Dynamics of pancreatic tissue cells in the rat exposed to long-term caerulein treatment: 1. Biochemical, morphological and morphometrical evaluations

This study was undertaken to evaluate whether hypertrophy and hyperplasia of the pancreatic acinar cells induced by caerulein remained after termination of the hormonal treatment. Rats received subcutaneous injections of saline or caerulein for 4 days and were killed immediately after termination of treatment or 2, 15 and 50 days later. Caerulein treatment induced significant increases in pancreatic weight and contents of DNA, RNA, protein, amylase and chymotrypsinogen along with an increased number of acinar cells per acinus and zymogen granules per acinar cells. During the post-treatment period, the caerulein-treated pancreas reverted to control values for their contents in proteins, enzymes and RNA and number of zymogen granules per acinar cell while the number of pancreatic cells remained constant as indicated by the absence of modification in total DNA content and acinar cells per acinus. During that same period, saline-treated pancreas exhibited constant growth. These morphological and biochemical data indicate that the already present and newly formed acinar cells can remain in place once the trophic stimulus is withdrawn and that they can adjust their cellular components and thus their digestive capacity to the circulating levels of endogenous cholecystokinin released in response to normal meals.     

RAPID RELEASE OF THE ZYMOGEN GRANULE PROTEIN BY OSMIUM TETROXIDE AND ITS RETENTION DURING FIXATION BY GLUTARALDEHYDE

Fixation by osmium tetroxide and glutaraldehyde of zymogen granules isolated from rat parotid and pancreas was investigated. Protein determinations showed that osmium tetroxide caused rapid release of most of the soluble protein of the granule during fixation in buffered isotonic sucrose. Such granules when examined in the electron microscope after shadow casting appeared quite flat, indicating that most of the contents had indeed been removed. Numerous damaged membranes of the granules were also observed. In contrast, zymogen granules fixed by glutaraldehyde and shadow cast essentially retained the spherical shape and the protein contents. The application of the shadow-casting technique in quantitative studies on the protein content of zymogen granules is discussed.     

FINE STRUCTURAL CHANGES IN RESPONSE TO HORMONAL STIMULATION OF THE PERFUSED CANINE PANCREAS

The dog pancreas isolated in situ was perfused with oxygenated dog blood and stimulated with pancreozymin, secretin, or both. There were no significant changes in the fine structure of the acinar, centroacinar, or duct cells attributable to the perfusion. Combined glutaraldehyde and osmium fixation gave good preservation of the secretory products of the acinar cell. Before stimulation, the lumen of the acini is filled with material similar in texture to the content of the zymogen granules, but of somewhat lower density. Release of secretion commonly takes place by coalescence of the limiting membrane of zymogen granules with the plasmalemma, but one granule opening at the surface may frequently be joined by others coalescing with its membrane and forming an interconnected series all with contents having the same texture as the released zymogen. Such a mechanism seems to permit a more rapid release of secretory product than discharge of individual granules. Pancreozymin stimulation caused marked depletion of zymogen granules, but no obvious changes in the Golgi apparatus. It is clear, therefore, that this hormone exerts its effect upon release of granules rather than upon their formation. Secretin stimulation of water and bicarbonate secretion caused a marked washing out of the luminal contents, but had little detectable effect on cellular structure.     

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.     

SULFATE METABOLISM IN PANCREATIC ACINAR CELLS

The metabolism of inorganic sulfate in pancreatic acinar cells was studied by electron microscope radioautography in mice injected with sulfate-³⁵S. Labeled sulfate was concentrated in the Golgi complex at 10 min. Within 30 min, much of the radioactive material had been transferred to condensing vacuoles. These were subsequently transformed into zymogen granules. By 4 hr after injection, some of the zymogen granules with radioactive contents were undergoing secretion, and labeled material was present in the pancreatic duct system. The Golgi complex in pancreatic acinar cells is known to be responsible for concentrating and packaging digestive enzymes delivered to it from the endoplasmic reticulum. Our work demonstrates that the Golgi complex in these cells is also engaged in the manufacture of sulfated materials, probably sulfated mucopolysaccharides, which are packaged along with the enzymes in zymogen granules and released with them into the pancreatic secretion.     

