The long term objective of this research is to further understand the molecular events associated with exocytosis. Rabbit and rat parotid glands will be used.
Specific aims i nclude an examination of phosphorylation of both plasma membrane and secretory granule membranes by cyclic nucleotide-dependent protein kinases and calcium-dependent kinases sensitive to either calmodulin or phosphatidylserine. The calcium binding and uptake characteristics of the plasma membranes and secretory granule membranes will be examined. The interaction of plasma membranes with secretory granules will then be examined. Plasma membranes will be converted into inside-out or rightside-out vesicles. These membranes will then be tested as to their sidedness by assays which detect activity unique to either the outside or inside aspect of the plasma membrane: ouabain binding and carbohydrate for the outside and actin inhibition of DNAse and cyclic AMP binding for the inside. Phosphorylation of secretory granule membranes and plasma membranes will then be investigated individually and together. Calcium binding to and uptake into the two structures will also be investigated as to rate and maximal binding to secretory granule membranes and plasma membranes. The influence of phosphorylation on calcium binding will be of major interest. The interaction of secretory granules with plasma membranes will be examined as to protein-protein interactions between membranes. Plasma membranes will be labeled with 125I in order to determine their location on sucrose gradients. Granules and membranes will be mixed under various conditions and cosedimentation will be followed with 125I-labelled membranes. Turbidity changes involving mixtures of granules with membranes will also be monitored with time. Electron microscopy will be used to monitor the interaction of granules with plasma membranes and will help confirm the interaction of membrane vesicles and secretory granules. Various factors will be tested as to their effect on granule-membrane interaction including calcium, synexin, and phosphorylation. The benefit from this research will be a better understanding of the elements involved in membrane aggregation and fusion in exocytosis. To understand and to alter gland function in disease states such as cystic fibrosis requires a basic knowledge of the cell biology of the glands.
| Dowd, F J; Pasieniuk, J A; Hand, A R et al. (1989) Characteristics of anion-stimulated Mg-ATPase from rat parotid gland secretory granules. Arch Oral Biol 34:167-76 |
