scholarly journals Sulfated compounds in the zymogen granules of the guinea pig pancreas

1978 ◽  
Vol 77 (2) ◽  
pp. 288-314 ◽  
Author(s):  
HA Reggio ◽  
GE Palade
Keyword(s):  
Guinea Pig ◽  
Gel Filtration ◽  
Secretory Proteins ◽  
Zymogen Granule ◽  
Zymogen Granules ◽  
Duct Epithelium ◽  
Granule Fraction ◽  
Em Autoradiography ◽  
Pancreatic Duct System

Sulfate incorporation into the guinea pig pancreas was investigated by light (LM) and electron microscope (EM) autoradiography using a system of minilobules incubated in vitro for 60 min in Krebs-Ringer bicarbonate medium (KRB) containing 35SO4(-2). In acinar cells, examined by EM autoradiography, the label was found concentrated over Golgi elements (including condensing vacuoles) and zymogen granules. 35SO4(-2) was also incorporated by the epithelial cells of the entire pancreatic duct system, the incorporation being surprisingly high in the epithelium of the major ducts. In all ductal epithelia, autoradiographic grains appeared over the Golgi complex and the plasmalemma. Since a contribution of duct epithelium to the sulfated compounds found in the discharged secretion could not be ruled out, a purified zymogen granule fraction was used as a source material for the isolation of sulfated compounds of acinar origin. The presence of 35S-radioactivity in the zymogen granules and condensing vacuoles of this fraction was ascertained by autoradiography (of sectioned pellets). From a lysate of this zymogen granule fraction, a soluble sulfated compound of low isoelectric point and high molecular weight was isolated by gel filtration under conditions that allowed its satisfactory separation from the bulk of the secretory proteins.

scholarly journals COMPOSITION OF CELLULAR MEMBRANES IN THE PANCREAS OF THE GUINEA PIG

1971 ◽  
Vol 49 (1) ◽  
pp. 109-129 ◽  
Author(s):  
J. Meldolesi ◽  
J. D. Jamieson ◽  
G. E. Palade
Keyword(s):  
Guinea Pig ◽  
Gradient Centrifugation ◽  
Secretory Proteins ◽  
Zymogen Granule ◽  
Marker Enzymes ◽  
Zymogen Granules ◽  
Terminal Web ◽  
Granule Fraction ◽  
Pancreatic Exocrine ◽  
Exocrine Cell

The subcellular components involved in the synthesis, transport, and discharge of secretory proteins in the guinea pig pancreatic exocrine cell have been isolated from gland homogenates by differential and gradient centrifugation. They include rough and smooth microsomes derived respectively from the rough endoplasmic reticulum and Golgi periphery, a zymogen granule fraction consisting mainly of mature zymogen granules and a smaller population of condensing vacuoles, and a plasmalemmal fraction. Membrane subfractions were obtained from the particulate components by treatment with mild (pH 7.8) alkaline buffers which extract the majority (>95%) of the content of secretory proteins, allowing the membranes to be recovered from the extracting fluid by centrifugation. The purity of the fractions was assessed by electron microscopy and by assaying marker enzymes for cross-contaminants. The rough and smooth microsomes were essentially free of mitochondrial contamination; the smooth microsomes contained <15% rough contaminants. The zymogen granule fraction and its derived membranes were free of rough microsomes and contained <3% contaminant mitochondria. The plasmalemmal fraction was heterogeneous as to origin (deriving from basal, lateral, and apical poles of the cell) and contained varying amounts of adherent fibrillar material arising from the basement membrane and terminal web. The lipid and enzymatic composition of the membrane fractions are described in the following reports.

scholarly journals INTRACELLULAR TRANSPORT OF SECRETORY PROTEINS IN THE PANCREATIC EXOCRINE CELL

1967 ◽  
Vol 34 (2) ◽  
pp. 597-615 ◽  
Author(s):  
James D. Jamieson ◽  
George E. Palade
Keyword(s):  
Intracellular Transport ◽  
Gradient Centrifugation ◽  
Secretory Proteins ◽  
Zymogen Granule ◽  
Zymogen Granules ◽  
Electron Microscopic ◽  
Intact Cells ◽  
Secretory Cycle ◽  
Granule Fraction ◽  
Electron Microscopic Radioautography

