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1
LAINÉ, Jean, and Denis LeBEL. "Efficient binding of regulated secretory protein aggregates to membrane phospholipids at acidic pH." Biochemical Journal 338, no. 2 (1999): 289–94. http://dx.doi.org/10.1042/bj3380289.
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Some regulated secretory proteins are thought to be targeted to secretory granules through an acidic-dependent aggregation in the trans-Golgi network. In this report we use pancreatic zymogens, a paradigm of regulated proteins, to test this hypothesis, because they qualitatively aggregate upon acidification in vitro. Pig zymogens were found to start to aggregate significantly at pH ∼ 6.0, a pH slightly lower than that at which rat zymogens aggregate, but still compatible with the pH of the cell-sorting compartments. When pig zymogen granule membranes were mixed with the zymogens in the aggregation assay, membranes that normally floated on 1 M sucrose were observed to be pelleted by the aggregating zymogens. Rat membranes were pelleted by pig zymogens and vice versa. Igs, typical constitutively secreted proteins, which needed chemical cross-linking to serve as an aggregated protein control, pelleted membranes almost independently of pH. Corresponding cross-linked zymogen-binding ability and pH dependence was unaffected by the chemical modification. Membranes treated with sodium carbonate, pH 11, or with protease K, were still pelleted by zymogens, suggesting that the aggregated zymogens bound to membrane lipids. This hypothesis was confirmed by the efficient pelleting of unilamellar vesicles composed of granule membrane lipids. Vesicles composed of single classes of phospholipids were also pelleted, but with various efficacies. We conclude that pancreatic zymogen aggregates, formed under the acidic conditions of the secretory pathway sorting compartments, have the capacity to bind firmly to membranes through their phospholipid constituents.
2
Antonin, Wolfram, Martin Wagner, Dietmar Riedel, Nils Brose, and Reinhard Jahn. "Loss of the Zymogen Granule Protein Syncollin Affects Pancreatic Protein Synthesis and Transport but Not Secretion." Molecular and Cellular Biology 22, no. 5 (2002): 1545–54. http://dx.doi.org/10.1128/mcb.22.5.1545-1554.2002.
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ABSTRACT Syncollin is a small protein that is abundantly expressed in pancreatic acinar cells and that is tightly associated with the lumenal side of the zymogen granule membrane. To shed light on the hitherto unknown function of syncollin, we have generated syncollin-deficient mice. The mice are viable and show a normal pancreatic morphology as well as normal release kinetics in response to secretagogue stimulation. Although syncollin is highly enriched in zymogen granules, no change was found in the overall protein content and in the levels of chymotrypsin, trypsin, and amylase. However, syncollin-deficient mice reacted to caerulein hyperstimulation with a more severe pancreatitis. Furthermore, the rates of both protein synthesis and intracellular transport of secretory proteins were reduced. We conclude that syncollin plays a role in maturation and/or concentration of zymogens in zymogen granules.
3
Arvan, P., and J. D. Castle. "Phasic release of newly synthesized secretory proteins in the unstimulated rat exocrine pancreas." Journal of Cell Biology 104, no. 2 (1987): 243–52. http://dx.doi.org/10.1083/jcb.104.2.243.
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Pancreatic lobules from fasted rats secrete pulse-labeled proteins in two phases comprising 15 and 85% of basal output, respectively. The first (0-6.5 h) is initially (less than or equal to 0.5 h) unstimulated by secretagogues, probably represents vesicular traffic of Golgi and post-Golgi origin (including condensing vaculoles/immature granules), and notably contains two groups of polypeptides with distinct release rates: zymogens (t1/2 approximately 2.4 h) and minor nonzymogens plus one unique zymogen (t1/2 approximately 1 h). The second phase (peak at 9-10 h) is stimulable, probably represents basal granule exocytosis (t1/2 approximately 5 h), and contains zymogens exclusively. Newly synthesized proteins released in both phases appear asynchronously, reiterating their asynchronous transport through intracellular compartments. Zymogens in both phases are secreted apically. The sorting of first from second phase zymogen release does not appear to be carrier-mediated, although the sorting of zymogens from other secretory proteins may use this process. Finally, data are presented that suggest that both secretory phases are subject to physiologic regulation.
4
Garcia-Montero, A. C., I. De Dios, A. I. Rodriguez, A. Orfao, and M. A. Manso. "Adrenalectomy induces a decrease in the light scatter properties and amylase content of isolated zymogen granules from rat pancreas as analyzed by flow cytometry." Journal of Endocrinology 147, no. 3 (1995): 431–40. http://dx.doi.org/10.1677/joe.0.1470431.
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Abstract The effect of glucocorticoid deprivation induced in male rats by adrenalectomy on the pancreatic zymogen granules was studied. Zymogen granules were purified from control, sham-operated and adrenalectomized animals studied 1, 3 and 7 days after surgery. The zymogen granules were characterized by flow cytometry, and in each granule the size (based on the forward or low angle light scatter (FSC) parameter), membrane complexity (based on side or 90° light scatter (SSC) parameter) and amylase content were evaluated. Amylase content/DNA ratio in pancreatic homogenates was also analyzed. The zymogen granules of the control rats were found to be distributed in two populations: a major one – R1 (95·45 ± 1·21%) – containing zymogen granules with a smaller mean size and complexity, and a minor population - R2 (4·45 ± 0·24%) – the granules of which had a mean size which was larger and more complex. At day +1 after adrenalectomy the zymogen granules were significantly (P<0·05) smaller than those of control animals. The R2 zymogen granules were similar to those from R1 as regards their size, but were more complex, suggesting that the immediate effect of glucocorticoid deprivation is to induce a depletion of the larger granules presumably belonging to the R2 population. The amount of amylase per granule did not vary at day +1 after adrenalectomy, although the amylase content/size ratio per granule was significantly (P<0·001) increased. This mechanism could be explained in terms of the existence of a bypass defined in the adrenalectomized animals between the granular content and cytosolic enzymes. Prolongation of the adrenalectomy period to 3 and 7 days resulted in a progressive increase in zymogen granule size and complexity, both parameters showing similar characteristics to those of the controls at day +7 after adrenalectomy. However, the percentage of zymogen granules within the R1 and R2 populations was clearly different from that of controls since the R2 population was much more numerous (11·25 ± 0·75% and 15·25 ± 1·15% (adrenalectomized rats at days +3 and +7 respectively) versus 4·45 ± 0·24% (controls)). An increase in the content of amylase per DNA was observed in adrenalectomized rats at day +1 although this transient effect cannot be related to glucocorticoid deprivation because it was also observed in sham-operated rats (day +1). However, a significant reduction, nearly 64%, in the amylase content/DNA ratio is produced by the absence of glucocorticoids 7 days after adrenalectomy and this is associated with a reduction in the content of amylase in each individual zymogen granule which reaches a minimum 3 days after adrenalectomy. It should be noted that, despite this, the enzyme concentration in each granule remains constant as there is a parallel decrease in the zymogen granule amylase content and size. Journal of Endocrinology (1995) 147, 431–440
5
Paquette, Jean, François A. Leblond, Marlyne Beattie, and Denis LeBel. "Reducing conditions induce a total degradation of the major zymogen granule membrane protein in both its membranous and its soluble form. Immunochemical quantitation of the two forms." Biochemistry and Cell Biology 64, no. 5 (1986): 456–62. http://dx.doi.org/10.1139/o86-064.
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The major protein of the pig pancreatic zymogen granule membrane is an integral glycoprotein of 92 × 103 daltons (Da) which amounts to 25% of the total proteins of this membrane. When zymogen granule membranes were prepared in presence of 5 mM dithiothreitol (DTT), this glycoprotein specifically vanished from the membrane preparation. During membrane purification two other fractions were produced out of the purified granules: a soluble fraction of zymogens referred to as granule content and a dense pellet. The possibility that DTT could release the 92-kDa protein from the membrane to these other fractions has been rejected. Altogether, addition of DTT during the lysis of the granules induced a total degradation of the 92-kDa protein. This hydrolysis could be inhibited by phenylmethylsulfonyl fluoride but not by N-α-p-tosyl-L-lysine chloromethyl ketone or L-1-tosylamide-2-phenylethylchloromethyl ketone. In the course of these experiments, using gel filtration of the granule content, it was found that the 92-kDa protein was also present in the granule content in the form of an aggregate of 300 kDa. A protease was present in this aggregate and could hydrolyse the 92-kDa protein upon addition of DTT. From immunoblotting studies and rocket immunoelectrophoresis, it was found that the soluble 92-kDa protein was antigenically similar to the membrane protein and that 44% of the immunoreactive glycoprotein of the granule was soluble in the content. A cross-reacting fragment of 65 kDa has been observed in all the fractions, yet at different levels. It is concluded that as much of the 92-kDa protein is soluble in the content as it is anchored in the membrane. The protease responsible for its degradation upon addition of DTT seems to be closely associated with the protein and could be involved in its posttranslational solubilization leading to its secretion.
