Robert A. Ronzio

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.

Comparative analysis of zymogen granule membrane polypeptides

Abstract Polypeptides of zymogen granule membranes from rat, beef, dog, pig, and rabbit pancreas were analyzed by polyacrylamide gel electrophoresis in 1% sodium dodecylsulfate. Granule membranes were characterized by a single major polypeptide species detected by the periodic acid-Schiff procedure. The relative mobilities of this component were similar for these mammals. The component was distinct from major polypeptides of mitochondrial or microsomal membranes. The zymogen granule membrane may, therefore, represent a highly specialized intracellular membrane.

Glycoprotein synthesis in the adult rat pancreas. I. Subcellular distributions of uridine diphosphate galactose: glycoprotein galactosyltransferase and thiamine pyrophospate phosphohydrolase.

Abstract 1. 1. UDPgalactose:glycoprotein galactosyltransferase in homogenates of adult rat pancreas catalyzed the transfer of galactose to desialylated submaxillary mucins. MnCl2 and Triton X-100 stimulated this reaction and the optimal pH was between 7 and 8. No transfer of galactose from UDPgalactose to N-acetylglucosamine was detected. 2. 2. Approx. 80% of the galactosyltransferase was associated with microsomes; less than 5% of the activity was soluble. The specific activity in the smooth microsomal subfraction was 8-fold greater than the homogenate. No activity was found in zymogen granules or zymogen granule membranes. 3. 3. Thiamine pyrophosphohydrolase was detected in all subcellular fractions. Approx. 60% of this activity was soluble; only 19% was associated with the microsomal fraction. The specific activity in the smooth microsomal subfraction was approx. 4-fold greater than the homogenate.

Purification and partial characterization of an integral membrane glycoprotein from zymogen granules of dog pancreas

Glycoproteins, long known to be functional constituents of cell surfaces, have recently been detected in intracellular membranes [ 1 ] . Membranes from the rough endoplasmic reticulum and the Golgi complex [2,3] and membranes from storage granules [4-61 of various secretory tissues, possess glycoproteins. Several functions for such intracellular, membrane-bound glycoproteins can be proposed. They may be organelle-specific and may function as determinants in membrane-membrane interactions, such as fusion. Alternatively, or additionally, membrane constituents may flow from the rough endoplasmic reticulum to the smooth endoplasmic reticulum and then to membrane-bound vesicles. Fusion of the vesicle membrane with the plasma membrane would provide a mechanism for the insertion of plasma membrane components [7,8]. According to this latter view, storage-granule membranes would contain precursors to plasma-membrane glycoproteins. The lack of detailed information regarding specific intracellular membrane glycoproteins has impeded exploration of these possibilities. Pancreatic zymogen granules are specialized organelles for the temporary storage of secretory proteins. Although zymogen granules arise from the Golgi complex and fuse with the plasma membrane during secretion, the molecular mechanisms underlying these

Glycoprotein synthesis in the adult rat pancreas: II. Characterization of Golgi-rich fractions

Abstract Golgi-rich fractions were prepared from homogenates of adult rat pancreas by discontinuous gradient centrifugation. These fractions were characterized by stacks of cisternae associated with large, irregular vesicles and were relatively free of rough microsomes, mitochondria, and zymogen granules. The Golgi-rich fractions contained 50% of the UDP-galactose: glycoprotein galactosyltransferase activity; the specific activity was 12-fold greater than the homogenate. Such fractions represented 2+ -adenosine triphosphatase. Zymogen granules and the Golgi-rich fractions were extracted with 0.2 m NaHCO3, pH 8.2, and the membranes were isolated by centrifugation. The glycoprotein galactosyltransferase could not be detected in granule membranes, while the specific activity in Golgi membranes was 25-fold greater than the homogenate. At least 35 polypeptide species were detected in Golgi membranes by polyacrylamide gel electrophoresis in 1% sodium dodecylsulfate. These ranged in molecular weight from 12,000 to