In the previous paper we described an in vitro system of guinea pig pancreatic slices whose secretory proteins can be pulse-labeled with radioactive amino acids. From kinetic experiments performed on smooth and rough microsomes isolated by gradient centrifugation from such slices, we obtained direct evidence that secretory proteins are transported from the cisternae of the rough endoplasmic reticulum to condensing vacuoles of the Golgi complex via small vesicles located in the periphery of the complex. Since condensing vacuoles ultimately become zymogen granules, it was of interest to study this phase of the secretory cycle in pulse-labeled slices. To this intent, a zymogen granule fraction was isolated by differential centrifugation from slices at the end of a 3-min pulse with leucine-14C and after varying times of incubation in chase medium. At the end of the pulse, few radioactive proteins were found in this fraction; after +17 min in chaser, its proteins were half maximally labeled; they became maximally labeled between +37 and +57 min. Parallel electron microscopic radioautography of intact cells in slices pulse labeled with leucine-3H showed, however, that zymogen granules become labeled, at the earliest, +57 min post-pulse. We assumed that the discrepancy between the two sets of results was due to the presence of rapidly labeled condensing vacuoles in the zymogen granule fraction. To test this assumption, electron microscopic radioautography was performed on sections of zymogen granule pellets isolated from slices pulse labeled with leucine-3H and subsequently incubated in chaser. The results showed that the early labeling of the zymogen granule fractions was, indeed, due to the presence of highly labeled condensing vacuoles among the components of these fractions.

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

1971 ◽  
Vol 48 (3) ◽  
pp. 503-522 ◽  
Author(s):  
James D. Jamieson ◽  
George E. Palade
Keyword(s):  
Osmotic Fragility ◽  
Secretory Process ◽  
Secretory Proteins ◽  
Zymogen Granule ◽  
Zymogen Granules ◽  
Inactive Form ◽  
Granule Membrane ◽  
Pancreatic Exocrine ◽  
Exocrine Cell

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.

Role of sulfated O-linked glycoproteins in zymogen granule formation

2002 ◽  
Vol 115 (14) ◽  
pp. 2941-2952 ◽  
Author(s):  
Robert C. De Lisle
Keyword(s):  
Acinar Cell ◽  
Secretory Granules ◽  
Sodium Chlorate ◽  
Pancreatic Acinar Cell ◽  
Secretory Proteins ◽  
Zymogen Granule ◽  
Acidic Ph ◽  
Zymogen Granules ◽  
Granule Formation

Packaging of proteins into regulated secretory granules is mediated by the mildly acidic pH of the trans Golgi network and immature secretory granules. This need for an acidic pH indicates that ionic interactions are important. The mouse pancreatic acinar cell contains four major sulfated glycoproteins,including the zymogen granule structural component Muclin. I tested the hypothesis that sulfation and the O-linked glycosylation to which the sulfates are attached are required for normal formation of zymogen granules in the exocrine pancreas. Post-translational processing was perturbed with two chemicals: sodium chlorate was used to inhibit sulfation and benzyl-N-acetyl-α-galactosaminide was used to inhibit O-linked oligosaccharide elongation. Both chemicals resulted in the accumulation in the Golgi region of the cell of large vacuoles that appear to be immature secretory granules, and the effect was much more extensive with benzyl-N-acetyl-α-galactosaminide than chlorate. Both chemical treatments inhibited basal secretion at prolonged chase times, and again benzyl-N-acetyl-α-galactosaminide had a greater effect than chlorate. In addition, benzyl-N-acetyl-α-galactosaminide, but not chlorate, totally inhibited stimulated secretion of newly synthesized proteins. These data provide evidence for a role of sulfated O-linked glycoproteins in protein condensation and maturation of zymogen granules. Under maximal inhibition of O-linked oligosaccharide biosynthesis, anterograde post-Golgi traffic in the regulated pathway is almost totally shut down, demonstrating the importance of these post-translational modifications in progression of secretory proteins through the regulated pathway and normal granule formation in the pancreatic acinar cell.