A cytochemical study on the pancreas of the guinea pig. I. Isolation and enzymatic activities of cell fractions

Pancreatic tissue, (guinea pig) homogenized in 0.88 M sucrose, was fractionated by differential centrifugation into a nuclear, zymogen, mitochondrial, microsomal, and final supernatant fraction. The components of the particulate fractions were identified with well known intracellular structures by electron microscopy. The fractions were analyzed for protein-N and RNA, and were assayed for RNase and trypsin-activatable proteolytic (TAPase) activity. The zymogen fraction accounted for 30 to 40 per cent of the total TAPase and RNase activities, and its specific enzymatic activities were 4 to 10 times higher than those of any other cell fraction. The zymogen fraction was cytologically heterogeneous; zymogen granules and mitochondria represented its main components. More homogeneous zymogen fractions, obtained by successive washing or by separation in a discontinuous density-gradient, had specific activities 2 to 4 times greater than the crude zymogen fractions. Chymotrypsinogen was isolated by column chromatography from pancreas homogenates and derived cell fractions. The largest amount was recovered in the zymogen fraction. The final supernatant had properties similar to those of the trypsin inhibitor described by Kunitz and Northrop.     

Regulated secretion of mature cathepsin B from rat exocrine pancreatic cells.

By indirect immunofluorescence and immunogold electron microscopy with an antibody that recognizes specifically the two forms of native mature rat cathepsin B (31 kDa and 5:25 kDa) but not the proenzyme, we detected cathepsin B not only in lysosomes of adult rat exocrine pancreatic cells but also in the trans Golgi condensing vacuoles, the zymogen granules and the pancreatic juice in the intralobular ducts. In contrast, immunocytochemistry with an antibody specific for rat cathepsin D showed the latter to be present in the same cells only in lysosomal compartments as expected. The same pattern of labeling with these two antibodies was found in the first zymogen granules to form in 17-day-old fetal rat pancreas. Counts of the extent of immunogold labeling of cathepsin B in the adult exocrine cells showed that the concentration of the enzyme was only two-fold higher in the lysosomal compartments than in the zymogen granules. To confirm these observations, rat pancreatic postnuclear supernatant (PNS), a fraction enriched in zymogen granules and rat pancreatic juice obtained by catheterization of the pancreatic duct, were subjected to 2D gel electrophoresis followed by immunoblotting with the cathepsin B antibody. All three samples contained a 31 kDa protein recognized by the antibody with a pI of about 4.5, the single chain mature form of cathepsin B. We then radiolabeled pancreatic PNS and zymogen granule fractions with benzyloxycarbonyl-Tyr[125I]-Ala-CHN2, an affinity label that covalently binds to the active sites of mature forms of both cathepsin B and cathepsin L. In both PNS and zymogen granule fractions this reagent labeled cathepsin B. Immunoprecipitation experiments showed that the antibody to cathepsin B recognized specifically both the single chain and the double chain mature forms of cathepsin B in the native state. Finally, Northern blots with a cDNA of rat cathepsin B showed that the concentration of cathepsin B mRNA in total pancreatic RNA increased following in vivo stimulation of the exocrine pancreatic cells with optimal doses of cerulein, a cholecystokinin analogue. We conclude that significant amounts of mature cathepsin B are secreted from exocrine pancreatic cells via the apical regulated exocytotic pathway, and we discuss this in terms of models for sorting of proteins to the cores of dense cored secretory granules.     

Selective fluorescence of zymogen granules of pancreatic acinar cells stained with hematoxylin and eosin

Following staining with hematoxylin and eosin Y, paraffin sections of mouse pancreas were examined by transmitted light, epifluorescence and confocal laser scanning microscopy. Light microscopy revealed that the nuclei of pancreatic acinar cells were located basally, while the apices of the cells appeared eosinophilic, although the secretory granules were difficult to visualize. Under violet-blue light excitation, the zymogen granules at the apices of the acinar cells showed strong yellowish fluorescence; the other part of the cytoplasm was only faintly fluorescent and the nuclei and the supporting tissues were nonfluorescent. Confocal laser scanning microscopy resulted in clear pictures of the zymogen granules and their distribution within the cell. The fluorescent emission of zymogen granules was certainly the result of eosin Y staining, because hematoxylin is not a fluorochrome and the zymogen granules are not autofluorescent. Staining with eosin Y alone, however, did not result in clear fluorescent images of zymogen granules or any other cellular structures. Our observation shows that the fluorescence emission of eosin Y allows easy and precise recognition of zymogen granules of pancreatic cells.     

A Cytochemical Study on the Pancreas of the Guinea Pig : I. Isolation and Enzymatic Activities of Cell Fractions

Pancreatic tissue, (guinea pig) homogenized in 0.88 M sucrose, was fractionated by differential centrifugation into a nuclear, zymogen, mitochondrial, microsomal, and final supernatant fraction. The components of the particulate fractions were identified with well known intracellular structures by electron microscopy. The fractions were analyzed for protein-N and RNA, and were assayed for RNase and trypsin-activatable proteolytic (TAPase) activity. The zymogen fraction accounted for 30 to 40 per cent of the total TAPase and RNase activities, and its specific enzymatic activities were 4 to 10 times higher than those of any other cell fraction. The zymogen fraction was cytologically heterogeneous; zymogen granules and mitochondria represented its main components. More homogeneous zymogen fractions, obtained by successive washing or by separation in a discontinuous density-gradient, had specific activities 2 to 4 times greater than the crude zymogen fractions. Chymotrypsinogen was isolated by column chromatography from pancreas homogenates and derived cell fractions. The largest amount was recovered in the zymogen fraction. The final supernatant had properties similar to those of the trypsin inhibitor described by Kunitz and Northrop.     

Selective fluorescence of zymogen granules of pancreatic acinar cells stained with hematoxylin and eosin.

Following staining with hematoxylin and eosin Y, paraffin sections of mouse pancreas were examined by transmitted light, epifluorescence and confocal laser scanning microscopy. Light microscopy revealed that the nuclei of pancreatic acinar cells were located basally, while the apices of the cells appeared eosinophilic, although the secretory granules were difficult to visualize. Under violet-blue light excitation, the zymogen granules at the apices of the acinar cells showed strong yellowish fluorescence; the other part of the cytoplasm was only faintly fluorescent and the nuclei and the supporting tissues were nonfluorescent. Confocal laser scanning microscopy resulted in clear pictures of the zymogen granules and their distribution within the cell. The fluorescent emission of zymogen granules was certainly the result of eosin Y staining, because hematoxylin is not a fluorochrome and the zymogen granules are not autofluorescent. Staining with eosin Y alone, however, did not result in clear fluorescent images of zymogen granules or any other cellular structures. Our observation shows that the fluorescence emission of eosin Y allows easy and precise recognition of zymogen granules of pancreatic cells.     

Selective fluorescence of zymogen granules of pancreatic acinar cells stained with hematoxylin and eosin

Following staining with hematoxylin and eosin Y, paraffin sections of mouse pancreas were examined by transmitted light, epifluorescence and confocal laser scanning microscopy. Light microscopy revealed that the nuclei of pancreatic acinar cells were located basally, while the apices of the cells appeared eosinophilic, although the secretory granules were difficult to visualize. Under violet-blue light excitation, the zymogen granules at the apices of the acinar cells showed strong yellowish fluorescence; the other part of the cytoplasm was only faintly fluorescent and the nuclei and the supporting tissues were nonfluorescent. Confocal laser scanning microscopy resulted in clear pictures of the zymogen granules and their distribution within the cell. The fluorescent emission of zymogen granules was certainly the result of eosin Y staining, because hematoxylin is not a fluorochrome and the zymogen granules are not autofluorescent. Staining with eosin Y alone, however, did not result in clear fluorescent images of zymogen granules or any other cellular structures. Our observation shows that the fluorescence emission of eosin Y allows easy and precise recognition of zymogen granules of pancreatic cells.     

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.     

An assessment of the role of protein kinase and zymogen granule phosphorylation during secretion by the rat exocrine pancreas.

1. The levels of protein kinase activity and zymogen granule phosphorylation were studied in the adult rat during stimulus-coupled secretion in vitro. 2. The specific activity of protein kinase associated with intact zymogen granules was 11 pmol [32P]phosphate transferred to histone per min per mg protein. Most of this activity was recovered in purified granule membranes. 2. The addition of 10(-6) M cyclic AMP to a mixture of zymogen granules and the postmicrosomal supernatant resulted in a 5-fold increase in protein kinase activity associated with zymogen granules. The adsorbed activity was eluted from granules by 0.15 M NaCl. Cyclic GMP did not promote protein kinase binding to isolated granules. 4. Incubation of tissues with carbachol (10(-5) M), pancreozymin (0.1 unit/ml), caerulein (10(-8) M) or dibutyryl cyclic AMP (2.10(-4) M) between 2.5 and 60 min did not increase the levels of protein kinase activity in isolated zymogen granules above control values. 5. Protein phosphorylation of zymogen granule membranes and granule content was not detectable in tissues incubated with carbachol, pancreozymin-C-octapeptide, or caerulein. 6. These results suggest that neither the phosphorylation of zymogen granule membrane protein nor the adsorption of protein kinase activity to zymogen granules is an obligatory step in secretion.     

Secretory apparatus assessed by analysis of pancreatic secretory stress protein expression in a rat model of chronic pancreatitis

Secretory stress proteins (SSP) are a family of proteins including isoforms of pancreatitis-associated protein (PAP) and pancreatic stone protein (PSP/reg). In vitro exposure to trypsin results in the formation of insoluble fibrillar structures. SSP are constitutively secreted into pancreatic juice at low levels. The WBN/Kob rat is a model for chronic pancreatitis, displaying focal inflammation, destruction of the parenchyma and changes in the architecture of the acinar cell; the synthesis and secretion of SSP are also increased. We have investigated the secretory apparatus by SSP immunohistochemistry at the light- and electron-microscopical (EM) levels. Immunocytochemistry of PSP/reg in Wistar control rats reveals low levels, with individual acinar cells exhibiting high immunoreactivity in zymogen granules. PAP is not detectable. In the WBN/Kob rat, PSP/reg and PAP immunoreactivity is markedly increased. Double immunofluorescence for PSP/reg and PAP I or II demonstrates that these proteins colocalize to the same cell. Acinar cells change their secretory architecture by fusion of zymogen granules and elongation of the fused organelles. The immunogold technique has demonstrated an increase of SSP in zymogen granules in WBN/Kob rats. PSP/reg-positive zymogen granules fuse to form elongated structures with fibrillar contents. An extensive PSP/reg-positive fibrillar network is established in the cytosol. Extracellular fibrils have been observed in several ductules. Thus, SSP-derived fibrils form concomitantly with acinar damage in the WBN/Kob rat. Based on the known tryptic cleavage site of SSP, the in vivo generation of fibrils is presumably the result of premature trypsin activation.     

Glycoprotein synthesis in the adult rat pancreas. IV. Subcellular distribution of membrane glycoproteins

Zymogen granule membranes from the rat exocrine pancreas displays distinctive, simple protein and glycoprotein compositions when compared to other intracellular membranes. The carbohydrate content of zymogen granule membrane protein was 5-10-fold greater than that of membrane fractions isolated from smooth and rough microsomes, mitochondria and a preparation containing plasma membranes, and 50-100-fold greater than the zymogen granule content and the postmicrosomal supernate. The granule membrane glycoprotein contained primarily sialic acid, fucose, mannose, galactose and N-acetylglucosamine. The levels of galactose, fucose and sialic acid increased in membranes in the following order: rough microsomes less than smooth microsomes less than zymogen granules. Membrane polypeptides were analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The profile of zymogen granule membrane polypeptides was characterized by GP-2, a species with an apparent molecular weight of 74 000. Radioactivity profiles of membranes labeled with [3H]glucosamine or [3H]leucine, as well as periodic acid-Schiff stain profiles, indicated that GP-2 accounted for approx. 40% of the firmly bound granule membrane protein. Low levels of a species similar to GP-2 were detected in membranes of smooth microsomes and the preparation enriched in plasma membranes but not in other subcellular fractions. These results suggest that GP-2 is a biochemical marker for zymogen granules. Membrane glycoproteins of intact zymogen granules were resistant to neuraminidase treatment, while those in isolated granule membranes were readily degraded by neuraminidase. GP-2 of intact granules was not labeled by exposure to galactose oxidase followed by reduction with NaB3H4. In contrast, GP-2 in purified granule membranes was readily labeled by this procedure. Therefore GP-2 appears to be located on the zymogen granule interior.     

Concanavalin a binding and cytodifferentiation in pancreatic acinar carcinoma of rat

The binding of concanavalin A to the plasmalemma of acinar carcinoma cells was characterized by electron microscopy utilizing horseradish peroxidase. Heavy labeling due to specific concanavalin A binding was detected on the plasmalemma of undifferentiated carcinoma cells lacking zymogen maturation, neoplastic cells of intermediate differentiation with only occasional zymogen granules, and highly differentiated acinar carcinoma cells containing numerous cytoplasmic zymogen granules. The plasmalemma of acinar carcinoma cells was also compared to the normal pancreatic acinar cell plasmalemma by measurement of specific ¹²⁵I-labeled concanavalin A binding. Although only about one-third of pancreatic acinar carcinoma cells demonstrate mature zymogen differentiation, the acinar carcinoma had a full complement of normal plasmalemma receptors for ¹²⁵I-labeled concanavalin A. It is concluded that, unlike normal pancreas, the presence of concanavalin A receptors on the plasmalemma of acinar carcinoma cells is not a specific membrane marker for differentiated cells containing zymogen granules.     

A combined stain for identifying epithelial cells of the gastric mucosa

New techniques are proposed for differentiating each type of gastric epithelial cell in the same tissue section. The techniques combine the following stains: paradoxical concanavalin A staining (PCS) to identify mucous neck cells, periodic acid Schiff-concanavalin A staining to distinguish mucous neck cells from surface mucous cells, and a modified Bowie's stain to demonstrate zymogen granules of chief cells. Feulgen hydrolysis preceding the Bowie stain was found to remove most of the nonspecific coloration encountered with the original Bowie method. The results obtained by the new sequences were as follows: Feulgen hydrolysis-PCS-Bowie staining: mucous neck cells stained brown and chief cell zymogen granules deep blue. The other mucin-secreting cells remained unstained; Feulgen hydrolysis-PAS-concanavalin A-Bowie staining: mucous neck cells stained brown, zymogen granules stained deep blue to purplish blue and surface mucous cells stained purplish red.     

Oxidative injury to isolated rat pancreatic acinar cells vs. isolated zymogen granules

This study compares the susceptibility of pancreatic acinar cells and zymogen granules against oxidative injury and analyzes the mechanisms involved. Zymogen granules and acinar cells, isolated from rat pancreas, were exposed to a reaction mixture containing xanthine oxidase, hypoxanthine, and chelated iron. Cell function and viability were assessed by various techniques. Trypsin activation was quantified by an Elisa for trypsinogen activating peptide. Integrity of granules was determined by release of amylase. The reaction mixture rapidly generated radicals as assessed by deoxyribose and luminol assays. This oxidative stress caused lysis of granules in a matter of minutes but significant cell death only after some hours. Nevertheless, radicals initiated intracellular vacuolization, morphological damage to zymogen granules and mitochondria, increase in trypsinogen activating peptide, and decrease in ATP already after 5–30 min. Supramaximal caerulein concentrations also caused rapid trypsin activation. Addition of cells but not of granules reduced deoxyribose oxidation, suggesting that intact cells act as scavengers. Caerulein pretreatment only slightly increased the susceptibility of cells but markedly that of granules. In conclusion, isolated zymogen granules are markedly more susceptible to oxidative injury than intact acinar cells, in particular, in early stages of caerulein pancreatitis. The results show that oxidative stress causes a rapid trypsin activation that may contribute to cell damage by triggering autodigestion. Zymogen granules and mitochondria appear to be important targets of oxidative damage inside acinar cells. The series of intracellular events initiated by oxidative stress was similar to changes seen in early stages of pancreatitis.     

A morphometric study of 24-hour variations in subcellular structures of the rat pancreatic acinar cell

Summary Subcellular structures of pancreatic acinar cells were examined at six evenly spaced time points in the 24-h period (light cycle: 06.00 h–18.00 h) in four Wistar male rats at each time point. At each sampling point, the area and circumference of acinar cell bodies and the area, number and circumference of their cytoplasmic organelles were measured using a semiautomatic computer system for morphometry and a point-counting method.The area, number and circumference-area ratio of the cytoplasmic organelles were subject to strong circadian variations, and the cellular area and circumference exhibited weak circadian variations. Variation pattern of the cytoplasmic organelles suggested an intracellular route for secretory proteins during a 24-h span. From the results it was possible to divide the 24-h period into three stages. 1. The resting or protein synthetic stage (00.00 h to 08.00h): the area of the rough surfaced endoplasmic reticulum (rER) was strongly increased, and that of zymogen granules was clearly decreased. 2. The granule accumulation stage (08.00h to 16.00h): the area of the rER was markedly decreased; that of zymogen granules was increased. 3. The secretion stage (16.00 h to 00.00): as a result of the release of zymogen granules from the acinar cell, the area of zymogen granules decreased, and that of the rER increased. The relationship between the area of the rER and zymogen granules varied in a reciprocal manner. Other cytoplasmic organelles, namely the Golgi complex, condensing vacuoles, mitochondria and lysosomes also varied prominently during the 24-h span, corresponding to variations in the rER and zymogen granules.     

Glycoprotein synthesis in the adult rat pancreas: IV. Subcellular distribution of membrane glycoproteins

Zymogen granule membranes from the rat exocrine pancreas displays distinctive, simple protein and glycoprotein compositions when compared to other intracellular membranes. The carbohydrate content of zymogen granule membrane protein was 5–10-fold greater than that of membrane fractions isolated from smooth and rough microsomes, mitochondria and a preparation containing plasma membranes, and 50–100-fold greater than the zymogen granule content and the postmicrosomal supernate. The granule membrane glycoprotein contained primarily sialic acid, fucose, mannose, galactose and $\text{N-}\text{acetylglucosamine}$. The levels of galactose, fucose and sialic acid increased in membranes in the following order: rough microsomes < smooth microsomes < zymogen granules.Membrane polypeptides were analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The profile of zymogen granule membrane polypeptide was characterized by GP-2, a species with an apparent molecular weight of 74 000. Radioactivity profiles of membranes labeled with [³H]glucosamine or [³H]leucine, as well as periodic acid-Schiff stain profiles, indicated that GP-2 accounted for approx. 40% of the firmly bound granule membrane protein. Low levels of a species similar to GP-2 were detected in membranes of smooth microsomes and the preparation enriched in plasma membranes but not in other subcellular fractions. These results suggest that GP-2 is a biochemical marker for zymogen granules.Membrane glycoproteins of intact zymogen granules were resistant to neuraminidase treatment, while those in isolated granule membranes were readily degraded by neuraminidase. GP-2 of intact granules was not labeled by exposure to galactose oxidase followed by reduction with NaB³H₄. In contrast, GP-2 in purified granule membranes was readily labeled by this procedure. Therefore GP-2 appears to be located on the zymogen granule interior.