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

1972 ◽  
Vol 55 (1) ◽  
pp. 1-18 ◽  
Author(s):  
Jacopo Meldolesi ◽  
Dario Cova
Keyword(s):  
Guinea Pig ◽  
Intracellular Transport ◽  
Plasma Membranes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein Composition ◽  
Zymogen Granule ◽  
Exocrine Cells ◽  
Acid Method ◽  
Two Systems ◽  
Pancreatic Exocrine

Two methods of polyacrylamide gel electrophoresis (the acid method of Eytan and Ohad and the Na dodecylsulfate (SDS) disc method of Maizel) have been used for analyzing the proteins of gel fractions isolated from the guinea pig pancreatic exocrine cells and in particular the proteins bound to the membranes involved in the synthesis, intracellular transport, and discharge of secretory enzymes: rough (RM) and smooth microsome (SM) membranes, zymogen granule (ZG) membranes, and plasma membranes (PM). Since in the two systems the electrophoretic mobility of proteins depends on different factors (size, shape, and net charge of molecules in the acid system; size only in the SDS system) a deeper insight into the protein composition of the fractions could be obtained. The gel patterns of RM, SM, and ZG membranes turned out to be accounted for mainly by segregated secretory enzymes (in rough microsomes also by ribosome proteins) and thus were found to share most of the bands. In contrast, with highly purified membrane fractions different patterns were obtained: RM and SM membrane proteins turn out to contain a large number of different proteins with molecular weights varying between ∼150,000 and 15,000 daltons. The pattern of ZG membranes was greatly different in the two systems: only two bands were separated by the acid method and as many as 23 by the SDS method. PM gave a rather complex pattern in either system. Both ZG membranes and PM were found to contain a large proportion of low molecular weight proteins. Nothing appears in common between the proteins of SM membranes (primarily of Golgi origin) and those of ZG membranes, while the latter and PM exhibit a certain degree of similarity. By amino acid analysis we found only slight differences: relative to the other fractions: RM membranes were higher in basic amino acids and ZG membranes contained a larger amount of methionine. Taken together with recent data on lipid composition and enzyme activities of the same fractions, these results indicate that the membranes of the pancreatic exocrine cells are chemically and functionally distinct, and hence do not mix randomly with one another during the transport of secretory products.

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

1967 ◽  
Vol 34 (2) ◽  
pp. 577-596 ◽  
Author(s):  
James D. Jamieson ◽  
George E. Palade
Keyword(s):  
Guinea Pig ◽  
Golgi Complex ◽  
Structural Damage ◽  
Intracellular Transport ◽  
Secretory Proteins ◽  
Zymogen Granule ◽  
Cell Fractionation ◽  
Secretory Cycle ◽  
Pancreatic Exocrine ◽  
Rough Er

It has been established by electron microscopic radioautography of guinea pig pancreatic exocrine cells (Caro and Palade, 1964) that secretory proteins are transported from the elements of the rough-surfaced endoplasmic reticulum (ER) to condensing vacuoles of the Golgi complex possibly via small vesicles located in the periphery of the complex. To define more clearly the role of these vesicles in the intracellular transport of secretory proteins, we have investigated the secretory cycle of the guinea pig pancreas by cell fractionation procedures applied to pancreatic slices incubated in vitro. Such slices remain viable for 3 hr and incur minimal structural damage in this time. Their secretory proteins can be labeled with radioactive amino acids in short, well defined pulses which, followed by cell fractionation, makes possible a kinetic analysis of transport. To determine the kinetics of transport, we pulse-labeled sets of slices for 3 min with leucine-14C and incubated them for further +7, +17, and +57 min in chase medium. At each time, smooth microsomes ( = peripheral elements of the Golgi complex) and rough microsomes ( = elements of the rough ER) were isolated from the slices by density gradient centrifugation of the total microsomal fraction. Labeled proteins appeared initially (end of pulse) in the rough microsomes and were subsequently transferred during incubation in chase medium to the smooth microsomes, reaching a maximal concentration in this fraction after +7 min chase incubation. Later, labeled proteins left the smooth microsomes to appear in the zymogen granule fraction. These data provide direct evidence that secretory proteins are transported from the cisternae of the rough ER to condensing vacuoles via the small vesicles of the Golgi complex.