6
Thrower, Edwin C., Alexander P. E. Diaz de Villalvilla, Thomas R. Kolodecik, and Fred S. Gorelick. "Zymogen activation in a reconstituted pancreatic acinar cell system." American Journal of Physiology-Gastrointestinal and Liver Physiology 290, no. 5 (2006): G894—G902. http://dx.doi.org/10.1152/ajpgi.00373.2005.
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Pathological activation of digestive zymogens within the pancreatic acinar cell initiates acute pancreatitis. Cytosolic events regulate this activation within intracellular compartments of unclear identity. In an in vivo model of acute pancreatitis, zymogen activation was detected in both zymogen granule-enriched and microsomal cellular fractions. To examine the mechanism of this activation in vitro, a reconstituted system was developed using pancreatic cytosol, a zymogen granule-enriched fraction, and a microsomal fraction. Addition of cytosol to either particulate fraction resulted in a prominent increase in both trypsin and chymotrypsin activities. The percentage of the pool of trypsinogen and chymotrypsinogen activated was about twofold and sixfold greater, respectively, in the microsomal than in the zymogen granule-enriched fraction. Activation of chymotrypsinogen but not trypsinogen was significantly enhanced by ATP (5 mM) but not by the inactive ATP analog AMP-PNP. The processing of procarboxypeptidase B to its mature form also demonstrated a requirement for ATP and cytosol. E64d, an inhibitor of cathepsin B, a thiol protease that can activate trypsin, completely inhibited trypsin activity but did not affect chymotrypsin activity or carboxypeptidase B generation. These studies demonstrate that both zymogen granule-enriched and microsomal fractions from the pancreas can support cytosol-dependent zymogen activation. A component of the activation of some zymogens, such as chymotrypsinogen and procarboxypeptidase, may depend on ATP but not on trypsin or cathepsin B.
7
Rothman, S. S., and C. Liebow. "Permeability of zymogen granule membrane to protein." American Journal of Physiology-Gastrointestinal and Liver Physiology 248, no. 4 (1985): G385—G392. http://dx.doi.org/10.1152/ajpgi.1985.248.4.g385.
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The evidence that the membrane of the pancreatic zymogen granule is permeable to its contained secretory proteins is outlined. Included is a discussion of the nature and characteristics of the equilibrium-dependent release of protein from isolated granules, the evidence for the permeability of the granule membrane to digestive enzyme protein in situ, and the seeming paradox that isolated granules release protein in medium similar to that thought to exist in the cell. The permeability hypothesis is reconsidered here in light of recent claims of stable nonpermeable granules.
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Lainé, J., G. Pelletier, G. Grondin, M. Peng, and D. LeBel. "Development of GP-2 and five zymogens in the fetal and young pig: biochemical and immunocytochemical evidence of an atypical zymogen granule composition in the fetus." Journal of Histochemistry & Cytochemistry 44, no. 5 (1996): 481–99. http://dx.doi.org/10.1177/44.5.8627005.
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To uncover the mechanisms involved in the biogenesis of secretory granules, we studied development of the exocrine pancreas in the pig from the fetus up to the mature animal by following the enzyme activities and expression (Northern blot) of five zymogens and GP-2, the major protein of the granule membrane. Fetal pancreas mainly contained chymotrypsinogen and barely detectable amounts of amylase, trypsin, lipase, and elastase. GP-2 was not notably expressed before the Day 21 of life. Ultrastructural examination of the fetal tissue embedded in Epon with osmium postfixation or in Lowicryl at -20 degrees C without postfixation showed dense granules with an irregular shape but also showed that most granules had uncondensed contents, with the aspect of immature granules, or had a dense core surrounded by light material. With immunogold cytochemistry, the concentration of chymotrypsinogen was directly associated with the acquisition of electron density by the granule matrix. These observations suggest that fetal granules have a slower rhythm of zymogen condensation and an irregular shape that could be due to the particular composition of the matrix and the absence of GP-2. We conclude that, in the exocrine pancreas, secretory granules can be formed under various conditions, even with a matrix containing a ratio of components very different from that of the normal mature animal.
9
NEZU, Akihiro, Akihiko TANIMURA, Takao MORITA, Kazuharu IRIE, Toshihiko YAJIMA, and Yosuke TOJYO. "Evidence that zymogen granules do not function as an intracellular Ca2+ store for the generation of the Ca2+ signal in rat parotid acinar cells." Biochemical Journal 363, no. 1 (2002): 59–66. http://dx.doi.org/10.1042/bj3630059.
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Rat parotid acinar cells lacking zymogen granules were obtained by inducing granule discharge with the β-adrenoceptor agonist isoproterenol. To assess whether zymogen granules are involved in the regulation of Ca2+ signalling as intracellular Ca2+ stores, changes in cytosolic free Ca2+ ion concentration ([Ca2+]i) were studied with imaging microscopy in fura-2-loaded parotid acinar cells lacking zymogen granules. The increase in [Ca2+]i induced by muscarinic receptor stimulation was initiated at the apical pole of the acinar cells, and rapidly spread as a Ca2+ wave towards the basolateral region. The magnitude of the [Ca2+]i response and the speed of the Ca2+ wave were essentially similar to those in control acinar cells containing zymogen granules. Western blot analysis of the inositol 1,4,5-trisphosphate receptor (IP3R) was performed on zymogen granule membranes and microsomes using anti-IP3R antibodies. The immunoreactivity of all three IP3Rs was clearly observed in the microsomal preparations. Although a weak band of IP3R type-2 was detected in the zymogen granule membranes, this band probably resulted from contamination by the endoplasmic reticulum (ER), because calnexin, a marker protein of the ER, was also detected in the same preparation. Furthermore, Western blotting and reverse transcriptase-PCR analysis failed to provide evidence for the expression of ryanodine receptors in rat parotid acinar cells, whereas expression was clearly detectable in rat skeletal muscle, heart and brain. These results suggest that zymogen granules do not have a critical role in Ca2+ signalling in rat parotid acinar cells.
10
Matthews, E. K., Jane Rogers, and D. B. McKay. "Control of secretory granule interaction and exocytosis in pancreatic cells." Bioscience Reports 7, no. 5 (1987): 435–42. http://dx.doi.org/10.1007/bf01362506.
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Application of the laser-based technique of photon correlation spectroscopy to an in vitro study of the ionic stability and interaction kinetics of zymogen granules isolated from rat exocrine pancreas is described here. In addition the separation from pancreatic acinar cell cytosol of a factor which stabilizes isolated zymogen granules and inhibits cation-induced granule aggregation is outlined. The basis of this action and the significance of the cytosolic inhibitory factor in the regulation of granule mobility and exocytosis in vivo is discussed.
11
Schmidt, K., H. Dartsch, D. Linder, H. F. Kern, and R. Kleene. "A submembranous matrix of proteoglycans on zymogen granule membranes is involved in granule formation in rat pancreatic acinar cells." Journal of Cell Science 113, no. 12 (2000): 2233–42. http://dx.doi.org/10.1242/jcs.113.12.2233.
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The secretory lectin ZG16p mediated the binding of aggregated zymogens to the granule membrane in pancreatic acinar cells. Using a recently established in vitro condensation-sorting assay, we now show that pretreatment of zymogen granule membranes (ZGM) with either sodium bicarbonate at pH 10 or with phosphatidyl inositol-specific phospholipase C (PI-PLC) reduced the binding efficiency of zymogens to the same extent, as distinct components were liberated from ZGM. Analysis of the composition of the bicarbonate extract revealed the presence of the secretory lectin ZG16p, the serpin ZG46p and the GPI-linked glycoprotein GP-2, together with several unknown proteins, and small amounts of lipase and carboxylester lipase. The unknown proteins detected in 2-D gels represented a group of acidic and basic protein spots, which were positive in a glycan staining reaction and were soluble in methanol. One protein spot of the acidic group and several of the basic group reacted with a monoclonal antibody directed against chondroitin sulfate, indicating that the proteins represented proteoglycans. A staining pattern similar to the glycan reaction was observed in immunoblots using a polyclonal antibody directed against the whole bicarbonate extract. Immunogold electron microscopy revealed that this antibody reacted with components in the periphery of zymogen granules and strongly stained ZGM in the pellet fraction of a standard in vitro condensation-sorting assay. The amino acid composition of isolated components of both the acidic and basic group showed similarities to aggrecan, a cartilage-specific proteoglycan, and to glycine-rich glycoproteins, respectively. We therefore conclude that a submembranous matrix on the ZGM composed of proteoglycans and glycoproteins is involved in granule formation in pancreatic acinar cells.
12
de Dios, I., AC Garcia-Montero, A. Orfao, and MA Manso. "Selective exocytosis of zymogen granules induces non-parallel secretion in short-term cholecystokinin-stimulated rats." Journal of Endocrinology 163, no. 2 (1999): 199–206. http://dx.doi.org/10.1677/joe.0.1630199.