Phosphorylation of a zymogen granule membrane polypeptide from rat pancreas

Secretion of digestive enzymes and proenzymes by the exocrine pancreas is stimulated by the polypeptide hormone, pancreozymin, and by acetylcholine [ 1,2] . Adenosine 3’:5’ cyclic monophosphate (cyclic AMP), butyryl derivatives of cyclic AMP and theophylline stimulated pancreatic secretion in vitro, suggesting that cyclic AMP is an intracellular mediator of these hormones [3]. The role of cyclic AMP in the pancreas is still not clear [4,5] . A possible function is the regulation of protein kinases. A soluble, cyclic-AMP dependent protein kinase was partially purified from beef pancreas [6] . Furthermore I-ambert et al. [7] observed that peptide hormonestimulated secretion in pancreatic tissue slices was coupled to increased phosphorylation of zymogen granules. We now report that purified zymogen granule membranes from rat pancreas phosphorylated a granule membrane polypeptide, distinct from the major polypeptide of this membrane [8,9]. This phosphorylation, apparently due to an endogenous protein kinase, was not stimulated by cyclic AMP. There was minimal stimulation by guanosine 3’: 5’ cyclic monophosphate (cyclic GMP).

Synthesis and subcellular distribution of heparan sulfate in the rat exocrine pancreas.

Abstract Tissue fragments and isolated acinar cells from adult rat pancreas rapidly incorporated inorganic 35SO42− into macromolecules. Secretion of labeled macromolecules could be stimulated by the secretagogue carbamylcholine chloride. The principal sulfated product synthesized by isolated acinar cells was purified by diethylaminoethyl cellulose chromatography and was identified as heparan sulfate by digestion with purified heparitinase and by deaminative cleavage with nitrous acid. The results of subcellular fractionation of pancreas tissue labeled in vitro with 35SO42− showed that sulfate-containing macromolecules were present both in soluble fractions and as tightly bound membrane components. Zymogen granules contained 14% of the total radioactivity incorporated; zymogen granule membranes, 3%; the soluble zymogen granule contents, 7%; microsomes, 30%; mitochondria, 14%; and the postmicrosomal supernate, 25%. Purified 35SO42−-labeled zymogen granule membranes had the highest specific radioactivity of any fraction examined. Degradative analysis with nitrous acid indicated that the major sulfated product present in all of the subcellular fractions was heparan sulfate.

A possible role of Golgi membrane-associated galactosyltransferase in the formation of zymogen granule glycoproteins.

Abstract A galactosyltransferase activity in smooth microsomes and Golgi membrane-rich fractions from rat pancreas glycosylated endogenous acceptors during incubation with UDP-[ 14 C]galactose in the absence of exogenous glycoproteins. To evaluate the role of this activity in secretion, the endogenous products were partially characterized. Galactose-labeled fractions were sequentially extracted in 0.2 m NaHCO 3 and 0.25 m NaBr to prepare membranes and soluble acceptors. Bound radioactivity was equally distributed between these two fractions. Analysis by polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicated that the particulate galactose-labeled polypeptides were distinct from the soluble galactose acceptors. Rabbit antisera against highly purified zymogen granule membranes precipitated approximately 40% of the radioactivity of the particulate fraction when solubilized in nonionic detergents. In polyacrylamide gels, the galactose-labeled species of the immunoprecipitate migrated with zymogen granule membrane glycoproteins. Rabbit antisera against secretory proteins cross-reacted with less than 5% of the galactose-labeled soluble acceptors. Mature zymogen granule membranes neither contained detectable galactosyltransferase activity nor served as galactosyltransferase acceptors. These results suggest that galactosyltransferase activity associated with membranes derived from the Golgi complex glycosylated zymogen granule membrane precursors. Analysis of [ 14 C]galactolipids did not implicate lipid intermediates in this process.

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.