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 COMPOSITION OF CELLULAR MEMBRANES IN THE PANCREAS OF THE GUINEA PIG

1971 ◽  
Vol 49 (1) ◽  
pp. 150-158 ◽  
Author(s):  
J. Meldolesi ◽  
J. D. Jamieson ◽  
G. E. Palade
Keyword(s):  
Electron Transport ◽  
Guinea Pig ◽  
Enzyme Activities ◽  
Transport Systems ◽  
Zymogen Granule ◽  
Electron Transport Chains ◽  
Microsomal Membranes ◽  
Pancreatic Exocrine ◽  
Exocrine Cell ◽  
Secretory Products

A comparative study of the enzymic activities of membrane fractions derived from guinea pig pancreatic homogenates has yielded the following results: Rough microsomal membranes (derived from the rough ER) have the reductase activities of the two microsomal electron transport systems but lack enzyme activities of Golgi-type (TPPase) and plasmalemmal-type (5'-nucleotidase, ß-leucyl naphthylamidase, Mg-ATPase). Smooth microsomal membranes (derived primarily from the Golgi complex), zymogen granule membranes, and plasmalemmal fractions possess overlapping enzyme activities of plasmalemmal type, in different relative concentrations for each fraction. In addition, the smooth microsomal membranes exhibit TPPase and ADPase activity and share with rough microsomes the reductase activities of the two electron transport chains. Taken together with recent data on the lipid composition of the same fractions (2), these results indicate that the membranes of the pancreatic exocrine cell are chemically and functionally distinct, and hence do not mix with one another during the transport of secretory products.

scholarly journals Fractionation of liver plasma membranes prepared by zonal centrifugation

1970 ◽  
Vol 116 (5) ◽  
pp. 833-842 ◽  
Author(s):  
W. H. Evans
Keyword(s):  
Adenosine Triphosphatase ◽  
Plasma Membranes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein Composition ◽  
Sucrose Density Gradient ◽  
Acetylneuraminic Acid ◽  
Rate Dependent ◽  
Junctional Complexes ◽  
Specific Activities ◽  
Isopycnic Centrifugation

1. Plasma membranes were isolated from crude nuclear sediments from mouse and rat liver by a rate-dependent centrifugation through a sucrose density gradient contained in the ‘A’ type zonal rotor. 2. The membranes were further purified by isopycnic centrifugation, and characterized enzymically, chemically and morphologically. 3. When the plasma-membrane fraction of sucrose density 1.17g/cm3 was dispersed in a tight-fitting homogenizer, two subfractions of densities 1.12 and 1.18 were obtained by isopycnic centrifugation. 4. The light subfraction contained 5′-nucleotidase, nucleoside diphosphatase, leucine naphthylamidase and Mg2+-stimulated adenosine triphosphatase activities at higher specific activities than unfractionated membranes. The heavy subfraction was deficient in the above enzymes but contained higher Na++K+-stimulated adenosine triphosphatase activity. 5. The light subfraction contained twice as much phospholipid and cholesterol, and three times as much N-acetylneuraminic acid relative to unit protein weight as the heavy subfraction. Polyacrylamide-gel electrophoresis indicated differences in protein composition. 6. Electron microscopy showed the light subfraction to be vesicular. The heavy subfraction contained membrane strips with junctional complexes in addition to vesicles.

scholarly journals Isolation and protein composition of membranes of neurosecretory vesicles and plasma membranes from the neural lobe of the bovine pituitary gland

1975 ◽  
Vol 148 (1) ◽  
pp. 57-65 ◽  
Author(s):  
H Vilhardt ◽  
R V Baker ◽  
D B Hope
Keyword(s):  
Plasma Membranes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Composition ◽  
Nerve Endings ◽  
Enzyme Assays ◽  
Neural Lobe ◽  
Pituitary Glands ◽  
Osmotic Lysis ◽  
Sucrose Gradient Ultracentrifugation