scholarly journals SYNTHESIS, INTRACELLULAR TRANSPORT, AND DISCHARGE OF SECRETORY PROTEINS IN STIMULATED PANCREATIC EXOCRINE CELLS

1971 ◽  
Vol 50 (1) ◽  
pp. 135-158 ◽  
Author(s):  
James D. Jamieson ◽  
George E. Palade
Keyword(s):  
Protein Synthesis ◽  
Golgi Complex ◽  
Intracellular Transport ◽  
Secretory Proteins ◽  
Cell Fractionation ◽  
Secretory Product ◽  
Zymogen Granules ◽  
Pancreatic Exocrine ◽  
Storage Granules

Our previous observations on the synthesis and transport of secretory proteins in the pancreatic exocrine cell were made on pancreatic slices from starved guinea pigs and accordingly apply to the resting, unstimulated cell. Normally, however, the gland functions in cycles during which zymogen granules accumulate in the cell and are subsequently discharged from it in response to secretogogues. The present experiments were undertaken to determine if secretory stimuli applied in vitro result in adjustments in the rates of protein synthesis and/or of intracellular transport. To this intent pancreatic slices from starved animals were stimulated in vitro for 3 hr with 0.01 mM carbamylcholine. During the first hour of treatment the acinar lumen profile is markedly enlarged due to insertion of zymogen granule membranes into the apical plasmalemma accompanying exocytosis of the granule content. Between 2 and 3 hr of stimulation the luminal profile reverts to unstimulated dimensions while depletion of the granule population nears completion. The acinar cells in 3-hr stimulated slices are characterized by the virtual complete absence of typical condensing vacuoles and zymogen granules, contain a markedly enlarged Golgi complex consisting of numerous stacked cisternae and electron-opaque vesicles, and possess many small pleomorphic storage granules. Slices in this condition were pulse labeled with leucine-3H and the route and timetable of intracellular transport assessed during chase incubation by cell fractionation, electron microscope radioautography, and a discharge assay covering the entire secretory pathway. The results showed that the rate of protein synthesis, the rate of drainage of the rough-surfaced endoplasmic reticulum (RER) compartment, and the over-all transit time of secretory proteins through the cells was not accelerated by the secretogogue. Secretory stimulation did not lead to a rerouting of secretory proteins through the cell sap. In the resting cell, the secretory product is concentrated in condensing vacuoles and stored as a relatively homogeneous population of spherical zymogen granules. By contrast, in the stimulated cell, secretory proteins are initially concentrated in the flattened saccules of the enlarged Golgi complex and subsequently stored in numerous small storage granules before release. The results suggest that secretory stimuli applied in vitro primarily affect the discharge of secretory proteins and do not, directly or indirectly, influence their rates of synthesis and intracellular transport.

Comparison of the ability to bind lipids of β-lactoglobulin and serum albumin of milk from ruminant and non-ruminant species

1993 ◽  
Vol 60 (1) ◽  
pp. 55-63 ◽  
Author(s):  
M. Dolores Pérez ◽  
Pilar Puyol ◽  
José Manuel Ena ◽  
Miguel Calvo
Keyword(s):  
Fatty Acids ◽  
Guinea Pig ◽  
Serum Albumin ◽  
Whey Protein ◽  
Whey Proteins ◽  
Gel Filtration ◽  
Ruminant Species ◽  
Β Lactoglobulin

