Protein kinase activity associated with pancreatic zymogen granules

Abstract

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.

Organellar Proteomics

“…Isolation of ZGs and Purification of ZG Membranes-A previously validated Percoll gradient procedure was utilized (7). Briefly rat pancreata were homogenized in ice-cold buffer containing 0.25 M sucrose, 25 mM MES, 2 mM EGTA (pH 6.0).…”

Dominant Negative Rab3D Mutants Reduce GTP-bound Endogenous Rab3D in Pancreatic Acini

“…Preparation of Zymogen Granules-A previously described method for purifying zymogen granules from rat pancreas was adopted (30). In brief, isolated mouse pancreatic acini were homogenized in 3 ml ice-cold homogenization buffer containing 0.3 M sucrose, 25 mM Pipes, 200 M CaCl 2 , 200 M MgCl 2 , and 2 mM EGTA (pH 6.8) supplemented with 0.02% w/v soybean trypsin inhibitor.…”

Channels and Transporters in Zymogen Granule Membranes and their Role in Granule Function: Recent Progress and a Critical Assessment

“…These methods yield fractions that are enriched 4-8-times in α-amylase compared to homogenate, which is close to the theoretical limit of purity (77). Yet, it is practically impossible to avoid contaminations by lysosomes (96), plasma membranes (110) or membranes from other (disrupted) organelles (see electron micrographs in references (14,110,128)). These contaminants are, however, a major draw-back for proteomic analyses (as well as for electrophysiological studies; see the paragraph on K + Channels) because even minimal contaminations by membranes originating from other organelles or plasma membranes may be prominent in a proteomic analysis of ZGM because of proteins that are highly expressed in these contaminating membranes.…”

“…Several proteomic studies have been published that used highly purified ZG membranes (ZGM) to identify cloned transporters and channel proteins (9,18,19,96). In most of these studies, granules were disrupted by different techniques and membranes were subjected to carbonate and/or bromide extraction, which is a standardized and reliable procedure to obtain pure membranes without peripheral proteins (14). A critical step in this isolation procedure represents the initial "purification" of ZG by either differential or continuous Percoll gradient centrifugation.…”