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Parallel studies on pancreatic enzyme secretion and zymogen granule enzyme composition have been carried out in rats subjected to infusion of cholecystokinin (CCK) (1.25 microgram/kg per h) over 30 min. Flow cytometric analysis showed a significant decrease in the mean value of granule size after CCK stimulation. The amount of trypsinogen stored in each individual zymogen granule was significantly lower at 30 min of CCK infusion, but no variation in intragranular amylase content was observed. As a result, the amylase/trypsinogen ratio was significantly increased in the zymogen granules that remained in the pancreas of rats stimulated with CCK for 30 min. A significantly greater proportion of trypsin than amylase was secreted after 30 min CCK infusion. Our results support the existence of different types of granules loaded with different proportions of enzymes. We conclude that short-term CCK stimulation induces the selective release of large granules containing a high proportion of trypsinogen, which leads to a non-parallelism of enzyme secretion.
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Gasser, K. W., J. DiDomenico, and U. Hopfer. "Potassium transport by pancreatic and parotid zymogen granule membranes." American Journal of Physiology-Cell Physiology 255, no. 6 (1988): C705—C711. http://dx.doi.org/10.1152/ajpcell.1988.255.6.c705.
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Zymogen granules that were stable at physiological conditions of pH, ionic strength, and temperature were isolated from the rat pancreas and parotid. The cation permeability of these granules was evaluated to characterize the mechanism of secretagogue-stimulated fluid secretion by acinar cells. Granule swelling and lysis provide a measure of the rate of cation transport, since the use of ionophore combinations such as tripropyltin and carbonyl cyanide 3-chlorophenylhydrazone (CCCP) will render cation conductance the rate-limiting step for salt influx. This technique supplies evidence for the existence of K+ conductance in the granule membrane. The pancreatic and parotid granules have a K+-selective conductance that is not inhibited by the K+ channel blockers barium, tetraethylammonium, quinidine, cesium, or 4-aminopyridine. Furthermore, the intragranular pH of pancreatic zymogen granules was measured to be approximately 6.5 and was identified as a factor that modulates the K+ conductance. Although the pancreatic and parotid granules were qualitatively identical, quantitatively the relative K+ transport rate constant was over twofold higher for the parotid than for the pancreatic granules. The zymogen granule K+ conductance may have an important role in active K+ secretion by exocrine glands, which is prominent in the parotid after stimulation with beta-adrenergic agents.
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MacLean, C. M., and J. M. Edwardson. "Fusion between rat pancreatic zymogen granules and plasma membranes. Modulation by a GTP-binding protein." Biochemical Journal 286, no. 3 (1992): 747–53. http://dx.doi.org/10.1042/bj2860747.
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At the moment, little is known about the molecular characteristics of the final step in the process of regulated exocytosis, i.e. the fusion of the membrane of a secretory vesicle with the plasma membrane. We have reconstituted this fusion event in vitro, using zymogen granules and plasma membranes from the exocrine pancreas of the rat. The membranes of zymogen granules were loaded with the lipid-soluble fluorescent probe octadecylrhodamine B, at a concentration that resulted in self-quenching of its fluorescence. The granules were then incubated with pancreatic plasma membranes at 37 degrees C, and fusion was measured through the dilution-dependent de-quenching of the fluorescence of the probe. Zymogen granules fused with pancreatic plasma membranes, but not with plasma membranes from liver or chromaffin cells; granules also fused with unlabelled granule membranes. The fusion of granules with plasma membranes was unaffected by variation of the Ca2+ concentration over a wide range, but fusion of granules with both plasma membranes and zymogen granule membranes was stimulated by GTP and, more potently, by guanosine 5′-[gamma-thio]triphosphate (GTP[S]). The effect of GTP[S] was to increase the extent of fusion occurring at low concentrations of plasma membranes, without affecting the maximum signal obtained at high membrane concentrations. Pre-incubation of the plasma membranes with GTP[S] also enhanced their ability to fuse with zymogen granules. Our results indicate that membrane fusion during exocytosis may be under the direct control of a GTP-binding protein.
15
Burnham, D. B., P. Munowitz, N. Thorn, and J. A. Williams. "Protein kinase activity associated with pancreatic zymogen granules." Biochemical Journal 227, no. 3 (1985): 743–51. http://dx.doi.org/10.1042/bj2270743.
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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.
16
Leblond, F. A., G. Viau, J. Lainé, and D. Lebel. "Reconstitution in vitro of the pH-dependent aggregation of pancreatic zymogens en route to the secretory granule: implication of GP-2." Biochemical Journal 291, no. 1 (1993): 289–96. http://dx.doi.org/10.1042/bj2910289.
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Regulated secretory proteins are thought to be sorted in the trans-Golgi network (TGN) via selective aggregation. To elucidate the biogenesis of the secretory granule in the exocrine pancreas, we reconstituted in vitro the conditions of pH and ions believed to exist in the TGN using the end product of this sorting process, the zymogen granule contents. Protein aggregation was dependent on pH (acidic) and on the presence of cations (10 mM Ca2+, 150 mM K+) to reproduce the pattern of proteins found in the granule. The constitutive secretory protein IgG was excluded from these aggregates. Zymogen aggregation correlated with the relative proportion of the major granule membrane protein GP-2 in the assay. These results show that the glycosylphosphatidylinositol-anchored protein GP-2 co-aggregates with zymogens in the acidic environment believed to exist in the pancreatic TGN, and thus suggest that GP-2 would function as a membrane anchor for zymogen aggregates, facilitating their entrapment in budding vesicles directed towards the regulated secretory pathway.
17
MARCINIAK, Stefan J., and J. Michael EDWARDSON. "Association of nucleoside diphosphate kinase with pancreatic zymogen granules: effects of local GTP generation on granule membrane characteristics." Biochemical Journal 316, no. 1 (1996): 99–106. http://dx.doi.org/10.1042/bj3160099.
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It is well established that both GTP-binding proteins and phosphoproteins are involved in the control of exocytosis in the exocrine pancreas. Exocytotic membrane fusion is stimulated by guanosine 5′-[γ-thio]triphosphate, and the phosphorylation states of several proteins, including at least one on the zymogen granule membrane, are known to change during exocytosis. We show here that a nucleoside diphosphate kinase is associated with the cytoplasmic face of pancreatic zymogen granules. This enzyme behaves as a phosphoprotein of apparent molecular mass 21 kDa on SDS/polyacrylamide gels, and is able to produce GTP by using ATP to phosphorylate endogenous GDP. GTP production by nucleoside diphosphate kinase is stimulated by the wasp venom peptide mastoparan, both through a direct action on the enzyme and through its ability to increase the availability of endogenous GDP. Two effects of the GTP produced by nucleoside diphosphate kinase are demonstrated: phosphorylation of a 37 kDa zymogen granule protein on histidine residues, and stimulation of the fusion of zymogen granules with pancreatic plasma membranes in vitro. These results suggest that granule-associated nucleoside diphosphate kinase is able to maintain local GTP concentrations, and raise the possibility that it might be involved in the control of exocytosis in the pancreatic acinar cell.
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Iida, H., S. Tanaka, and Y. Shibata. "Small GTP-binding protein, Rab6, is associated with secretory granules in atrial myocytes." American Journal of Physiology-Cell Physiology 272, no. 5 (1997): C1594—C1601. http://dx.doi.org/10.1152/ajpcell.1997.272.5.c1594.
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Rab proteins, a subfamily of small GTP-binding proteins, have been shown to play key roles in regulation of vesicular traffic in eukaryotic cells. In this study, we have intended to identify, the atrial granule-associated Rab proteins that seem to be required for formation or intracellular transport of the granules. Atrial granules contained at least four small GTP-binding proteins, and we have demonstrated by biochemical analysis that one of the small GTP-binding proteins associated with the atrial granules is a Rab6 protein (Rab6p). Rab6p was also detected in highly purified zymogen granules of pancreatic exocrine gland. Immunogold electron microscopy performed on ultrathin cryosections of rat auricle revealed that Rab6p was associated with the atrial granule membranes. Association of Rab6p with the atrial granule membranes was also confirmed by immunodiffusion electron microscopy in agarose-embedded atrial granules. These data indicate that Rab6p is associated with the atrial granules and that it might function in the intracellular traffic of the secretory granules in the atrial myocytes.
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De Lisle, R. C., and K. S. Isom. "Expression of sulfated gp300 and changes in glycosylation during pancreatic development." Journal of Histochemistry & Cytochemistry 44, no. 1 (1996): 57–66. http://dx.doi.org/10.1177/44.1.8543783.
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The pancreatic zymogen granule membrane protein gp300 is the major sulfated glycoprotein of the mouse acinar cell and has been proposed to be an important structural component of the zymogen granule membrane. A prediction of this proposed function is that gp300 expression should be coordinately regulated with the digestive enzymes and appearance of zymogen granules during differentiation of acinar cells in fetal development. By Western blots and immunolocalization with a polyclonal antiserum to gp300, we found that gp300 protein expression paralleled expression of amylase and the appearance of zymogen granules in differentiating acinar cells. Lectin blots were performed to assess the glycoconjugate composition of gp300 during development. Using the fucose binding lectin Ulex europaeus I, we found that gp300 acquires this carbohydrate only postnatally, temporally correlated with weaning. In addition, gp300 showed complex changes during postnatal development in reactivity with the galactose binding lectin peanut agglutinin (PNA) and the sialic acid binding lectin Maackia amuresis (MAA). Levels of reactivity of PNA and MAA were reciprocal, suggesting that sialylation of galactose (which can block peanut agglutinin binding) was not constant on gp300 during development.
20
De Lisle, R. C., and J. A. Williams. "Zymogen granule acidity is not required for stimulated pancreatic protein secretion." American Journal of Physiology-Gastrointestinal and Liver Physiology 253, no. 6 (1987): G711—G719. http://dx.doi.org/10.1152/ajpgi.1987.253.6.g711.
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It has been demonstrated recently by acridine orange fluorescence that pancreatic zymogen granules are acidic in situ, with respect to the cytoplasm. To evaluate the relationship between the acidic intragranular pH and hormone-stimulated secretion, mouse pancreatic acini were treated with lysosomotropic agents to collapse the zymogen granule pH gradient. Methylamine, monensin, and chloroquine collapsed the granule pH gradient as evidenced by a disappearance of acridine orange fluorescence. Cholecystokinin octapeptide (CCK-8)-stimulated acinar amylase secretion was unaffected in the presence of up to 30 mM methylamine and slightly enhanced in the presence of 0.3-10 microM monensin or 3-300 microM chloroquine. Acini were also preincubated for 15 min before addition of either CCK-8 or bombesin with concentrations of the lysosomotropic agents that dissipated the granule acridine orange fluorescence within this time. With preincubation, basal amylase release was unaffected, while stimulated secretion was slightly enhanced by all three lysosomotropic agents. Monensin and methylamine caused vacuolization of Golgi and lysosomal membranes and inhibition of intracellular transport of newly synthesized proteins. Chloroquine affected lysosomes similarly but had little effect on Golgi membranes or on intracellular protein transport. We also demonstrate that parotid secretory granules are acidic in situ by the acridine orange technique. Thus acidified secretory granules may be a general feature of exocrine secretory granules, but the acid pH is not requisite for the final steps in protein secretion from isolated pancreatic acini.
21
Takuma, T., T. Ichida, K. Okumura, Y. Sasaki, and M. Kanazawa. "Effects of valinomycin on osmotic lysis of zymogen granules and amylase exocytosis from parotid acini." American Journal of Physiology-Gastrointestinal and Liver Physiology 264, no. 5 (1993): G895—G901. http://dx.doi.org/10.1152/ajpgi.1993.264.5.g895.
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The role of osmotic swelling of the secretory granules in adenosine 3',5'-cyclic monophosphate (cAMP)-mediated amylase exocytosis was evaluated by use of isolated zymogen granules and saponin-permeabilized acini of the rat parotid gland. The osmotic lysis of the isolated granules was markedly enhanced by the addition of valinomycin (> 10(-9) M) in the presence of isosmotic KSCN or KI medium. However, valinomycin (up to 10(-5) M) did not increase the granule lysis in KCl medium, although the granules were slightly less stable in KCl medium than in K2SO4 or potassium gluconate medium. Guanosine 5'-O-(3-thiotriphosphate) did not affect the granule lysis. Valinomycin alone had no effect on amylase release from saponin-permeabilized parotid acini incubated in KCl medium, but completely abolished cAMP-mediated amylase release in all K+ media used. The inhibition was clearly detected at 0.1 microM valinomycin in KCl medium, not blocked by the addition of 1 mM MgATP to the medium, and was greatly reduced in NaCl medium. cAMP-evoked amylase release was completely inhibited by SCN- and I- (permeant anions), the mean inhibitory dosages of which were approximately 25 and 50 mM, respectively. These results suggest that 1) the membrane of parotid zymogen granules has no detectable Cl- channels responsible for osmotic swelling of the granules, and 2) increase in K+ or anion conductance of the granule does not enhance but inhibits cAMP-mediated amylase exocytosis from parotid acini.
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De Lisle, R. C., and U. Hopfer. "Electrolyte permeabilities of pancreatic zymogen granules: implications for pancreatic secretion." American Journal of Physiology-Gastrointestinal and Liver Physiology 250, no. 4 (1986): G489—G496. http://dx.doi.org/10.1152/ajpgi.1986.250.4.g489.
Full textAbstract:
Zymogen granules from rat pancreas were prepared on a 40% Percoll gradient at free calcium levels less than 0.2 microM. We have previously shown [Am. J. Physiol. 246 (Gastrointest. Liver Physiol. 9)] that zymogen granules prepared by this method are stable in vitro for more than 1 h in "physiological buffers." The electrolyte permeabilities of the zymogen granule membrane were investigated to determine the basis for this stability. Ionic permeabilities were estimated from rates of osmotic lysis and measured as decrease in optical density (OD) of granule suspensions. OD correlated linearly with lysis, as indicated by release of amylase, except for the highest and lowest 10% of the OD of intact granules. Lysis of freshly isolated granules was slow in Na+ or K+ salt solutions (e.g., t1/2 approximately 3 h for Cl-) but was accelerated 5- to 50-fold when cation ionophores were present simultaneously. This behavior indicates that zymogen granules have low endogenous permeabilities to the cations Na+ and K+, but are highly permeable to a variety of anions. Both anion conductance and anion-exchange pathways were found. The relative selectivity of the anion conductance pathway was SCN- greater than Br- approximately NO-3 greater than SO2-(4) greater than acetate- approximately Cl- greater than isethionate-. The relative selectivity sequence for anion/-OH- exchange was acetate- greater than SCN- greater than Br- approximately NO-3 approximately Cl- much greater than isethionate- greater than SO2-(4). The anion transport blocker DIDS blocked the electrogenic pathway with a half-maximal effectiveness at approximately 2 microM. DIDS had little effect on the anion-exchange pathway.(ABSTRACT TRUNCATED AT 250 WORDS)
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Gasser, K. W., J. DiDomenico, and U. Hopfer. "Secretagogues activate chloride transport pathways in pancreatic zymogen granules." American Journal of Physiology-Gastrointestinal and Liver Physiology 254, no. 1 (1988): G93—G99. http://dx.doi.org/10.1152/ajpgi.1988.254.1.g93.
Full textAbstract:
The membrane permeability of pancreatic zymogen granules was evaluated in vitro with granules isolated from rats in different secretory states: 1) untreated, 2) pretreated with a muscarinic antagonist, 3) pretreated with a muscarinic and an adrenergic antagonist, 4) pretreated as in 3 and then stimulated with the secretagogue cholecystokinin 4 min before death, and 5) pretreated as in 3 and then stimulated with the secretagogue secretin 4 min before death. Granules isolated from untreated rats had variable ionic permeabilities but in general possessed both chloride conductance and electroneutral exchange pathways with low permeabilities to alkali metal ions. In contrast, granules from animals pretreated with secretory antagonists had very low ion permeabilities to both inorganic anions, such as chloride, and alkali metal ions. Injection of the peptide secretagogues cholecystokinin or secretin resulted in a relatively fast (within 4 min) activation or induction of high chloride permeabilities through both chloride conductance and chloride/hydroxide (or chloride/bicarbonate) exchange pathways. In addition, the secretagogues increased the cation permeability of the granule membrane, which exhibited a distinct potassium selectivity. Chloride conductance has been postulated to play a major role in fluid secretion coupled to exocytosis of macromolecules [R. C. DeLisle and U. Hopfer, Am. J. Physiol. 250 (Gastrointest. Liver Physiol. 13): G489-G496, 1986]. These results demonstrate that granules may actively participate in the secretory process and suggest that some of the physiological targets in the cascade of events leading to secretion are anion and cation transporters in the zymogen granule membrane.
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Rindler, Michael J., Chong-feng Xu, Iwona Gumper, Nora N. Smith, and Thomas A. Neubert. "Proteomic Analysis of Pancreatic Zymogen Granules: Identification of New Granule Proteins." Journal of Proteome Research 6, no. 8 (2007): 2978–92. http://dx.doi.org/10.1021/pr0607029.
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De Lisle, Robert C. "Role of sulfated O-linked glycoproteins in zymogen granule formation." Journal of Cell Science 115, no. 14 (2002): 2941–52. http://dx.doi.org/10.1242/jcs.115.14.2941.
Full textAbstract:
Packaging of proteins into regulated secretory granules is mediated by the mildly acidic pH of the trans Golgi network and immature secretory granules. This need for an acidic pH indicates that ionic interactions are important. The mouse pancreatic acinar cell contains four major sulfated glycoproteins,including the zymogen granule structural component Muclin. I tested the hypothesis that sulfation and the O-linked glycosylation to which the sulfates are attached are required for normal formation of zymogen granules in the exocrine pancreas. Post-translational processing was perturbed with two chemicals: sodium chlorate was used to inhibit sulfation and benzyl-N-acetyl-α-galactosaminide was used to inhibit O-linked oligosaccharide elongation. Both chemicals resulted in the accumulation in the Golgi region of the cell of large vacuoles that appear to be immature secretory granules, and the effect was much more extensive with benzyl-N-acetyl-α-galactosaminide than chlorate. Both chemical treatments inhibited basal secretion at prolonged chase times, and again benzyl-N-acetyl-α-galactosaminide had a greater effect than chlorate. In addition, benzyl-N-acetyl-α-galactosaminide, but not chlorate, totally inhibited stimulated secretion of newly synthesized proteins. These data provide evidence for a role of sulfated O-linked glycoproteins in protein condensation and maturation of zymogen granules. Under maximal inhibition of O-linked oligosaccharide biosynthesis, anterograde post-Golgi traffic in the regulated pathway is almost totally shut down, demonstrating the importance of these post-translational modifications in progression of secretory proteins through the regulated pathway and normal granule formation in the pancreatic acinar cell.
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Chen, Ying, Jennifer D. Warner, David I. Yule, and David R. Giovannucci. "Spatiotemporal analysis of exocytosis in mouse parotid acinar cells." American Journal of Physiology-Cell Physiology 289, no. 5 (2005): C1209—C1219. http://dx.doi.org/10.1152/ajpcell.00159.2005.
Full textAbstract:
Exocrine cells of the digestive system are specialized to secrete protein and fluid in response to neuronal and/or hormonal input. Although morphologically similar, parotid and pancreatic acinar cells exhibit important functional divergence in Ca2+ signaling properties. To address whether there are fundamental differences in exocytotic release of digestive enzyme from exocrine cells of salivary gland versus pancreas, we applied electrophysiological and optical methods to investigate spatial and temporal characteristics of zymogen-containing secretory granule fusion at the single-acinar cell level by direct or agonist-induced Ca2+ and cAMP elevation. Temporally resolved membrane capacitance measurements revealed that two apparent phases of exocytosis were induced by Ca2+ elevation: a rapidly activated initial phase that could not be resolved as individual fusion events and a second phase that was activated after a delay, increased in a staircaselike fashion, was augmented by cAMP elevation, and likely reflected both sequential compound and multivesicular fusion of zymogen-containing granules. Optical measurements of exocytosis with time-differential imaging analysis revealed that zymogen granule fusion was induced after a minimum delay of ∼200 ms, occurred initially at apical and basolateral borders of acinar cells, and under strong stimulation proceeded from apical pole to deeper regions of the cell interior. Zymogen granule fusions appeared to coordinate subsequent fusions and produced persistent structures that generally lasted several minutes. In addition, parotid gland slices were used to assess secretory dynamics in a more physiological context. Parotid acinar cells were shown to exhibit both similar and divergent properties compared with the better-studied pancreatic acinar cell regarding spatial organization and kinetics of exocytotic fusion of zymogen granules.
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Larina, Olga, Purnima Bhat, James A. Pickett, et al. "Dynamic Regulation of the Large Exocytotic Fusion Pore in Pancreatic Acinar Cells." Molecular Biology of the Cell 18, no. 9 (2007): 3502–11. http://dx.doi.org/10.1091/mbc.e07-01-0024.
Full textAbstract:
Loss of granule content during exocytosis requires the opening of a fusion pore between the secretory granule and plasma membrane. In a variety of secretory cells, this fusion pore has now been shown to subsequently close. However, it is still unclear how pore closure is physiologically regulated and contentious as to how closure relates to granule content loss. Here, we examine the behavior of the fusion pore during zymogen granule exocytosis in pancreatic acinar cells. By using entry of high-molecular-weight dyes from the extracellular solution into the granule lumen, we show that the fusion pore has a diameter of 29–55 nm. We further show that by 5 min after granule fusion, many granules have a closed fusion pore with evidence indicating that pore closure is a prelude to endocytosis and that in granules with a closed fusion pore the chymotrypsinogen content is low. Finally, we show that latrunculin B treatment promotes pore closure, suggesting F-actin affects pore dynamics. Together, our data do not support the classical view in acinar cells that exocytosis ends with granule collapse. Instead, for many granules the fusion pore closes, probably as a transition to endocytosis, and likely involving an F-actin–dependent mechanism.
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Otani, Taiichi, Sergei M. Chepilko, James H. Grendell, and Fred S. Gorelick. "Codistribution of TAP and the granule membrane protein GRAMP-92 in rat caerulein-induced pancreatitis." American Journal of Physiology-Gastrointestinal and Liver Physiology 275, no. 5 (1998): G999—G1009. http://dx.doi.org/10.1152/ajpgi.1998.275.5.g999.
Full textAbstract:
The pathological activation of zymogens within the pancreatic acinar cell plays a role in acute pancreatitis. To identify the processing site where activation occurs, antibodies to the trypsinogen activation peptide (TAP) were used in immunofluorescence studies using frozen sections from rat pancreas. Saline controls or animals receiving caerulein in amounts producing physiological levels of pancreatic stimulation demonstrated little or no TAP immunoreactivity. However, after caerulein hyperstimulation (5 μg ⋅ kg−1 ⋅ h−1) for 30 min and the induction of pancreatitis, TAP immunoreactivity appeared in a vesicular, supranuclear compartment that demonstrated no overlap with zymogen granules. The number of vesicles and their size increased with time. After 60 min of hyperstimulation with caerulein, most of the TAP reactivity was localized within vacuoles ≥1 μm that demonstrated immunoreactivity for the granule membrane protein GRAMP-92, a marker for lysosomes and recycling endosomes. Pretreatment with the protease inhibitor FUT-175 blocked the appearance of TAP after hyperstimulation. These studies provide evidence that caerulein hyperstimulation stimulates trypsinogen processing to trypsin in distinct acinar cell compartments in a time-dependent manner.
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WÄSLE, Barbara, Matthew TURVEY, Olga LARINA, et al. "Syncollin is required for efficient zymogen granule exocytosis." Biochemical Journal 385, no. 3 (2005): 721–27. http://dx.doi.org/10.1042/bj20041064.
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Syncollin is a 13 kDa protein that is present in the exocrine pancreas, where the majority of the protein is tightly attached to the luminal surface of the zymogen granule membrane. We have addressed the physiological role of syncollin by studying the phenotype of syncollin KO (knockout) mice. These mice show pancreatic hypertrophy and elevated pancreatic amylase levels. Further, secretagogue-stimulated amylase release from pancreatic lobules of syncollin KO mice was found to be reduced by about 45% compared with wild-type lobules, and the delivery of newly synthesized protein to zymogen granules was delayed, indicating that the mice have a pancreatic secretory defect. As determined by two-photon imaging, the number of secretagogue-stimulated exocytotic events in acini from syncollin KO mice was reduced by 50%. This reduction was accounted for predominantly by a loss of later, ‘secondary’ fusion events between zymogen granules and other granules that had already fused with the plasma membrane. We conclude that syncollin is required for efficient exocytosis in the pancreatic acinar cell, and that it plays a particularly important role in compound exocytosis.
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Niederau, C., J. H. Grendell, and S. S. Rothman. "Characteristics of rat pancreatic zymogen granules prepared by different methods." American Journal of Physiology-Gastrointestinal and Liver Physiology 251, no. 3 (1986): G421—G429. http://dx.doi.org/10.1152/ajpgi.1986.251.3.g421.
Full textAbstract:
Zymogen granules isolated from tissue homogenates by differential centrifugation in isotonic sucrose solutions show substantial release of digestive enzyme when suspended in isotonic NaCl and in sucrose solutions at pH values above neutrality. A recent study reported a new method for isolating granules, involving the use of a complex homogenization medium and a Percoll gradient that was claimed to produce "stable" granules, i.e., granules that do not release their content in salt solutions and at pH values at or above neutrality. In the present study, we compare granules prepared in both ways, particularly in terms of their tendency to release amylase in isotonic ionic solutions and as a function of pH. The relative absence of amylase release from granules isolated by the new technique was found to be attributable to simple differences in the details of the experimental procedures that were used and not to actual differences in the characteristics of the two granule preparations. For example, previous studies with granules prepared in sucrose solutions reported substantial salt-induced release at 37 degrees C, whereas the recent study reporting the absence of salt-induced release from granules obtained from a Percoll gradient was done at 24 degrees C. Under the identical experimental conditions as used in the present study, little amylase release was seen at 24 degrees C for granules isolated by either technique, but substantial release was seen for both at 37 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)
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Groblewski, Guy E., Mutsumi Yoshida, Hongren Yao, John A. Williams, and Stephen A. Ernst. "Immunolocalization of CRHSP28 in exocrine digestive glands and gastrointestinal tissues of the rat." American Journal of Physiology-Gastrointestinal and Liver Physiology 276, no. 1 (1999): G219—G226. http://dx.doi.org/10.1152/ajpgi.1999.276.1.g219.
Full textAbstract:
The 28-kDa (on SDS-PAGE) Ca2+-regulated heat stable protein (CRHSP28) was recently purified as novel phosphoprotein in exocrine pancreas, since it undergoes an immediate increase in serine phosphorylation when acini are stimulated with Ca2+-mobilizing agonists. Examination of CRHSP28 protein expression in rat revealed that most was highly expressed in pancreas and other morphologically related exocrine tissues, including the parotid, lacrimal, and submandibular glands. Immunofluorescence staining in pancreas indicated that CRHSP28 was specifically concentrated in zymogen granule-rich areas in the apical cytoplasm of acinar cells. Lack of colocalization with pancreatic lipase in dual immunofluorescence studies confirmed localization of CRHSP28 to the area immediately surrounding the granules. Western analysis of pancreatic zymogen granule membrane proteins indicated CRHSP28 was not associated with the granules following their purification. A similar pattern of apical cytoplasmic secretory granule staining was noted in lacrimal and submandibular glands. CRHSP28 protein was also expressed at relatively high levels in mucosal epithelial cells of the stomach and small intestine. CRHSP28 was found in the supranuclear apical cytoplasm of cells lining the small intestinal crypts, including Paneth cells, and was abundant in the cytoplasm of goblet cells. In the stomach, strong CRHSP28 staining was seen in mucus-secreting cells in the upper portion of the gastric glands and in the apical, granule-rich cytoplasm of chief cells located in the lower portions of the glands. Dual labeling with anti-H+-K+-ATPase demonstrated a comparatively lower expression of CRHSP28 in parietal cells. Collectively, the high relative expression of CRHSP28 in various secretory cell types within the digestive system, together with its intracellular localization surrounding the acinar cell secretory granules, strongly supports a role for CRHSP28 in Ca2+-mediated exocrine secretion.
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MacLean, C. M., G. J. Law, and J. M. Edwardson. "Stimulation of exocytotic membrane fusion by modified peptides of the rab3 effector domain: re-evaluation of the role of rab3 in regulated exocytosis." Biochemical Journal 294, no. 2 (1993): 325–28. http://dx.doi.org/10.1042/bj2940325.
Full textAbstract:
We have shown previously that fusion between pancreatic zymogen granules and plasma membranes is stimulated by a peptide corresponding to the putative effector domain of rab3. Here we show that this stimulatory effect persists when the amino acid sequence of the peptide is substantially modified. We also show that an antibody raised against rab3a recognizes a protein of appropriate size on the zymogen-granule membrane, but has no effect on membrane fusion. We suggest that rab3 is not directly involved in the control of this membrane fusion event, and that the peptides are stimulating fusion by a mechanism unrelated to rab3.
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Lebel, Denis, and Jean Paquette. "Structure of the pig pancreatic GP-2: role of intramolecular disulfides in the resistance to proteolysis." Biochemistry and Cell Biology 67, no. 6 (1989): 281–87. http://dx.doi.org/10.1139/o89-042.
Full textAbstract:
GP-2 is the major membrane glycoprotein characteristic of the pancreatic zymogen granule membrane. When granules are lysed in the presence of DTT, GP-2 becomes completely and specifically degraded. This proteolysis was reproducible with the same characteristics in the purified granule membrane. The protease was purified from this source using hydrophobic interaction chromatography. The proteolytic activity was identified as a 29-kDa protein because, in a reconstituted system containing both the purified GP-2 and the 29-kDa protein, the proteolytic degradation of GP-2 was sensitive to the same spectrum and concentrations of inhibitors or reducing agents as in the membrane. The activity was characteristic of a serine protease. It was also shown that GP-2 only becomes sensitive to proteolytic digestion when its disulfide bonds are reduced, and that DTT does not activate the protease. Seven intramolecular disulfide bonds were identified on GP-2. All of them are located in a 65-kDa tryptic fragment that is very resistant to exogenous proteases under nonreducing conditions. Because of the quite specific degradation of GP-2 under reducing conditions, we believe that the 29-kDa protease must be closely associated with GP-2 on the membrane. This protease could be responsible, in part, for the solubilization of the GP-2 from the membrane into the zymogen granule content and its resulting secretion by the pancreas.Key words: GP-2, zymogen granule, disulfide, exocrine, pancreas, secretion.
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Frenette, Gilles, Jean Y. Dubé, Jean R. Marcotte, and Roland R. Tremblay. "Arginine esterase from isolated dog prostate secretory granules is fully active enzymatically." Canadian Journal of Physiology and Pharmacology 63, no. 12 (1985): 1603–7. http://dx.doi.org/10.1139/y85-264.
Full textAbstract:
We have isolated secretory granules from dog prostate homogenates and have determined whether a major portion of arginine esterase was localized in this fraction and if it was enzymatically active. Secretory granules were purified by density gradient centrifugation on sucrose, metrizamide, or Percoll. A major proportion of whole prostate homogenate arginine esterase was found in the granule fractions. Furthermore, the specific enzymatic activity in the granules was similar to the one observed in seminal plasma. No evidence could be found for the existence of significant amount of a zymogen inactive form of arginine esterase. These results suggest that arginine esterase could be active within the secretory granules in vivo and that it could hydrolyze protein substrates contained in this organelle.
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Kleene, R., J. Zdzieblo, K. Wege, and H. F. Kern. "A novel zymogen granule protein (ZG29p) and the nuclear protein MTA1p are differentially expressed by alternative transcription initiation in pancreatic acinar cells of the rat." Journal of Cell Science 112, no. 15 (1999): 2539–48. http://dx.doi.org/10.1242/jcs.112.15.2539.
Full textAbstract:
Using a polyclonal antibody against purified zymogen granule membrane components from rat pancreas a cDNA coding for the 29 kDa protein (ZG29p) was identified by immunoscreening of a hormonally stimulated pancreas cDNA library. Western blot analysis suggests that ZG29p is a pancreas-specific protein and immunofluorescence shows that ZG29p is mainly associated with zymogen granules. Analysis of subcellular fraction applying immunoblotting revealed that ZG29p was localized mainly in the soluble fraction of zymogen granules and in a Golgi- and RER-enriched fraction, but was absent from the cytosol. In isolated zymogen granule content ZG29p was associated with protein complexes containing amylase as main constituent. The cDNA coding for ZG29p is homologous to the C-terminal region of the candidate metastasis-associated gene mta1. Northern blot analysis and RT-PCR showed that no MTA1 mRNA is present in pancreas from fasted rats and in the rat pancreas carcinoma cell line AR4-2J in its protodifferentiated state. Although no ZG29p specific mRNA was seen in the northern blot analysis, RT-PCR showed that ZG29p was expressed under both non-stimulated and stimulated conditions. The expression of MTA1 was up-regulated in the pancreas by endogenous cholecystokinin release and in AR4-2J after induction of cellular differentiation by dexamethasone. Western blotting and immunofluorescense studies indicated that MTA1p is localized in the nucleus in all tissues studied. Using genomic DNA in PCR analysis it was shown that two short introns are present flanking the sequences of the 5′end of ZG29p cDNA. One intron contains consensus elements required for pancreas specific transcription initiation, suggesting that MTA1 and ZG29 are differentially expressed by alternative transcription initiation in the pancreas. The localisation of MTA1p in the nucleus of most cell types could signify a general role in gene regulation, while the cell type specific and exclusive expression of ZG29p in pancreatic acinar cells could indicate a role in granule formation.
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Manso, Manuel A., Alberto Orfao, M. Dolores Tabernero, Secundino Vicente, and Isabel De Dios. "Changes in Both the Membrane and the Enzyme Content of Individual Zymogen Granules are Associated with Sodium Taurocholate-Induced Pancreatitis in Rats." Clinical Science 94, no. 3 (1998): 293–301. http://dx.doi.org/10.1042/cs0940293.
Full textAbstract:
1. After monitoring the changes associated with necrotizing acute pancreatitis in rats from early stages to 24 h after infusion of 5% sodium taurocholate in the choledocus, we characterized by flow cytometry the zymogen granules that still remained in the pancreas 18 h after sodium taurocholate infusion in order to explore whether alterations in the enzyme content and/or in the composition of the granule membrane could be related to the intracellular mechanisms involved in the development of necrotizing acute pancreatitis. 2. Significant increases in the haematocrit, plasma and peritoneal exudate amylase levels and oedema were observed from the third hour after 5% sodium taurocholate infusion onwards. Additionally, cell alterations such as hypergranulation, dilatation of the endoplasmic reticulum and autophagic vacuoles were found 3 and 6 h after infusion. DNA decrease, degranulation and necrosis were observed from 12 h after sodium taurocholate infusion onwards. 3. Flow cytometric measurements of zymogen granules isolated from rat pancreas 18 h after 5% sodium taurocholate infusion revealed a significant decrease in their internal complexity without major changes in their size. Double staining of granules with Tetragonolobus purpureus lectin, which specifically binds l-fucose and specific anti-trypsinogen or anti-amylase antisera, showed that rats with induced pancreatitis have decreased amounts of l-fucose in the membrane glycoconjugates and lower enzyme content (70% and 30% less for trypsinogen and amylase respectively). 4. A decrease in l-fucose in the membrane together with membrane abnormalities observed by electron microscopy in zymogen granules isolated 18 h after sodium taurocholate infusion indicate an altered synthesis of new granules or lysis of preformed zymogen granules which would favour differential loss of granular enzymes, mainly tripsinogen, which in turn could increase the severity of disease.
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Ishihara, Yukio, Takashi Sakurai, Taizou Kimura, and Susumu Terakawa. "Exocytosis and movement of zymogen granules observed by VEC-DIC microscopy in the pancreatic tissue en bloc." American Journal of Physiology-Cell Physiology 279, no. 4 (2000): C1177—C1188. http://dx.doi.org/10.1152/ajpcell.2000.279.4.c1177.
Full textAbstract:
The dynamic aspects of exocytosis, especially in the normal acinar tissue en bloc, have remained unclear. We visualized exocytosis directly in the tissue of the exocrine pancreas of rodents by video-enhanced contrast-differential interference contrast (VEC-DIC) microscopy to investigate various exocytosis-related rates and the relationship between the movement of granules and exocytotic responses. Stimulation of the tissue with bethanechol or cholecystokinin caused many of the zymogen granules in the apical pole to disappear abruptly. The exocytotic transients of individual granules were completed in 0.48–0.65 s. Granules destined to participate in the exocytotic response moved randomly at velocities of ∼0.06 μm/s or less during stimulation. In the tissue preparation, granules located far from the apical pole frequently moved back and forth for 1–7 μm without showing exocytosis. Colchicine suppressed this movement and the late phase of the secretory response. Real-time (VEC-DIC) observation of granule dynamics revealed that the initial step of exocytosis was not coupled directly with the microtubule-dependent translocation but with a continuous, slow Brownian fluctuation of granules.
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Ohnishi, H., S. A. Ernst, N. Wys, M. McNiven, and J. A. Williams. "Rab3D localizes to zymogen granules in rat pancreatic acini and other exocrine glands." American Journal of Physiology-Gastrointestinal and Liver Physiology 271, no. 3 (1996): G531—G538. http://dx.doi.org/10.1152/ajpgi.1996.271.3.g531.
Full textAbstract:
Rab3 proteins are members of the family of Ras-like monomeric GTP-binding proteins that have been implicated in secretion in neuronal cells. Although an isoform of Rab3 has been assumed to exist in pancreatic acini, its identity has not yet been established. We now report that Rab3D is present in rat pancreatic acini and is localized to the zymogen granule membrane. Reverse transcription-polymerase chain reaction (PCR) was used with primers based on mouse Rab3D to amplify Rab3D from rat pancreas. The PCR product without primer sites consisted of 580 base pairs and was 94% identical to the mouse Rab3D cDNA sequence previously cloned from adipocytes. Western blotting with a polyclonal antiserum raised against Rab3D-specific carboxyterminal amino acids identified Rab3D in rat pancreatic acini and revealed its concentration on zymogen granule membranes. Immunocytochemistry of pancreatic lobules showed that Rab3D localized to the apical region in a pattern similar to amylase. Confocal fluorescence microscopy of lobules double immunolabeled with antibodies to Rab3D and the granule membrane marker protein glycoprotein-2 (GP-2) revealed a similar localization of these proteins to zymogen granules. Immunocytochemistry also revealed the presence of Rab3D in chief and enterochromaffin-like cells in the stomach, acinar cells in lacrimal and parotid gland, and Paneth cells in the intestine. These results show that Rab3D is expressed in rat pancreatic acini and other exocrine secretory cells. Its location implies it may be involved in regulated exocytosis.
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Freedman, S. D., K. Sakamoto, and G. A. Scheele. "Nonparallel secretion of GP-2 from exocrine pancreas implies luminal coupling between acinar and duct cells." American Journal of Physiology-Gastrointestinal and Liver Physiology 267, no. 1 (1994): G40—G51. http://dx.doi.org/10.1152/ajpgi.1994.267.1.g40.
Full textAbstract:
The in vivo and in vitro secretion of glycoprotein-2 (GP-2), a glycosyl phosphatidylinositol (GPI)-anchored protein from the rat exocrine pancreas, was characterized. GP-2 was secreted in a nonparallel manner compared with amylase, a marker of secretory enzymes. Attenuated GP-2 secretion correlated with hormones that stimulated exocytosis in acinar cells. Augmented GP-2 secretion correlated with hormones that stimulated fluid and bicarbonate secretion from ductal elements. Immunofluorescence studies identified an enriched pool of GP-2 tightly bound to the apical membranes of acinar cells in addition to zymogen granules. This non-zymogen granule pool appears to represent the source of GP-2 released from acinar cells in a nonparallel manner. With the use of dispersed pancreatic acini largely devoid of ductal elements, GP-2 release was found to be augmented by alkaline pH. Thus GP-2 secretion appears to be modulated by two discrete cellular processes: 1) delivery of prereleased GP-2 within zymogen granules to the ductal lumen by exocytic mechanisms and 2) enzymatic release of GPI-anchored GP-2 from the luminal membranes, a kinetic process that appears to be regulated by secretin- or carbachol-induced secretion of bicarbonate.
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Tang, L. H., I. M. Modlin, T. A. Caulfield, and J. R. Goldenring. "A novel serine-specific kinase activity associated with exocrine secretory granules." American Journal of Physiology-Gastrointestinal and Liver Physiology 269, no. 4 (1995): G481—G489. http://dx.doi.org/10.1152/ajpgi.1995.269.4.g481.
Full textAbstract:
Zymogen secretion from exocrine cells involves an exocytotic process that is highly regulated by the modification of cytoplasmic components at different cellular levels. In the present studies, purified secretory granules were prepared from rabbit gastric chief cells, rat pancreatic acinar cells, and parotid glands to characterize a Mg(2+)-dependent protein kinase activity. In chief cell granules, endogenous pepsinogen, a fortuitous substrate, was phosphorylated at optimal Mg2+ and K+ concentrations of 40 and 50 mM, respectively. Adenosine 3',5'-cyclic monophosphate, Ca2+, and calmodulin had no significant effects on the kinase activity. In contrast, Mn2+ or Zn2+ inhibited the kinase activity. In addition to pepsinogen, the exogenous substrates casein, myelin basic protein, and lysine-rich histone were also phosphorylated by the granule-associated kinase. All substrates were exclusively phosphorylated on serine residues. ATP, but not GTP, served as the donor in the phosphorate transfer reaction. Casein kinase (CK) inhibitors CKI-7 and dibromoribofuransylbenzimidazole at concentrations (10 microM) that significantly inhibited CK activities in the tissue homogenate failed to inhibit the granule-associated kinase activity. The kinase activity was localized to the granule membrane and could be removed from the membrane with either 5 mM EDTA or alkaline carbonate extraction. Furthermore, protease digestion sensitivity revealed that the kinase was localized on the cytoplasmic face of the granules. Our results therefore indicate that the secretory granules of exocrine gastric chief cells, pancreatic acini, and parotid acini possess a unique serine-specific protein kinase activity. The cytoplasmic orientation of the kinase activity suggests a possible role in vesicle processing or the exocytotic process.
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Viau, G., J. Laine, F. Levenez, et al. "Evidence for an apical, nonregulated protein secretion in pig exocrine pancreas." American Journal of Physiology-Gastrointestinal and Liver Physiology 267, no. 5 (1994): G764—G771. http://dx.doi.org/10.1152/ajpgi.1994.267.5.g764.
Full textAbstract:
Secretory proteins are segregated into two pathways out of the trans-Golgi network of regulated secretory cells. To identify proteins specifically secreted by pathways other than the one leading to zymogen granule exocytosis in the exocrine pancreas, conscious permanently cannulated pigs were perfused with atropine to inhibit the regulated fusion of granules. Atropine almost totally inhibited the protein secretion after 1 h of perfusion. The secretion of GP-2, a glycosyl phosphatidylinositol-anchored protein of the zymogen granule membrane, was partially inhibited but was never totally abolished by atropine perfusion. The pattern of proteins secreted under atropine was almost totally different. Soluble GP-2 was the major secretory product. Its specific activity increased 60 times over its normal level in all other conditions. This secretion clearly originated from nonregulated pathways. Results suggest that during the atropine block the apical plasmalemma could be the source of the released GP-2 and that the sustained nature of this release is compatible with a replenishment of the plasmalemma with GP-2 by the continuous exocytosis of vesicles from the nonregulated pathways.
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Nadin, C. Y., J. Rogers, S. Tomlinson, and J. M. Edwardson. "A specific interaction in vitro between pancreatic zymogen granules and plasma membranes: stimulation by G-protein activators but not by Ca2+." Journal of Cell Biology 109, no. 6 (1989): 2801–8. http://dx.doi.org/10.1083/jcb.109.6.2801.
Full textAbstract:
The molecular details of the final step in the process of regulated exocytosis, the fusion of the membrane of the secretory granule with the plasma membrane, are at present obscure. As a first step in an investigation of this membrane fusion event, we have developed a cell-free assay for the interaction between pancreatic zymogen granules and plasma membranes. We show here that plasma membranes are able to trigger the release of the granule contents, and that this effect is specific to pancreatic membranes, involves membrane fusion, requires membrane proteins, and is stimulated by activators of G-proteins but not by Ca2+. The assay is simple, reliable, and rapid, and should permit the identification of proteins that are involved in the exocytotic fusion event.
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Hammel, Ilan, and Debbie Anaby. "Imaging of zymogen granules in fully wet cells. Evidence for restricted mechanism of granule growth." Microscopy Research and Technique 70, no. 9 (2007): 790–95. http://dx.doi.org/10.1002/jemt.20467.
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Giovannucci, David R., David I. Yule, and Edward L. Stuenkel. "Optical measurement of stimulus-evoked membrane dynamics in single pancreatic acinar cells." American Journal of Physiology-Cell Physiology 275, no. 3 (1998): C732—C739. http://dx.doi.org/10.1152/ajpcell.1998.275.3.c732.
Full textAbstract:
Stimulation of pancreatic acinar cells induces the release of digestive enzymes via the exocytotic fusion of zymogen granules and activates postfusion granule membrane retrieval and receptor cycling. In the present study, changes in membrane surface area of rat single pancreatic acinar cells were monitored by cell membrane capacitance ( C m) measurements and by the membrane fluorescent dye FM1-43. When measured with the C mmethod, agonist treatment evoked a graded, transient increase in acinar cell surface area averaging 3.5%. In contrast, a 13% increase in surface area was estimated using FM1-43, corresponding to the fusion of 48 zymogen granules at a rate of 0.5 s−1. After removal of FM1-43 from the surface-accessible membrane, a residual fluorescence signal was shown by confocal microscopy to be localized in endosome-like structures and confined to the apical regions of acinar cells. The development of an optical method for monitoring the membrane turnover of single acinar cells, in combination with measurements of C m changes, reveals coincidence of exocytotic and endocytotic activity in acinar cells after hormonal stimulation.
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HODEL, Alois, and J. Michael EDWARDSON. "Targeting of the zymogen-granule protein syncollin in AR42J and AtT-20 cells." Biochemical Journal 350, no. 3 (2000): 637–43. http://dx.doi.org/10.1042/bj3500637.
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Syncollin is a 13-kDa protein associated with the membranes of pancreatic zymogen granules. Here we determine the in situ localization of syncollin in pancreatic acinar cells from adult and neonatal rats, and study the targeting of green fluorescent protein-(GFP-) and His6-tagged syncollin chimaeras in model exocrine and endocrine secretory cells. Immunocytochemical analysis of the distribution of syncollin in fully differentiated and neonatal acinar cells revealed a granular pattern that corresponded with that of the zymogen-granule markers synaptobrevin 2 and amylase. In fully differentiated acinar cells syncollin-positive vesicles were detected in the apical region of the cells, whereas in neonatal acinar cells they were found clustered near the cell nucleus. Both GFP- and His6-tagged syncollin entered the secretory pathway when transiently expressed in AR42J or AtT-20 cells. Syncollin-GFP was found predominantly in amylase-positive granules in AR42J cells and in adrenocorticotrophic hormone- (ACTH-) positive granules in AtT-20 cells. Syncollin-GFP was also present in the Golgi complex in AR42J cells. Syncollin-His6 became localized in ACTH-containing granules in the neuritic processes of AtT-20 cells. In AR42J cells syncollin-His6 did not co-localize with amylase, but was detected in acidic vesicles. These results show that the exocrine protein syncollin contains intrinsic cell-type-independent targeting information that is retained in both exocrine and endocrine cells after fusion to the GFP tag. In contrast, His6-tagged syncollin is efficiently targeted to secretory granules only in AtT-20 cells and not in AR42J cells.
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Niederau, Claus, Rebecca W. Van Dyke, Bruce F. Scharschmidt, and James H. Grendell. "Rat pancreatic zymogen granules." Gastroenterology 91, no. 6 (1986): 1433–42. http://dx.doi.org/10.1016/0016-5085(86)90197-6.
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Takahashi, Kenichi, Hirosato Mashima, Kouichi Miura, Takahsi Goto, and Hirohide Ohnishi. "Rab7 Localizes to Zymogen Granule Membrane in Pancreatic Acinar Cells and Contributes to Maturation of Zymogen Granules but Not to Exocytosis." Gastroenterology 152, no. 5 (2017): S900. http://dx.doi.org/10.1016/s0016-5085(17)33075-5.
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LeBel, Denis, та Marlyne Beattie. "Identification of the catalytic subunit of the ATP diphosphohydrolase by photoaffinity labeling of high-affinity ATP-binding sites of pancreatic zymogen granule membranes with 8-azido-[α-32P]ATP". Biochemistry and Cell Biology 64, № 1 (1986): 13–20. http://dx.doi.org/10.1139/o86-003.
Full textAbstract:
Photoaffinity labeling has been performed on pancreatic zymogen granule membranes using 8-azido-[α-32P]ATP (8-N3-ATP). Proteins of 92, 67, 53, and 35 kdaltons (kDa) were specifically labeled. ATP (100 μM) inhibited very strongly the labeling with 8-N3-ATP, while ADP was much less potent, AMP and cAMP being inefficient. The apparent constants for 8-N3-ATP binding were in the micromolar concentration range for the four labeled proteins. Without irradiation, 8-N3-ATP was a competitive inhibitor (Ki = 2.66 μM) for the hydrolysis of ATP by the ATP diphosphohydrolase. The optimal conditions for the photolabeling of the 92- and 53-kDa proteins were pH 6.0 in presence of divalent cations. On the other hand the 67- and 35-kDa proteins required an alkaline pH and the addition of EDTA in the photolabeling medium. No proteins could be labeled on intact zymogen granules, showing that all the high-affinity ATP-binding sites of the membrane were located at the interior of the granule. Both the 92- and 53-kDa glycoproteins could bind to concanavalin A–Sepharose and be extracted in the detergent phase in the Triton X-114 phase separation system. These latter properties are typical of integral membrane proteins. In addition, the 53-kDa labeled protein was sensitive to endo-β-N-acetylglucosaminidase digestion. Photolabeling with 8-N3-ATP of two different preparations of purified ATP diphosphohydrolase also led to the labeling of a 53-kDa protein. Thus among the four proteins labeled with 8-N3-ATP on the pancreatic zymogen granule membrane, the 53-kDa integral membrane glycoprotein was shown to bear the catalytic site of the ATP diphosphohydrolase.
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Jacob, Michèle, Jean Lainé, and Denis LeBel. "Specific interactions of pancreatic amylase at acidic pH. Amylase and the major protein of the zymogen granule membrane (GP-2) bind to immobilized or polymerized amylase." Biochemistry and Cell Biology 70, no. 10-11 (1992): 1105–14. http://dx.doi.org/10.1139/o92-156.
Full textAbstract:
Regulated secretory proteins are thought to be sorted in the trans-Golgi network towards the secretory granule via acidic aggregation. In the exocrine pancreas, amylase is one of the major zymogens. It is a basic protein of pI 8.6 and does not precipitate in acidic conditions. To identify the mechanism by which amylase aggregates in the acidic cisternæ of the pancreatic trans-Golgi network, we have developed an in vitro model in which amylase was fixed to plastic microtiter plates. The fixed amylase was probed with two ligands: amylase itself and GP-2, the major protein of the zymogen granule membrane. Biotinylated amylase bound to fixed amylase in a strict pH-dependent manner with optimal binding between pH 5.0 and 5.7. The affinity of binding was in the nanogram range (Kd ≈ 20.0 ng/mL) at pH 5.5. Acid binding of amylase was not reversible by incubation at neutral pH, nor could it be displaced by native amylase. GP-2 binding to fixed amylase was also pH dependent with optimal binding between pH 5.0 and 5.7. As for amylase, it was not reversible by incubation at neutral pH. GP-2 binding sites on fixed amylase appeared to be different from those of biotinylated amylase. While native and biotinylated amylase did not bind to GP-2, polymerized amylase precipitated GP-2 at acidic pH. Taken together these data suggest that slight modifications are sufficient to reveal on the amylase molecule binding sites for GP-2 and for amylase itself. These new binding capacities acquired at acidic pH could be involved in the cascade of reactions that lead to the in vivo formation of the immature secretory granule.Key words: regulated secretion, sorting, granules, trans-Golgi network.
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Green, D. P. "Mechanical coupling of zymogen granule membrane with the granule core." Biophysical Journal 58, no. 6 (1990): 1557–58. http://dx.doi.org/10.1016/s0006-3495(90)82500-5.
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