Absence of the major zymogen granule membrane protein, GP2, does not affect pancreatic morphology or secretion

The majority of digestive enzymes in humans are produced in the pancreas where they are stored in zymogen granules before secretion into the intestine. GP2 is the major membrane protein present in zymogen granules of the exocrine pancreas. Numerous studies have shown that GP2 binds digestive enzymes such as amylase, thereby supporting a role in protein sorting to the zymogen granule. Other studies have suggested that GP2 is important in the formation of zymogen granules. A knock-out mouse was generated for GP2 to study the impact of the protein on pancreatic function. GP2-deficient mice displayed no gross signs of nutrient malab-sorption such as weight loss, growth retardation, or diarrhea. Zymogen granules in the GP2 knock-out mice appeared normal on electron microscopy and contained the normal complement of proteins excluding GP2. Primary cultures of pancreatic acini appropriately responded to secretagogue stimulation with the secretion of digestive enzymes. The course of experimentally induced pancreatitis was also examined in the knock-out mice because proteins known to associate with GP2 have been found to possess a protective role. When GP2 knock-out mice were subjected to two different models of pancreatitis, no major differences were detected. In conclusion, GP2 is not essential for pancreatic exocrine secretion or zymogen granule formation. It is unlikely that GP2 serves a major intracellular role within the pancreatic acinar cell and may be functionally active after it is secreted from the pancreas.     

Modulating zymogen granule formation in pancreatic AR42J cells

Zymogen granules (ZG) are specialized organelles in the exocrine pancreas which allow digestive enzyme storage and regulated secretion. To investigate ZG biogenesis, cargo sorting and packaging, suitable cellular model systems are required. Here, we demonstrate that granule formation in pancreatic AR42J cells, an acinar model system, can be modulated by altering the growth conditions in cell culture. We find that cultivation of AR42J cells in Panserin^? 401, a serum-free medium, enhances the induction of granule formation in the presence or absence of dexamethasone when compared to standard conditions including serum. Biochemical and morphological studies revealed an increase in ZG markers on the mRNA and protein level, as well as in granule size compared to standard conditions. Our data indicate that this effect is related to pronounced differentiation of AR42J cells. To address if enhanced expression of ZG proteins promotes granule formation, we expressed several zymogens and ZG membrane proteins in unstimulated AR42J cells and in constitutively secreting COS-7 cells. Neither single expression nor co-expression was sufficient to initiate granule formation in AR42J cells or the formation of granule-like structures in COS-7 cells as described for neuroendocrine cargo proteins. The importance of our findings for granule formation in exocrine cells is discussed.     

Effects of GP2 expression on secretion and endocytosis in pancreatic AR4-2J cells

GP2 is the major membrane protein present in secretory granules of the exocrine pancreas. GP2's function is unknown, but a role in digestive enzyme packaging or secretion from secretory granules has been proposed. In addition, GP2 has been proposed to influence endocytosis and membrane recycling following stimulated secretion. Adenovirus-mediated GP2 overexpression in the rat pancreatic cell line AR4-2J was used to study its impact on digestive enzyme secretion and membrane recycling. Immunoelectron microscopy showed that GP2 and amylase co-localized in secretory granules in infected AR4-2J cells. CCK-8 stimulation resulted in a fourfold increase in amylase secretion with or without GP2 expression. GP2 expression also did not influence endocytosis following CCK-8 stimulation. Thus, GP2 expression in AR4-2J cells does not affect amylase packaging in secretory granules or stimulated secretion. GP2 expression also does not influence membrane recycling in response to stimulated stimulation in AR4-2J cells.     

Aquaporin 1 is important for maintaining secretory granule biogenesis in endocrine cells

