Student Theses and Dissertations
Date of Award
1973
Document Type
Thesis
Degree Name
Doctor of Philosophy (PhD)
Thesis Advisor
George Palade
Keywords
secretion granules, rabbit parotid gland, in vitro labeling, exocytosis, membrane and content proteins, biosynthesis timing
Abstract
An in vitro incubation system which allows the labeling and isolation of secretion granules - and their subsequent resolution into membrane and content subfractions - has been developed by using dissected lobules of rabbit parotid glands. The system can be used to study a series of aspects related to the last phases of the secretory cycle (concentration, storage, discharge). The work reported in this thesis characterizes the system and presents data concerning the relative rates of synthesis of membrane and content proteins. The dissected lobules can be maintained in vitro in a tissue culture medium for 9 to 10 hr during which period their acinar cells retain their organization, synthesize proteins at a constant rate, and discharge their secretory proteins by exocytosis upon adrenergic stimulation. Autoradiographic experiments have shown that the acinar cells of the system transport newly synthesized secretory proteins along an intracellular pathway (rough ER → Golgi complex → (condensing vacuole) → secretion granules) similar to that already described for the pancreatic exocrine cells (Jamieson and Palade, 1967a,b) but at a slower rate, especially for the last steps of the operation. Under these conditions it was possible to recognize by morphological and kinetic criteria an immature secretion granule stage preceding the final storage stage (secretion granule). These experiments indicate the conditions needed to label the content of the secretion granules. A reasonably pure secretion granule fraction has been isolated from parotid tissue homogenates by a new procedure which prevents granule aggregation by avoiding pelleting and by using EDTA in the suspension media. Purification of the fraction is obtained by flotation of membranous debris and sedimentation of the granules in a discontinuous sucrose density gradient. The fraction has been shown to be morphologically and cytochemically homogeneous as assessed by electron microscopy and enzymatic assays. Since it accounts for ~15% of the amylase activity of the original homogenate, it is considered representative of the total granule population of the gland. Secretion granules were lysed by gradual exposure to hypotonic media, and the lysate subsequently was resolved into content and membrane subfractions by centrifugation. The latter subfraction appeared as a homogeneous population of flattened and infolded vesicles. When assessed for residual granule content by enzymatic assays, the membrane subfraction was found to contain low, but assayable, levels of amylase activity (~0.03% of the total granule complement). Contamination by the entire spectrum of secretory proteins, especially by those species having estimated molecular weights between 95,000 and 130,000 daltons was detected when gel electropherograms of membrane subfractions were compared to gel electropherograms of: 1) mixtures of membrane and labeled content subfractions, and 2) secretory proteins released from granules by the physiologic process of exocytosise The membrane and content subfractions obtained from secretion granules of lobules that had been labeled with a mixture of [14C]-amino acids during in vitro incubation, have been used to compare biosynthetic rates (assumed to be proportional to specific radioactivities) of polypeptides of granule membranes and content at two different levels: 1) specific radioactivities of unresolved mixtures, and 2) specific radioactivities of individual gel bands of the subfractions. The first level proved unreliable due to the magnitude and variability of content adsorption to the membranes. The results of the comparison at the more refined, second level have shown that all membrane polypeptides analyzed, with the exception of a majority component of estimated molecular weight 40,000 daltons, have specific radioactivities substantially (two- to tenfold) lower than those of content polypeptides destined for secretion. This has been taken as indicative that polypeptides of the granule membrane are not synthesized pari passu with those of the secretory content, but rather predate the packaged exportable products. The major membrane polypeptide having a specific radioactivity comparable to that of the content polypeptides represents one of the most intriguing findings of this thesis. It may represent a modifier required to convert a Golgi membrane into a secretion granule membrane, i.e., a membrane competent for exocytosis.
License and Reuse Information
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Recommended Citation
Castle, John David, "Studies on the Secretory Process of Rabbit Parotid" (1973). Student Theses and Dissertations. 537.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/537
Comments
A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy