Purified Zymogen Granules Research Articles

ZnT1 (SLC30A1) and ZnT2 (SLC30A2) regulate zinc efflux through two different pathways in pancreatic acinar cells

Pancreatic acinar cells play an important role in zinc excretion. We have found that dietary Zn intake regulates the expression of ZnT1 and ZnT2 in the pancreas. The mechanisms with which ZnT1 and ZnT2 mediate Zn efflux in pancreatic acinar cells have not been characterized. Here, we report Zn deficiency caused a significantly lowered Zn concentration in both the cytoplasm and zymogen granule compartments of acinar cells. Western blot analysis also showed a decrease in the association of both ZnT1 and ZnT2 with the purified zymogen granule membrane fraction. In addition, in pancreatic AR42J cells, ZnT1 was found to be tightly regulated by intracellular Zn levels. In contrast, ZnT2 expression was upregulated by dexamethasone, which induces cell differentiation, and increases both secretory organelle development and secretion. Both ZnT1 and ZnT2 were down-regulated in Zn deficient cell cultures, obtained through the use of Zn chelating reagents, TPEN, DTPA, or dialysis against dipicolinic acid. In ZnT1 over-expressing transiently transfected AR42J cells, a lower intracellular Zn level and higher Zn efflux rate was found through 65Zn loss. Transient transfection of ZnT2 and over-expression resulted in more sequestered intracellular Zn, with a normal 65Zn efflux rate. In conclusion, ZnT1 and ZnT2 regulate Zn output through two different pathways in pancreatic acinar cells. Supported by NIH Grant DK 31127.

Expression of the two‐pore domain potassium channel, TASK‐2, in rat pancreatic acinar cell zymogen granule membrane

Ion flux across pancreatic secretory vesicle membranes contributes to exocytotic secretion: mediating vesicular swelling involved in “kiss and run” exocytosis, promoting solubilization and release of vesicle content, and contributing to intravesicular pH changes. The present study implicates the pH sensitive TASK‐2 channel in one or more of these processes: TASK‐2 expression was documented and localized to zymogen granule membranes and secretion shown to be modulated by TASK‐2 selective drugs. TASK‐2 mRNA expression was demonstrated using SYBR Green real time PCR. Total RNA was isolated from purified acinar cells obtained by Percoll density gradient centrifugation of collagenase‐dispersed cells. Reverse transcription was performed on 1 μg of DNase‐treated total RNA using random decamer primers. TASK‐2‐specific forward and reverse primers amplified a single product from the cDNA based on dissociation curve analysis. Western blot analysis using an anti‐TASK‐2 antibody (Alomone) revealed two immunoreactive protein bands in a crude microsomal fraction migrating as a doublet at 54 and 55 kD (the predicted mass of rTASK‐2 is 58 kD); identical protein bands were obtained in Western blots of purified zymogen granule membranes. Confocal microscopy revealed that immunoreactivity was cytoplasmic and primarily localized to the apical pole. Finally, CCK‐induced amylase secretion from diced pancreatic tissue fragments was increased 100% by the TASK‐2 agonist, halothane, without affecting basal release and completely inhibited by the TASK‐2 inhibitor, clofilium, without affecting basal secretion.

Subcellular localization of fumarase in mammalian cells and tissues

Fumarase, a mitochondrial matrix protein, is previously indicated to be present in substantial amounts in the cytosol as well. However, recent studies show that newly synthesized human fumarase is efficiently imported into mitochondria with no detectable amount in the cytosol. To clarify its subcellular localization, the subcellular distribution of fumarase in mammalian cells/tissues was examined by a number of different methods. Cell fractionation using either a mitochondria fraction kit or extraction with low concentrations of digitonin, detected no fumarase in a 100,000 g supernatant fraction. Immunofluorescence labeling with an affinity-purified antibody to fumarase and an antibody to the mitochondrial Hsp60 protein showed identical labeling pattern with labeling seen mainly in mitochondria. Detailed studies were performed using high-resolution immunogold electron microscopy to determine the subcellular localization of fumarase in rat tissues, embedded in LR White resin. In thin sections from kidney, liver, heart, adrenal gland and anterior pituitary, strong and specific labeling due to fumarase antibody was only detected in mitochondria. However, in the pancreatic acinar cells, in addition to mitochondria, highly significant labeling was also observed in the zymogen granules and endoplasmic reticulum. The observed labeling in all cases was completely abolished upon omission of the primary antibody indicating that it was specific. In a western blot of purified zymogen granules, a fumarase-antibody cross-reactive protein of the same molecular mass as seen in the mitochondria was present. These results provide evidence that fumarase in mammalian cells/tissues is mainly localized in mitochondria and significant amounts of this protein are not present in the cytosol. However, these studies also reveal that in certain tissues, in addition to mitochondria, this protein is also present at specific extramitochondrial sites. Although the cellular function of fumarase at these extramitochondrial locations is not known, the appearance/localization of fumarase outside mitochondria may help explain how mutations in this mitochondrial protein can give rise to a number of different types of cancers.

Exocrine pancreatic secretion of phospholipid, menaquinone-4, and caveolin-1 in vivo

The exocrine pancreas releases secretory products essential for nutrient assimilation. In addition to digestive enzymes, the release of lipoprotein-like particles containing the membrane trafficking protein caveolin-1 from isolated pancreatic explants has been reported. The present study examined: (1) if gastrointestinal hormones induce the apical secretion of phospholipid in vivo and (2) a potential association of caveolin-1 and the lipid-soluble vitamin K analog menaquinone-4 (MK-4) with these structures. Analysis of isolated acinar cells, purified zymogen granules, and pancreatic juice collected in vivo indicated the presence a caveolin-1 immunoreactive protein that was acutely released in response hormone stimulation. Chloroform-extracted fractions of pancreatic juice also contained high concentrations of MK-4 that was secreted in parallel to protein and phospholipid. The presence of caveolin-1 and MK-4 in the phospholipid fraction of pancreatic juice places these molecules in the secretory pathway of exocrine cells and suggests a physiological role in digestive enzyme synthesis and/or processing.

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

Two dominant negative mutants of Rab3D, N135I and T36N were recently reported to inhibit the early phase of regulated amylase secretion from mouse pancreatic acini (Chen, X., Edwards, J. A., Logsdon, C. D., Ernst, S. A., and Williams, J. A. (2002) J. Biol. Chem. 277, 18002-18009). Immunocytochemical studies showed that while the wild-type Rab3D localized to zymogen granules, the two dominant negative mutants did not localize to granules and were primarily in the basolateral regions of the cell. The present study, therefore, evaluated the potential mechanisms by which the dominant negative mutants might act. An affinity precipitation assay based on the property of the Rab3 effector Rim1 to interact only with GTP-bound Rab3D was developed. 78.9 +/- 4.5% of total endogenous Rab3D was found in the GTP-bound form. Overexpression of HA-tagged Rab3D, and its Q81L, N135I, and T36N mutants had no effect on the total amount of endogenous Rab3D. However, the dominant negative mutants, T36N and N135I, reduced GTP-bound endogenous Rab3D by 70.0 +/- 3.5% and 72.7 +/- 1.2%, respectively, while the wild-type Rab3D and Q81L mutant had no effect. Triton X-114 phase separation and cell fractionation studies showed that dominant negative Rab3D mutants did not alter isoprenylation or membrane association of endogenous Rab3D. The dominant negative Rab3D did not affect the amount of endogenous Rab3D on purified zymogen granules as assessed by either Western blotting or immunocytochemistry, but reduced the GTP-bound form by 78.6 +/- 3.3%. The two dominant negative Rab3D mutants, therefore, interfere with endogenous Rab3D function by blocking the GDP/GTP exchange but not zymogen granule targeting of endogenous Rab3D.

Localization of mitochondrial 60-kD heat shock chaperonin protein (Hsp60) in pituitary growth hormone secretory granules and pancreatic zymogen granules.

We used quantitative immunogold electron microscopy and biochemical analysis to evaluate the subcellular distribution of Hsp60 in rat tissues. Western blot analysis, employing both monoclonal and polyclonal antibodies raised against mammalian Hsp60, shows that only a single 60-kD protein is reactive with the antibodies in brain, heart, kidney, liver, pancreas, pituitary, spleen, skeletal muscle, and adrenal gland. Immunogold labeling of tissues embedded in the acrylic resin LR Gold shows strong labeling of mitochondria in all tissues. However, in the anterior pitutary and in pancreatic acinar cells, Hsp60 also localizes in secretory granules. The labeled granules in the pituitary and pancreas were determined to be growth hormone granules and zymogen granules, respectively, using antibodies to growth hormone and carboxypeptidase A. Immunogold labeling of Hsp60 in all compartments was prevented by preadsorption of the antibodies with recombinant Hsp60. Biochemically purified zymogen granules free of mitochondrial contamination are shown by Western blot analysis to contain Hsp60, confirming the morphological localization results in pancreatic acinar cells. In kidney distal tubule cells, low Hsp60 reactivity is associated with infoldings of the basal plasma membrane. In comparison, the plasma membrane in kidney proximal tubule cells and in other tissues examined showed only background labeling. These findings raise interesting questions concerning translocation mechanisms and the cellular roles of Hsp60.

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.

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.

Cytoplasmic dynein and dynactin as likely candidates for microtubule-dependent apical targeting of pancreatic zymogen granules

The critical role of microtubules in vectorial delivery of post-Golgi carrier vesicles to the apical cell surface has been established for various polarized epithelial cell types. In the present study we used secretory granules of the rat and chicken pancreas, termed zymogen granules, as model system for apically bound post-Golgi carrier vesicles that underlie the regulated exocytotic pathway. We found that targeting of zymogen granules to the apical cell surface requires an intact microtubule system which contains its colchicine-resistant organizing center and, thus, the microtubular minus ends close to the apical membrane domain. Purified zymogen granules and their membranes were found to be associated with cytoplasmic dynein intermediate and heavy chain and to contain the major components of the dynein activator complex, dynactin, i.e. p150Glued, p62, p50, Arp1, and beta-actin. Kinesin heavy chain and the kinesin receptor, 160 kD kinectin, were not detected as components of zymogen granules. Immunofluorescence staining showed a zymogen granule-like distribution for dynein and dynactin (p150Glued, p62, p50, Arpl) in the apical cytoplasm, whereas kinesin and kinectin were largely concentrated in the basal half of the cells in a pattern similar to the distribution of calreticulin, a component of the endoplasmic reticulum. Secretory granules of non-polarized chromaffin cells of the bovine adrenal medulla, that are assumed to underlie microtubular plus end targeting from the Golgi apparatus to the cell periphery, were not found to be associated with dynein or dynactin. To our knowledge, this is the first demonstration of major components of the dynein-dynactin complex associated with the membrane of a biochemically and functionally well-defined organelle which is considered to underlie a vectorial minus end-driven microtubular transport critically involved in precise delivery of digestive enzymes to the apically located acinar lumen.

Heterotrimeric G-protein Gq/11 Localized on Pancreatic Zymogen Granules Is Involved in Calcium-regulated Amylase Secretion

The heterotrimeric G-protein Gq/11 was identified on pancreatic acinar zymogen granules and its function in calcium-regulated exocytosis was examined. Western blotting showed alphaq/11, but not alphas or alphao, to be localized to the zymogen granule membrane along with G-protein beta-subunit; all three alpha subunits were present in a plasma membrane fraction and the alphaq/11 signal was 30-fold more enriched in the plasma membrane as compared with granule membrane. Neither CCK receptors nor alpha subunits of the sodium pump, both plasma membrane markers were present on granule membranes. Immunohistochemistry of pancreatic lobules showed that alphaq/11 localized to the zymogen granule-rich apical region of acinar cells together with a much stronger signal at the basolateral plasma membrane. When the substance-P-related peptide GPAnt-2a, an antagonist of Gq/11, was introduced into streptolysin-O permeabilized acini to bypass the plasma membrane, the amylase release induced by 10 microM free calcium was potentiated in a concentration-dependent manner. By contrast, another substance-P-related peptide, GPAnt-1, an antagonist of Go and Gi, showed no effect on calcium-induced amylase release from permeabilized acini. GPAnt-2a peptide also exerted an inhibitory effect on the total GTPase activity of the purified zymogen granules and a larger inhibitory effect on the GTPase activity of the Gq/11 protein immunopurified from zymogen granules. GPAnt-1, however, did not inhibit GTPase activity of either zymogen granules or immunopurified Gq/11. These results suggest that GPAnt-2a peptide augmented calcium-induced amylase release from permeabilized acini by inhibiting GTPase activity of the Gq/11 protein on zymogen granules. We conclude that Gq/11 protein on zymogen granules plays a tonic inhibitory role in calcium-regulated amylase secretion from pancreatic acini.

Small GTP-binding protein, Rab6, is associated with secretory granules in atrial myocytes.

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.

Nucleoside phosphatase activities on pig pancreas zymogen granule membranes analyzed by nondenaturing polyacrylamide gel electrophoresis

The membrane of the pancreatic zymogen granule plays an important part in the sequence of storage, transport and exocytosis of digestive enzymes. While much is known on stimulus-secretion coupling, very little is understood about how the storage organelles move in the cytoplasm to the luminal plasma membrane and why and how they fuse with it to release the contents. It is assumed that nucleoside phosphatases are involved in these energy consuming processes. Pancreatic zymogen granule membranes contain one major glycoprotein, GP-2, and a few minor proteins all with unknown functions. In order to identify functions we have purified zymogen granule membranes from pig pancreas, solubilized the proteins under non-denaturing conditions with the detergent CHAPS and characterized the extracted proteins by polyacrylamide gel electrophoresis, histochemistry and lectins. Three major protein bands, often fused in one broad band, revealed enzymatic activity for adenosine-, cytidine-, inositol- and guanidinedi- and triphosphates by the precipitation of liberated phosphate by Pb(NO 3) 2. This activity was sensitive to known ATP diphosphohydrolase inhibitors. The band with activity arises from a 92 kDa glycoprotein. A different narrow band showed monophosphatase activity for AMP, GMP, IMP and CMP. Some of the activities were inhibited by different lectins, indicating glycosyl groups near the active site. Electron microscopical cytochemistry confirmed a nucleoside phosphatase activity on granule membranes. Our results show for the first time that the nucleoside phosphatase activity of the zymogen granule membranes is carried by a 92 kDa glycoprotein, probably the known self-associating form of GP-2. The hydrolysis of tri- and diphosphate nucleotides could provide the energy required by exocytosis.

Identification of Rab 5 But Not Rab 3A in Rat Pancreatic Zymogen Granule Membranes

Low molecular weight GTP-binding (LMWG) proteins are known to be present on secretory organelles and a role for Rab 3A in exocytosis of synaptic vesicles has been postulated. Pancreatic zymogen granules possess multiple LMWG proteins which were shown to be on the outside of the granules by pronase susceptability. Rab 3A, however, could not be identified in rat pancreatic acinar cells or on zymogen granules by immunoblots or by northern blots or PCR amplification although all three techniques demonstrated its presence in rat brain, RINm5F and AR42J cells. Immunoblots revealed expression of Rab 5, Rab 7, Rab 11 and ARF in acini. Of these, Rab 5 was identified in purified zymogen granule membranes where it may be involved in the endocytotic retrieval of granule membrane following exocytosis.

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 ∼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.

Characterization of phospholipase A 2 and acyltransferase activities in purified zymogen granule membranes

Phospholipase A 2 and acyltransferase activities were identified in membranes associated with purified pancreatic zymogen granules. In homogenate and granule membranes, phospholipase activity was linearly related to protein concentration and was Ca 2+-dependent with an alkaline pH optimum. The Ca 2+ sensitivity was observed over the range of concentrations through which intracellular ionic Ca 2+ is elevated by physiological stimuli in intact cells. Intact zymogen granules and granule membranes also demonstrated reacylating activity in the presence and absence of an exogenous acceptor. Reacylating activity was related to the concentration of lyosphospholipid added and was optimally activated at alkaline pH. A more rapid rate of reacylation was observed when [ 14C)] arachidonoyl CoA was employed as the donor molecule rather than [ 3H]arachidonate (plus coenzyme A); this suggests the absence of acyl-CoA synthetase in the purified granule membranes. We conclude that granule membrane phospholipase A 2 and acyltransf erases may be involved in arachidonic acid turnover in exocrine pancreas and perhaps in membrane fusion events associated with exocytosis.

Localization of lysosomal and digestive enzymes in cytoplasmic vacuoles in caerulein-pancreatitis

Intracellular localization and enzymatic activities of lysosomal enzymes (cathepsin B, N-acetyl-beta-glucosaminidase, and beta-glucuronidase) were studied in control rats and after induction of caerulein pancreatitis. In control rats high enzymatic activities were found in the postnuclear 1000 g fraction (purified zymogen granules). The corresponding subcellular fraction in pancreatitis animals additionally contained larger secretory vacuoles and autophagosomes and revealed a marked increase in lysosomal enzyme activities. Immunolabelling studies at the ultrastructural level for trypsinogen and cathepsin B demonstrated a colocalization of lysosomal and digestive enzymes in zymogen granules in healthy controls. After induction of pancreatitis immunolabelling still demonstrated a colocalisation of cathepsin B and trypsinogen in secretory granules and newly formed Golgi-derived secretory vacuoles. Concomitantly appearing autophagosomes were, however, only labelled for cathepsin B. It is concluded that segregation of lysosomal and digestive enzymes is incomplete in normal acinar cells resulting in a colocalization in zymogen granules. In pancreatitis colocalization in secretory granules is maintained, whereas only lysosomal enzymes were sufficiently transferred into autophagic vacuoles. No indication for impaired mechanisms of molecular sorting of lysosomal and digestive enzymes in caerulein-induced pancreatitis was found.

Calcium binding properties of purified zymogen granule membrane of pig pancreas. Evidence for calcium binding proteins

Ca 2+ binding properties of purified zymogen granule membranes of pig pancreas have been measured: Binding increased linearly with Ca 2+ concentration in the medium up to the micromolar range; in the millimolar range a sharp rise in binding capacity was observed. Binding increased with pH both at low and high concentrations of Ca 2+. It was insensitive to Na + and K + ions at concentrations up to 100 mM. Mg 2+ was inhibitory in the millimolar range whereas La 2+ and Tb 3+ were inhibitory in the micromolar range. The Ca 2+ binding components of zymogen granule membranes were identified by two methods: (1) by measuring 45Ca 2+ binding after counter-ion electrophoresis and (2) by Stain's-all (forms a complex with Ca 2+ binding proteins absorbing maximally at 600 nm), after SDS-polyacrylamide gel electrophoresis. The first method, counter-ion electrophoresis, indicated that most of the 45Ca 2+ was associated with an acidic band which could be subsequently subfractionated by SDS-polyacrylamide gel electrophoresis in five bands: 66, 57, 30, 27 and 22.5 kDa. The second method, Stain's-all, revealed six positive polypeptides after SDS-polyacrylamide gel electrophoresis of native zymogen granule membranes' two were unreactive after neuraminidase treatment (130 and 92 kDa, respectively), whereas four other bands were still reactive (66, 57, 43, 30 kDa, respectively.) Ca 2+ binding was also measured on intact zymogen granules: the binding capacity was higher than for zymogen granule membranes. Among the Ca 2+ binding proteins of the zymogen granule membrane only one is apparently located on the granule external surface: the 30 kDa polypeptide. If Ca 2+ directly facilitates fusion of zymogen granules with plasma membrane by a Ca 2+-protein interaction, then this protein is a presumptive candidate to play such a key role.

Protein kinase activity associated with pancreatic zymogen granules.

Purified zymogen granules were prepared from rat pancreas by using an iso-osmotic Percoll gradient. In the presence of [gamma-32P]ATP, phosphorylation of several granule proteins was induced by Ca2+, most notably a Mr-13 000 protein, whereas addition of cyclic AMP was without effect. When phosphatidylserine was also added, Ca2+ increased the phosphorylation of additional proteins, with the largest effect on a protein of Mr 62 000. Purified granules were also able to phosphorylate exogenous substrates. Ca2+-induced phosphorylation of lysine-rich histone was enhanced over 3-fold in the presence of phosphatidylserine, and cyclic AMP-activated protein kinase activity was revealed with mixed histone as substrate. The concentrations of free Ca2+ and cyclic AMP required for half-maximal phosphorylation of both endogenous and exogenous proteins were 1-3 microM and 57 nM respectively. Treatment of granules with 0.25 M-KCl resulted in the release of phosphatidylserine-dependent kinase activity into a high-speed granule supernatant. In contrast, granule-protein substrates of Ca2+-activated kinase activity were resistant to KCl extraction, and in fact were present in purified granule membranes. Kinase activity activated by cyclic AMP was not extracted by KCl treatment. It is concluded that phosphorylation of integral membrane proteins in the zymogen granule can be induced by one or more Ca2+-activated protein kinases. Such a reaction is a potential mechanism by which exocytosis may be regulated in the exocrine pancreas by Ca2+-mediated secretagogues.

Isolation and partial characterization of a complex of several (pro-) endopeptidases from whole porcine pancreatic extract and from purified zymogen granules

Pancreatic zymogen granules contain exportable proteins at a very high concentration. The mechanism leading to this condensation is unknown. On the other hand, it is known that aqueous extract of pancreatic acetone powder precipitates at low ionic strength and acidic pH. The precipitable fraction is called ‘euglobulin’ in the literature. We thought that euglobulin could serve as a simplified model to study the condensation of some of the pancreatic exportable proteins. We have compared quantitatively and qualitatively the composition of euglobulins prepared from porcine pancreatic acetone powder and from lysates of purified pancreatic zymogen granules. They were found to be nearly identical, consisting of glycoprotein(s) and/or proteoglycan(s) associated to a proesterase activity, chymotrypsinogens C and D and proelastase. We conclude therefore, that the interactions between the constituents of euglobulin must be specific, since they can occur in the complex protein mixture of the whole organ to a similar extent as in the zymogen granules themselves. We have tried to identify the nature of these specific interactions. We were able to demonstrate that neither the granule membranes, nor the high-molecular-weight proteoglycan present in the granules (Reggio, H.A. and Palade, C.E. (1978) J. Cell. Biol. 77, 288–314) were responsible for the observed aggregation. Electrostatic interactions between acidic and basic proteins (Thomson, A. and Denniss, I.S. (1976) Biochim. Biophys. Acta 429, 581–590) were demonstrated between proelastase and chymotrypsinogens C and D. However, the possible roles of the glycoprotein(s) and/or proteoglycan(s) in the condensation process remain unknown.

Evidence for the lack of classical secretory phospholipase A 2 in guinea-pig pancreas

Several lipolytic enzymes from guinea-pig pancreas have been determined in a soluble extract and in a purified zymogen granule fraction. The positional specificity of phospholipolytic enzymes was detected using phospholipids bearing various radioactive labels. It is shown that guinea-pig pancreatic extracts are able to release both fatty acids from phosphatidylcholine, but with more efficiency towards the fatty acid occupying the 1-position of sn-glycerol. Evidence is given that guinea-pig pancreas lacks the classical secretory phospholipase A 2 and that phospholipid digestion is achieved through the sequential action of phospholipase A 1 and lysophospholipase.

Some properties of a Ca2+- and (or) Mg2+-requiring nucleoside di- and tri-phosphatase(s) associated with the membranes of rat pancreatic zymogen granules.

Membranes of rat pancreatic zymogen granules have been purified and found to be essentially free of contamination by mitochondria, microsomes, and plasma membranes. They possessed an acid phosphatase activity which derived probably from lysed lysosomes contaminating the purified zymogen granules from which the membranes were prepared. These membranes were found to contain a strong Ca2+- and (or) Mg2+-requiring activity toward all nucleoside di- and tri-phosphates. Various data support the tentative conclusion that a single protein catalyzes the hydrolysis of the nucleoside di- and tri-phosphates. This protein appears to be intrinsic with its active site localized on the internal face of the membranes.