Aquaporins (AQPs), a family of water channels expressed in epithelial cells, function to transport water in a bidirectional manner to facilitate transepithelial fluid absorption and secretion. Additionally, AQP1 and AQP5 are found in pancreatic zymogen granules and synaptic vesicles and are involved in vesicle swelling and exocytosis in exocrine cells and neurons. Here, we show AQP1 is in dense-core secretory granule (DCSG) membranes of endocrine tissue: pituitary and adrenal medulla. The need for AQP1 in endocrine cell function was examined by stable transfection of AQP1 antisense RNA into AtT20 cells, a pituitary cell line, to down-regulate AQP1 expression. These AQP1-deficient cells showed more than 60% depletion of DCSGs and significantly decreased DCSG protein levels, including proopiomelanocotin/pro-ATCH and prohormone convertase 1/3, but not non-DCSG proteins. Pulse-chase studies revealed that whereas DCSG protein synthesis was unaffected, approximately 50% of the newly synthesized proopiomelanocortin was degraded within 1 h. Low levels of ACTH were released upon stimulation, indicating that the small number of DCSGs that were made in the presence of the residual AQP1 were functionally competent for exocytosis. Analysis of anterior pituitaries from AQP1 knockout mice showed reduced prohormone convertase 1/3, carboxypeptidase E, and ACTH levels compared to wild-type mice demonstrating that our results observed in AtT20 cells can be extended to the animal model. Thus, AQP1 is important for maintaining DCSG biogenesis and normal levels of hormone secretion in pituitary endocrine cells.     

Processing of pro-Muclin and divergent trafficking of its products to zymogen granules and the apical plasma membrane of pancreatic acinar cells

Proteins are sorted and packaged into regulated secretory granules at the trans Golgi network but how such granules form is poorly understood. We are studying Muclin, the major sulfated protein of the mouse pancreatic acinar cell, and what its role may be in zymogen granule formation. Muclin behaves as a peripheral membrane protein localized to the lumen of the zymogen granule but the cDNA for this protein predicts it is a type I membrane protein with a short, 16-amino-acid, cytosolic tail (C-Tail). Using domain-specific antibodies, we demonstrate that Muclin is derived from a precursor, pro-Muclin, which is cleaved to produce Muclin and an approximately 80-kDa membrane glycoprotein (p80). Incubation of pulse-labeled cells at < or = 22 degrees C to block exit from the trans Golgi network also blocks cleavage of pro-Muclin but not sulfation, a trans Golgi network event, suggesting that cleavage occurs in a post-Golgi compartment. After cleavage the two products of pro-Muclin diverge with Muclin remaining in the regulated secretory pathway and p80 trafficking to the apical plasma membrane, presumably via the constitutive-like pathway. When transfected into exocrine AR42J cells, Muclin labeling is perinuclear and in large sub-plasma membrane puncta. Transiently transfected AR42J cells have greater immunolabeling for amylase than nontransfected cells, suggesting a role for Muclin in cargo accumulation in the regulated secretory pathway. A construct with the C-Tail deleted targets to small diffusely-distributed puncta and without the large sub-plasma membrane structures. Thus, the C-Tail is required for proper Muclin targeting. When transfected into neuroendocrine AtT-20 cells Muclin is not colocalized with ACTH in cell processes, and it appears to be constitutively trafficked to the plasma membrane, suggesting that Muclin has exocrine-specific information. We present a working model for pro-Muclin as a Golgi cargo receptor for exocrine secretory granule formation at the trans Golgi network.     

Rab3D regulates amylase levels,not agonist-induced amylase release,in AR42J cells

Rab3D is a low molecular weight GTP-binding protein that associates with secretory granules in exocrine cells. AR42J cells are derived from rat pancreatic exocrine tumor cells and develop an acinar cell-like phenotype when treated with dexamethasone (Dex). In the present study, we examined the role of Rab3D in Dex-treated AR42J cells. Rab3D expression and localization were analyzed by subcellular fractionation and immunoblotting. The role of Rab3D was examined by overexpressing myc-labeled wild-type-Rab3D and a constitutively active form of Rab3D (Rab3D-Q81L) in AR42J cells. We found that Rab3D is predominantly membrane-associated in AR42J cells and co-localizes with zymogen granules (ZG). Following CCK-8-induced exocytosis, amylase-positive ZGs appeared to move towards the periphery of the cell and co-localization between Rab3D and amylase was less complete when compared to basal conditions. Overexpression of WT, but not mutant Rab3D, resulted in an increase in cellular amylase levels. Overexpression of mutant and WT Rab3D did not affect granule morphology, CCK-8-induced secretion, long-term (48 hr) basal amylase release or granule density. We conclude that Rab3D is not involved in agonist-induced exocytosis in AR42J cells. Instead, Rab3D may regulate amylase content in these cells.     

The major zymogen granule membrane protein GP-2 in the rat pancreas is not involved in granule formation.

The major membrane protein of zymogen granules in the rat pancreas is a glycoprotein of 78 kDa (GP-2), which is inserted into the membrane via a glycosyl-phosphatidylinositol (GPI) anchor. GP-2 occurs in both, a membrane-attached and a soluble form. Due to its specific luminal orientation and its quantitative contribution to the zymogen granule membrane, GP-2 has been postulated to play an important role in sorting of digestive enzymes into the granule and in the formation of the granule as a storage organelle. We have tested this hypothesis in the rat pancreas under three different functional conditions, where both the rates of enzyme/isoenzyme synthesis change drastically, and new zymogen granules form at a high rate: a) during prolonged hormonal stimulation of the adult rat pancreas, b) during the differentiation of AR4-2J cells induced by dexamethasone in vitro, and c) during embryonic development and early postnatal life, when gene expression is modulated due to the differentiation program. Both, GP-2 mRNA levels and the rate of GP-2 biosynthesis were quantitated and compared to the immunohistochemical localization of this protein in tissue sections. Under all three functional conditions, significant changes could be demonstrated at the level of digestive enzyme gene expression, but no concomitant modulation of GP-2 expression was observed. GP-2 mRNA is absent from the embryonic pancreas and for the first time is expressed after birth with a significant increase during the period of weaning. Furthermore, GP-2 mRNA and protein levels are not modulated by hormonal stimulation, either in the adult pancreas or in AR4-2J cells in culture. Therefore, we conclude that GP-2, in spite of its quantitative contribution to the zymogen granule membrane, is not involved in enzyme protein sorting or granule formation. Alternative functions for GP-2 are discussed.     

Monoclonal antibody to a common antigen of secretory granule membranes: intracellular localization and recycling of the antigen after secretion.

Monoclonal antibody (MAb) 170-5 was generated to the secretory granule membrane of rat parotid acinar cells. The MAb recognized integral membrane glycoproteins (SG 170 antigen) localized on the luminal side of the secretory granules with N-linked carbohydrates, molecular weights 92, 84, 76, 69, and 65 KD. Immunohistochemical studies indicated that the SG 170 antigen was found in the secretory granules of both exocrine and endocrine cells and in the lysosomes of various cells in the rat. Immunoelectron microscopy with immunogold revealed that the antigen was present on the membrane of the secretory granules, lysosomes, the Golgi vesicles, and condensing vacuoles in pancreatic and parotid acinar cells and in AR42J rat pancreatic tumor cells; the Golgi stacks exhibited no immunoreaction. The common localization of the antigen in the secretory granule membranes indicated that this antigen may play an essential role in regulated secretion. Employing HRP-labeled MAb 170-5, we followed the retrieval of the antigen after exocytosis in AR42J cells. The MAb was internalized specifically with antigen-mediated endocytosis. It was transported to endosomes, subsequently to the trans-Golgi network, and then packaged into secretory granules. However, the Golgi stacks revealed no uptake of the labeled antibody.     

Chromogranin B (secretogranin I), a neuroendocrine-regulated secretory protein, is sorted to exocrine secretory granules in transgenic mice.

Chromogranin B (CgB, secretogranin I) is a secretory granule matrix protein expressed in a wide variety of endocrine cells and neurons. Here we generated transgenic mice expressing CgB under the control of the human cytomegalovirus promoter. Northern and immunoblot analyses, in situ hybridization and immunocytochemistry revealed that the exocrine pancreas was the tissue with the highest level of ectopic CgB expression. Upon subcellular fractionation of the exocrine pancreas, the distribution of CgB in the various fractions was indistinguishable from that of amylase, an endogenous constituent of zymogen granules. Immunogold electron microscopy of pancreatic acinar cells showed co-localization of CgB with zymogens in Golgi cisternae, condensing vacuoles/immature granules and mature zymogen granules; the ratio of immunoreactivity of CgB to zymogens being highest in condensing vacuoles/immature granules. CgB isolated from zymogen granules of the pancreas of the transgenic mice aggregated in a mildly acidic (pH 5.5) milieu in vitro, suggesting that low pH-induced aggregation contributed to the observed concentration of CgB in condensing vacuoles. Our results show that a neuroendocrine-regulated secretory protein can be sorted to exocrine secretory granules in vivo, and imply that a key feature of CgB sorting in the trans-Golgi network of neuroendocrine cells, i.e. its aggregation-mediated concentration in the course of immature secretory granule formation, also occurs in exocrine cells although secretory protein sorting in these cells is thought to occur largely in the course of secretory granule maturation.     

A secretion granule membrane protein (GRAMP 92) is found in non-granule membranes including those of the endocytic pathway.

GRAMP 92, a secretion granule-associated membrane protein, has been identified in exocrine and endocrine storage granule membranes using a monoclonal antibody against rat parotid secretion granule membranes. This integral membrane glycoprotein has a M(r) of 92,000 in pancreatic zymogen granule membranes, and is slightly smaller in endocrine granule membranes. In both cases, deglycosylation produces core proteins of M(r) 52,000, that have identical peptide fingerprints. Unlike the slightly smaller zymogen granule membrane glycoprotein GP-2, GRAMP 92 does not appear to be bound to the membrane by a glycophosphatidyl inositol anchor, is not found on the plasma membrane and is not released into the secretion. Within acinar cells, low levels of antigen are observed immunocytochemically over the membranes of most granules. Antigen is highly concentrated on small vesicles that are closely apposed to (and possibly interact with) granules. As well, antigen is localized to organelles in the Golgi and basolateral regions that are part of the endocytic pathway. In hepatocytes a glycoprotein similar if not identical to GRAMP 92 marks the endocytic pathway including lysosomes. These findings indicate that GRAMP 92 is a widely distributed endocytic component and suggest that cells specialized for regulated secretion may adapt such components for storage granule function. Granule-associated GRAMP 92-rich membranes may link the exocytotic and endocytic pathways.     

Immunocytochemical localization of secretory proteins in bovine pancreatic exocrine cells

The bovine exocrine pancreatic cell produces a variety of enzymes and proenzymes for export. Biochemical studies by Greene L.J., C.H. Hirs, and G.E. Palade (J. Biol. Chem. 1963. 238:2054) have shown that the mass proportions of several of these proteins in resting pancreatic juice and zymogen granule fractions are identical. In this study we have used immunocytochemical techniques at the electron microscope level to determine whether regional differences exist in the bovine gland with regard to production of individual secretory proteins and whether specialization of product handling occurs at the subcellular level. The technique used is a modification of one previously reported (McLean, J.D., and S.J. Singer. 1970. Proc. Natl. Acad. Sci U.S.A. 69:1771) in which immunocytochemical reagents are applied to thin sections of bovine serum albumin-imbedded tissue and zymogen granule fractions. A double antibody technique was used in which the first step consisted of rabbit F(ab')2 antibovine secretory protein and the detection step consisted of sheep (F(ab')2 antirabbit F(ab')2 conjugated to ferritin. The results showed that all exocrine cells in the gland, and all zymogen granules and Golgi cisternae in each cell, were qualitatively alike with regard to their content of secretory proteins examined (trypsinogen, chymotrypsinogen A, carboxypeptidase A, RNase, and DNase). The data suggest that these secretory proteins are transported through the cisternae of the Golgi complex where they are intermixed before copackaging in zymogen granules; passage through the Golgi complex is apparently obligatory for these (and likely all) secretory proteins, and is independent of extent of glycosylation, e.g., trypsinogen, a nonglycoprotein vs. DNase, a glycoprotein.     

A lysophospholipase specific for exocrine pancreatic cells is stored in zymogen granules and secreted into pancreatic juice

We separated by two-dimensional (2D) gel electrophoresis the content of isolated rat zymogen granules and from the gel excised a protein of apparent MW 77,500 and an isoelectric point of about 4.7. A rabbit antiserum against this previously uncharacterized rat zymogen granule protein recognized two cDNA clones in a rat pancreas expression library. The cDNA inserts of these two clones had sequences showing perfect homology to the published cDNA sequence of rat pancreatic lysophospholipase. The antiserum recognized only a single protein, lysophospholipase, on one and two-dimensional immunoblots of rat pancreas homogenates and isolated zymogen granules. The antiserum did not react with any protein in homogenates of rat liver, spleen, adrenal, parotid, and prostate tissue. The zymogen granule protein of the guinea pig, previously identified as Lipase 1, was recognized specifically by the antiserum against rat lysophospholipase. This guinea pig protein can now be regarded as lysophospholipase. The same protein was demonstrated in the transformed rat acinar cell line AR4-2J, where both the rate of total enzyme synthesized and the amount of mRNA increased following treatment with dexamethasone. Immunogold labeling established that pancreatic lysophospholipase is restricted exclusively to exocrine cells where it occurs only in compartments of the exocytotic pathway. It could also be detected in pancreatic juice in the ducts of the tissue. Finally, we have shown that lysophospholipase is not related to the zymogen granule membrane protein GP2. This work establishes that lysophospholipase is a normal member of the set of soluble enzymes and proenzymes that are stored in zymogen granules and secreted into pancreatic juice.     

The pancreatic membrane protein GP-2 localizes specifically to secretory granules and is shed into the pancreatic juice as a protein aggregate

GP-2 is the major secretory granule membrane glycoprotein of the exocrine pancreas and appears in the pancreatic juice in a modified sedimentable form. We have localized GP-2 in the rat pancreas at the electron microscopic level using affinity-purified antibodies and found it to be concentrated in the zymogen granules and in the acinar lumen. Label was also present on the apical and basolateral plasma membranes but prior treatment of the sections with periodate to eliminate the contribution of highly antigenic oligosaccharide moieties reduced substantially the staining of the basolateral surface. Approximately 45% of the GP-2 in the granules was not membrane-associated but appeared instead in the granule lumen. Parallel biochemical characterization of GP-2 in isolated secretory granules demonstrated that 60% fractionated with the membranes after granule lysis while 40% remained in the content fraction. Unlike the membrane-associated form of the protein, which is linked to the membrane via glycosyl-phosphatidylinositol (GPI), GP-2 in the content did not enter the detergent phase upon Triton X-114 extraction; nor was it sedimentable at 200,000g, as is characteristic of the form collected in the pancreatic juice. In addition, GP-2 in the pancreatic juice was recovered in the aqueous phase during Triton X-114 extraction and yet remained sedimentable after detergent extraction, demonstrating that its ability to remain in large aggregates was independent of lipid. These results are consistent with a life cycle for the protein that begins with synthesis of a membrane-associated precursor that can be converted by lipolytic or proteolytic cleavage to a soluble form within the zymogen granule. Further modification to a sedimentable form may then occur in the pancreatic juice.     

Regulated apical secretion of zymogens in rat pancreas. Involvement of the glycosylphosphatidylinositol-anchored glycoprotein GP-2, the lectin ZG16p, and cholesterol-glycosphingolipid-enriched microdomains

We examined the role of glycosphingolipid- and cholesterol-enriched microdomains, or rafts, in the sorting of digestive enzymes into zymogen granules destined for apical secretion and in granule formation. Isolated membranes of zymogen granules from pancreatic acinar cells showed an enrichment in cholesterol and sphingomyelin and formed detergent-insoluble glycolipid-enriched complexes. These complexes floated to the lighter fractions of sucrose density gradients and contained the glycosylphosphatidylinositol (GPI)-anchored glycoprotein GP-2, the lectin ZG16p, and sulfated matrix proteoglycans. Morphological and pulse-chase studies with isolated pancreatic lobules revealed that after inhibition of GPI-anchor biosynthesis by mannosamine or the fungal metabolite YW 3548, granule formation was impaired leading to an accumulation of newly synthesized proteins in the Golgi apparatus and the rough endoplasmic reticulum. Furthermore, the membrane attachment of matrix proteoglycans was diminished. After cholesterol depletion or inhibition of glycosphingolipid synthesis by fumonisin B1, the formation of zymogen granules as well as the formation of detergent-insoluble complexes was reduced. In addition, cholesterol depletion led to constitutive secretion of newly synthesized proteins, e.g. amylase, indicating that zymogens were missorted. Together, these data provide first evidence that in polarized acinar cells of the exocrine pancreas GPI-anchored proteins, e.g. GP-2, and cholesterol-sphingolipid-enriched microdomains are required for granule formation as well as for regulated secretion of zymogens and may function as sorting platforms for secretory proteins destined for apical delivery.     

CONDENSING VACUOLE CONVERSION AND ZYMOGEN GRANULE DISCHARGE IN PANCREATIC EXOCRINE CELLS: METABOLIC STUDIES

We have examined, in the pancreatic exocrine cell, the metabolic requirements for the conversion of condensing vacuoles into zymogen granules and for the discharge of the contents of zymogen granules. To study condensing vacuole conversion, we pulse labeled guinea pig pancreatic slices for 4 min with leucine-³H and incubated them in chase medium for 20 min to allow labeled proteins to reach condensing vacuoles. Glycolytic and respiratory inhibitors were then added and incubation continued for 60 min to enable labeled proteins to reach granules in control slices. Electron microscope radioautography of cells or of zymogen granule pellets from treated slices showed that a large proportion of prelabeled condensing vacuoles underwent conversion in the presence of the combined inhibitors. Osmotic fragility studies on zymogen granule suspensions suggest that condensation may result from the aggregation of secretory proteins in an osmotically inactive form. Discharge was studied using an in vitro radioassay based on the finding that prelabeled zymogen granules can be induced to release their labeled contents to the incubation medium by carbamylcholine or pancreozymin. Induced discharge is not affected if protein synthesis is blocked by cycloheximide for up to 2 hr, but is strictly dependent on respiration. The data indicate that transport and discharge do not require the pari passu synthesis of secretory or nonsecretory proteins (e.g. membrane proteins), suggesting that the cell may reutilize its membranes during the secretory process. The energy requirements for zymogen discharge may be related to the fusion-fission of the granule membrane with the apical plasmalemma.     

Loss of the zymogen granule protein syncollin affects pancreatic protein synthesis and transport but not secretion

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.     

The secretory granule protein syncollin localizes to HL-60 cells and neutrophils.

The secretory granule protein syncollin was first identified in the exocrine pancreas where a population of the protein is associated with the luminal surface of the zymogen granule membrane. In this study we provide first morphological and biochemical evidence that, in addition to its pancreatic localization, syncollin is also present in neutrophilic granulocytes of rat and human origin. By immunohistological studies, syncollin was detected in neutrophilic granulocytes of the spleen. Furthermore, syncollin is expressed by the promyelocytic HL-60 cells, where it is stored in azurophilic granules and in a vesicular compartment. These findings were confirmed by fractionation experiments and immunoelectron microscopy. Treatment with a phorbol ester triggered the release of syncollin indicating that in HL-60 cells it is a secretory protein that can be mobilized upon stimulation. A putative role for syncollin in host defense is discussed.     

Large-scale purification of calf pancreatic zymogen granule membranes.

A protocol for isolating milligram quantities of highly purified zymogen granule membranes from calf pancreas was developed. The method provides a fivefold enriched zymogen granule fraction that is virtually free from major isodense contaminants, such as mitochondria and erythrocytes. Isolated granules are osmotically stable in isosmotic KCl buffers with half-lives between 90 and 120 min. They display specific ion permeabilities that can be demonstrated using ionophore probes to override intrinsic control mechanisms. A Cl- conductance, a Cl-/anion exchanger, and a K+ conductance are found in the zymogen granule membrane, as previously reported for rat pancreatic, rat parotid zymogen granules, and rabbit pepsinogen granules. Lysis of calf pancreatic secretory granules in hypotonic buffers and subsequent isolation of pure zymogen granule membranes yield about 5-10 mg membrane protein from approximately 1000 ml pancreas homogenate. The purified zymogen granule membranes are a putative candidate for the rapid identification and purification of epithelial Cl- channels and regulatory proteins, since they contain fewer proteins than plasma membranes.     

Analysis of ZAPs,Zymogen Granule Membrane Associated Proteins,in the Regulated Exocytosis of the Pancreas

To characterize molecules involved in the intracellular sorting and regulated exocytosis of digestive enzymes in the pancreas, proteins that are specifícially associated with the zymogen granule membranes were analyzed. Zymogen granules, the major secretory organelles in the pancreas, were highly purified. SDS-PAGE analysis found at least 7 protein components in the zymogen granule membranes including ZAP (zymogen granule membrane associated protein) 75, 54, 47, 36, 32, 29, 25 (numbers refer to their apparent kDas). ZAP75 is identical to the glycophosphatidylinositol (GPI)-anchored protein, GP2. Partial amino acid sequencing of ZAP47 and ZAP36 found similarities to a preprocarboxypeptidase B and annexins, respectively. The method we used was a useful tool for structural analysis of the members of ZAPs.