest to cell biologists particularly with respect to the mechanisms of protein synthesis, intracellular transport, and discharge of secretory proteins (1)(2)(3)(4)(5)(6)(7)(8)(9)(10) . Although a considerable amount of biochemical data is now available, no attempt has been made to introduce stereological methods as an aid in the interpretation of studies concerned with the relationships of cellular ultrastructure to biochemical function .…”

Section: Introductionmentioning

confidence: 99%

Stereological Analysis of the Guinea Pig Pancreas

Bolender

1974

233

“…This apparently common feature remains to be tested by determining whether these major components in granule membranes prepared from the parotid or other sources are or are not bona fide membrane proteins. Such a situation should be contrasted with that found for other intracellular membranes for which the number of resolvable bands is considerably higher and in which majority components have seldom been detected (19,36,48).…”

Section: Castle Et Al Secretion Granules O F the Rabbit Parotid Glanmentioning

confidence: 64%

“…zymogen granules of the exocrine pancreas [44,48] and chromaffin granules of the adrenal medulla [70]) seems to indicate that membranes specialized for storage of exportable products have a relatively simple composition, that is, are resolved into a limited number of Coomassie brilliant blue-stainable bands and contain a few polypeptide species which constitute a large fraction of the total protein as estimated from stain distribution in the entire profile. This apparently common feature remains to be tested by determining whether these major components in granule membranes prepared from the parotid or other sources are or are not bona fide membrane proteins.…”

Section: Castle Et Al Secretion Granules O F the Rabbit Parotid Glanmentioning

confidence: 99%

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

Castle¹,

Jamieson²,

Palade³

1975

104

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...