Protein kinase activity associated with pancreatic zymogen granules.
Purified zymogen granules were prepared from rat pancreas by using an iso-osmotic Percoll gradient. In the presence of [gamma-32P]ATP, phosphorylation of several granule proteins was induced by Ca2+, most notably a Mr-13 000 protein, whereas addition of cyclic AMP was without effect. When phosphatidylserine was also added, Ca2+ increased the phosphorylation of additional proteins, with the largest effect on a protein of Mr 62 000. Purified granules were also able to phosphorylate exogenous substrates. Ca2+-induced phosphorylation of lysine-rich histone was enhanced over 3-fold in the presence of phosphatidylserine, and cyclic AMP-activated protein kinase activity was revealed with mixed histone as substrate. The concentrations of free Ca2+ and cyclic AMP required for half-maximal phosphorylation of both endogenous and exogenous proteins were 1-3 microM and 57 nM respectively. Treatment of granules with 0.25 M-KCl resulted in the release of phosphatidylserine-dependent kinase activity into a high-speed granule supernatant. In contrast, granule-protein substrates of Ca2+-activated kinase activity were resistant to KCl extraction, and in fact were present in purified granule membranes. Kinase activity activated by cyclic AMP was not extracted by KCl treatment. It is concluded that phosphorylation of integral membrane proteins in the zymogen granule can be induced by one or more Ca2+-activated protein kinases. Such a reaction is a potential mechanism by which exocytosis may be regulated in the exocrine pancreas by Ca2+-mediated secretagogues.
D000818:Animals; D002498:Centrifugation; D003594:Cytoplasmic Granules / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000378:metabolism*; D006657:Histones / Q000378:metabolism; D066298:In Vitro Techniques; D008297:Male; D008854:Microscopy, Electron; D010179:Pancreas / Q000201:enzymology*; D010766:Phosphorylation; D011189:Potassium Chloride; D011494:Protein Kinases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains
D004792:Enzyme Precursors; D006657:Histones; D011506:Proteins; D011189:Potassium Chloride; D011494:Protein Kinases