SummaryThe interaction of sheep, horse, pig, human and guinea-pig whey proteins with fatty acids has been studied. Using gel filtration and autoradiography, it was found that sheep β-lactoglobulin and serum albumin from all species had the ability to bind fatty acids in vitro. Sheep β-lactoglobulin, isolated from milk, had ˜ 0·5 mol fatty acids bound per mol monomer protein, and albumin from sheep, horse and pig contained ˜ 4·5, 2·9 and 4·7 mol fatty acids/mol protein respectively. However, β-lactoglobulin from horse and pig milk had neither fatty acids physiologically bound nor the ability to bind them in vitro. Albumin was the only whey protein detected with bound fatty acids in these species as well as in human and guinea pig. This suggests that the ability of ruminant β-lactoglobulin to bind fatty acids was not shared by the same protein of non-ruminants.

scholarly journals Initiation and processing in vitro of the primary translation products of guinea-pig caseins

1979 ◽  
Vol 184 (2) ◽  
pp. 261-267 ◽  
Author(s):  
R K Craig ◽  
P A J Perera ◽  
A Mellor ◽  
A E Smith
Keyword(s):  
Guinea Pig ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Secretory Proteins ◽  
Post Translational Modifications ◽  
Microsomal Membranes

1. Guinea-pig caseins synthesized in a mRNA-directed wheat-germ cell-free protein-synthesizing system represent the primary translation products, even though they appear to be of lower molecular weight when analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in parallel with caseins isolated from guinea-pig milk. 2. Identification of the N-terminal dipeptide of the primary translational product of caseins A, B and C and alpha-lactalbumin showed that all shared a common sequence, which was identified as either Met-Arg or Met-Lys. 3. Procedures utilizing methionyl-tRNAfMet or methionyl-tRNAMet in the presence or absence of microsomal membranes during translation provide a rapid method of distinguishing between N-terminal processing of peptides synthesized in vitro and other post-translational modifications (glycosylation, phosphorylation), which also result in a change in mobility of peptides when analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 4. The results demonstrate that guinea-pig caseins, in common with most other secretory proteins, are synthesized with transient N-terminal ‘signal’-peptide extensions, which are cleaved during synthesis in the presence of microsomal membranes.

scholarly journals Association of nucleoside diphosphate kinase with pancreatic zymogen granules: effects of local GTP generation on granule membrane characteristics

1996 ◽  
Vol 316 (1) ◽  
pp. 99-106 ◽  
Author(s):  
Stefan J. MARCINIAK ◽  
J. Michael EDWARDSON
Keyword(s):  
Plasma Membranes ◽  
Direct Action ◽  
Nucleoside Diphosphate Kinase ◽  
Pancreatic Acinar Cell ◽  
Zymogen Granule ◽  
Zymogen Granules ◽  
Cytoplasmic Face ◽  
Granule Membrane ◽  
Stimulation Of

It is well established that both GTP-binding proteins and phosphoproteins are involved in the control of exocytosis in the exocrine pancreas. Exocytotic membrane fusion is stimulated by guanosine 5′-[γ-thio]triphosphate, and the phosphorylation states of several proteins, including at least one on the zymogen granule membrane, are known to change during exocytosis. We show here that a nucleoside diphosphate kinase is associated with the cytoplasmic face of pancreatic zymogen granules. This enzyme behaves as a phosphoprotein of apparent molecular mass 21 kDa on SDS/polyacrylamide gels, and is able to produce GTP by using ATP to phosphorylate endogenous GDP. GTP production by nucleoside diphosphate kinase is stimulated by the wasp venom peptide mastoparan, both through a direct action on the enzyme and through its ability to increase the availability of endogenous GDP. Two effects of the GTP produced by nucleoside diphosphate kinase are demonstrated: phosphorylation of a 37 kDa zymogen granule protein on histidine residues, and stimulation of the fusion of zymogen granules with pancreatic plasma membranes in vitro. These results suggest that granule-associated nucleoside diphosphate kinase is able to maintain local GTP concentrations, and raise the possibility that it might be involved in the control of exocytosis in the pancreatic acinar cell.

scholarly journals Cell fractionation studies on the guinea pig pancreas. Redistribution of exocrine proteins during tissue homogenization.

