The long-term objective is to gain insight into the mechanism of exocytosis, especially to identify polypeptides involved in the interaction of secretion granule- and plasma membranes and to trace the dynamic behavior of membrane components during the discharge process. The focus is on acinar cells comprising the parotid and pancreatic exocrine glands of rats. Highly purified preparations of granule- and plasma membranes from both tissues obtained by cellular fractionation will be used for compositional studies.
Specific aims : 1) Gamma glutamyl transferase (Gamma GT, EC2.3.2.2) has been identified as a common component of secretory membranes. Anti Gamma GT antibodies will be used, first to isolate Gamma GT antigens (including the active enzyme and a suspected proenzyme) from granule- and plasma membranes for comparative structural analysis and second, to examine the distribution of antigens kinetically in intact tissue upon secretory stimulation. 2) Studies of specific components marking secretory membranes will be extended to dipeptidyl peptidase IV (EC 3.4.14) and neutral metalloendopeptidase (EC3.4.24.11), investigated initially as enzyme activities, to test for possible interrelationships of their dynamic behavior and function to Gamma GT. 3) Heterologous antisera raised against parotid granule membranes will be used in more general analyses to identify a catalog of polypeptides common to granule and plasma membranes of parotid and pancreas and to secretory membranes of other cell types and to compare the apparent different fates of granule membrane glycoproteins and non-glycosylated polypeptides upon exocytosis. 4) Anti Gamma GT and antimembrane antibodies will also be used in attempts to isolate by immunoadsorption an apical plasmalemmal subfraction and granule membrane reinternalized post-discharge, respectively. Such fractions will facilitate a quantitative and comprehensive description of the dynamic behavior of granule membrane components during secretion. 5) In the long range individual polypeptides suspected to participate in a general secretory mechanism will be amplified through use of recombinant DNA technology to obtain sufficient quantities for detailed structural and biochemical studies. These approaches emphasize common elements of secretory function at a level of refinement not attempted previously. They may aid in forming a basis against which to evaluate secretory disorders that characterize Cystic Fibrosis.
| Brand, S H; Laurie, S M; Mixon, M B et al. (1991) Secretory carrier membrane proteins 31-35 define a common protein composition among secretory carrier membranes. J Biol Chem 266:18949-57 |
| Marino, C R; Castle, J D; Gorelick, F S (1990) Regulated phosphorylation of secretory granule membrane proteins of the rat parotid gland. Am J Physiol 259:G70-7 |