1. Homogenates of neural lobes of bovine pituitary glands were fractionated by differential ultracentrifugation. 2. Neurosecretory vesicles were isolated by sucrose-gradient ultracentrifugation and membranes were obtained after hypo-osmotic lysis of the particles. 3. A method is described for the isolation of a preparation of purified neuronal plasma membranes by using a fraction enriched in nerve endings as a starting material. 4. The purity of the subcellular fractions was estimated by enzyme assays and by examination with the electron microscope. 5. On the basis of the results it was estimated that neuronal plasma membranes constitute more than 30% of the protein of the nerve endings and neurosecretory vesicles more than 45% of the total amount of protein in the homogenate. 6. The proteins of membranes of neurosecretory vesicles and of plasma membranes were solubilized by means of sodium dodecyl sulphate. Polyacrylamide-gel electrophoresis of such preparations showed that both membranes contained a large number of proteins, including three glycoproteins.

scholarly journals STEREOLOGICAL ANALYSIS OF THE GUINEA PIG PANCREAS

1974 ◽  
Vol 61 (2) ◽  
pp. 269-287 ◽  
Author(s):  
Robert P. Bolender
Keyword(s):  
Guinea Pig ◽  
Plasma Membranes ◽  
Membrane Surface ◽  
Quantitative Information ◽  
Secretory Process ◽  
Zymogen Granules ◽  
Plasma Membrane Vesicles ◽  
Cytoplasmic Matrix ◽  
Exocrine Cells ◽  
Exocrine Cell

A stereological model which provides detailed quantitative information on the structure of the fasted, nonstimulated gland has been developed for the guinea pig pancreas. The model consists of morphologically defined space and membrane compartments which were used to describe the general composition of the tissue and the specific components of exocrine cells. The results are presented, where appropriate, relative to a cubic centimeter of pancreas, a cubic centimeter of exocrine cell cytoplasm, and to the volume of an average exocrine cell. The exocrine cells, accounting for 82% of the pancreas volume, consisted of 54% cytoplasmic matrix, 22% rough-surfaced endoplasmic reticulum (RER), 8.3% nuclei, 8.1% mitochondria, 6.4% zymogen granules, and 0.7% condensing vacuoles. Their total membrane surface area was distributed as follows: 60% RER, 21% mitochondria, 9.9% Golgi apparatus, 4.8% plasma membranes, 2.6% zymogen granules, 1.8% plasma membrane vesicles, and 0.4% condensing vacuoles. The application of this model to the study of membrane movements associated with the secretory process is discussed within the framework of an analytical approach.

scholarly journals DYNAMICS OF CYTOPLASMIC MEMBRANES IN GUINEA PIG PANCREATIC ACINAR CELLS

1974 ◽  
Vol 61 (1) ◽  
pp. 1-13 ◽  
Author(s):  
J. Meldolesi
Keyword(s):  
Molecular Weight ◽  
Membrane Proteins ◽  
Guinea Pig ◽  
Intracellular Transport ◽  
Sodium Dodecyl ◽  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Secretory Proteins ◽  
Zymogen Granule ◽  
Double Labeling

The rate of synthesis and the turnover of cytoplasmic membrane proteins were determined in the acinar cells of guinea pig pancreas with the aim of investigating the mechanisms by which the intracellular transport of secretion products occurs. These cells are highly specialized toward protein secretion. By means of in vitro pulse-chase experiments and in vivo double-labeling experiments, using radioactive L-leucine as the tracer, it was found that the turnover of secretory proteins is much faster than that of all membranes involved in their transport (rough and smooth microsome and zymogen granule membranes). Sodium dodecyl sulfate-polyacrylamide disk gel electrophoresis of membrane proteins revealed that in each of these membranes there is a marked heterogeneity of turnover; generally the high molecular weight polypeptides have a shorter half-life than the low molecular weight polypeptides. These data indicate that the membranes participating in the intracellular transport of secretory proteins are not synthesized concomitantly with the latter. Rather, they are probably reutilized in several successive secretory cycles. The possible relevance of these findings to other secretory systems is discussed.

scholarly journals Ca++-dependent disassembly and reassembly of occluding junctions in guinea pig pancreatic acinar cells. Effect of drugs.