1978 ◽  
Vol 78 (1) ◽  
pp. 110-130 ◽  
Author(s):  
G A Scheele ◽  
G E Palade ◽  
A M Tartakoff
Keyword(s):  
Guinea Pig ◽  
Intracellular Transport ◽  
Cell Fraction ◽  
Cell Fractionation ◽  
Zymogen Granules ◽  
Cell Compartments ◽  
Intracellular Compartments ◽  
Primary Location ◽  
Double Label

A double-label protocol was used to estimate the extent of leakage and relocation artifacts that affect exocrine pancreatic proteins in cell fractionation experiments. Guinea pig pancreatic lobules were pulsed in vitro with a mixture of 14C-amino acids to enable the lobules to produce and process endogenously labeled exocrine proteins. At the end of the pulse (10 min) or after an appropriate chase interval, the lobules were homogenized in 0.3 M sucrose to which a complete mixture of 3H-labeled exocrine pancreatic proteins was added as an exogenous tracer. The distribution of both labels was studied in each cell fraction of interest at the level of TCA-insoluble proteins and individual exocrine proteins resolved by using a two-dimensional gel system. Based on the premises that the exogenous and endogenous label behave identically during homogenization-fractionation and that all endogenously labeled exocrine proteins found in the postmicrosomal supernate come from intracellular compartments ruptured during tissue homogenization, a series of equations was derived to quantitate leakage and adsorption and to define the ratio of endogenous label still in its primary location to total label (primary location index or PLI) for each cell fraction. Leakage was found to be uniform for all exocrine proteins, but unequal in extent from different cell compartments (condensing vacuoles is greater than zymogen granules is greater than rough endoplasmic reticulum) ; it increased with exposure to shearing forces especially in the case of zymogen granules and condensing vacuoles, and was substantially reduced from rough microsomes by adding 10 mM KCl to the homogenization media. Relocation of exogenous label by adsorption to other subcellular components was extensive (approximately 55%), uneven (free polysomes is greater than rough microsomes is greater than smooth microsomes and zymogen granules), preferential (cationic proteins are massively adsorbed to ribosomes and membranes, resulting in a complementary enrichment of the post-microsomal supernate with anionic exocrine proteins), and reversible (with successive 50-100 mM KCl washes). After correction for adsorption and leakage, the kinetics of intracellular transport derived from cell fractionation data were found to be nearly identical to those obtained from quantitative autoradiographic studies.

Nonparallel secretion of GP-2 from exocrine pancreas implies luminal coupling between acinar and duct cells

1994 ◽  
Vol 267 (1) ◽  
pp. G40-G51 ◽  
Author(s):  
S. D. Freedman ◽  
K. Sakamoto ◽  
G. A. Scheele
Keyword(s):  
Exocrine Pancreas ◽  
Acinar Cells ◽  
Zymogen Granule ◽  
Zymogen Granules ◽  
Pancreatic Acini ◽  
Glycosyl Phosphatidylinositol ◽  
Cellular Processes ◽  
Rat Exocrine Pancreas

The in vivo and in vitro secretion of glycoprotein-2 (GP-2), a glycosyl phosphatidylinositol (GPI)-anchored protein from the rat exocrine pancreas, was characterized. GP-2 was secreted in a nonparallel manner compared with amylase, a marker of secretory enzymes. Attenuated GP-2 secretion correlated with hormones that stimulated exocytosis in acinar cells. Augmented GP-2 secretion correlated with hormones that stimulated fluid and bicarbonate secretion from ductal elements. Immunofluorescence studies identified an enriched pool of GP-2 tightly bound to the apical membranes of acinar cells in addition to zymogen granules. This non-zymogen granule pool appears to represent the source of GP-2 released from acinar cells in a nonparallel manner. With the use of dispersed pancreatic acini largely devoid of ductal elements, GP-2 release was found to be augmented by alkaline pH. Thus GP-2 secretion appears to be modulated by two discrete cellular processes: 1) delivery of prereleased GP-2 within zymogen granules to the ductal lumen by exocytic mechanisms and 2) enzymatic release of GPI-anchored GP-2 from the luminal membranes, a kinetic process that appears to be regulated by secretin- or carbachol-induced secretion of bicarbonate.

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