1978 ◽  
Vol 79 (1) ◽  
pp. 156-172 ◽  
Author(s):  
J Meldolesi ◽  
G Castiglioni ◽  
R Parma ◽  
N Nassivera ◽  
P De Camilli
Keyword(s):  
Guinea Pig ◽  
Pancreatic Acinar Cells ◽  
Antimycin A ◽  
Intramembrane Particles ◽  
Exocrine Cells ◽  
Occluding Junctions ◽  
Ca Ions ◽  
Pancreatic Exocrine

Incubation of guinea pig pancreatic lobules in Ca++-free Krebs-Ringer bicarbonate solution (KRB) containing 0.5 mM ethylene glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA) results in the progressive fragmentation of the occluding zonulae (ZO) with formation of multiple discrete junctions (fasciae occludentes) localized in the lateral and lumenal plasmalemma. After 1--2 h of such incubation, most ZO appear completely disassembled. This results in the disappearance of the heterogeneity in density of intramembrane particles on the P-fracture faces of the basolateral and lumenal plasmalemma. If Ca++ ions are reintroduced into the incubation fluid at this point, continous zonulae reform around the apices of the cells; in contrast, the density of intramembrane particles (imp) at the lumenal plasmalemma remains the same as in the basolateral region, at least for 3 h after Ca++ reintroduction. When added to the incubation fluid, cycloheximide (at a dose known to inhibit protein synthesis greater than 95%) and cytochalasin B (at doses which disrupt microfilaments and modify the cell shape) had no effect on the organization of ZO, on their disassembly in Ca++-free, EGTA medium, or on their Ca++-dependent reformation. Likewise, the organization and disassembly of ZO were unaffected by colchicine; however, after treatment with the latter drug the reassembly was defective, with formation of strand networks on the lateral surface and incomplete segregation of the lumenal region. Antimycin A, on the other hand, when added to the Ca++-EGTA medium, induced a large proliferation of long, infrequently anastomosed junctional strands, usually arranged to form ribbons, festoons, and other bizarre arrays. The possible relationship of these in vitro findings to the in vivo biogenesis and turnover of occluding junctions is discussed. It is suggested that the impairment of reassembly of zonulae by colchicine might be correlated with the disorder induced by the drug on the general organization of pancreatic exocrine cells. Moreover, antimycin A could act by promoting the aggregation of a pool of free junctional strand components (or precursors) that might exist normally in pancreatic exocrine cells.

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

1968 ◽  
Vol 39 (3) ◽  
pp. 580-588 ◽  
Author(s):  
James D. Jamieson ◽  
George E. Palade
Keyword(s):  
Protein Synthesis ◽  
Intracellular Transport ◽  
Secretory Proteins ◽  
Zymogen Granule ◽  
Cell Fractionation ◽  
Maximum Inhibition ◽  
Granule Fraction ◽  
Pancreatic Exocrine ◽  
Exocrine Cell ◽  
Acinar Lumen

Experiments have been carried out to determine whether intracellular transport of pancreatic secretory proteins is obligatorily coupled to protein synthesis or whether it is a separable process which can be independently regulated. To this intent, guinea pig pancreatic slices were pulse labeled with leucine-3H for 3 min and incubated post-pulse for 37 min in chase medium containing cycloheximide up to concentrations sufficient to inhibit protein synthesis by 98%. In controls, newly synthesized secretory proteins are transported over this interval to condensing vacuoles of the Golgi complex. Since the latter are recovered in the zymogen granule fraction upon cell fractionation, intracellular transport was assayed by measuring the amount of protein radioactivity found in the zymogen granule fraction after a (3 + 37) min incubation. The results indicated that at maximum inhibition of protein synthesis (5 x 10-4 M cycloheximide), transport proceeded with an efficiency ∼80% of control. Parallel radioautographic studies on intact slices confirmed these data and further indicated that all the steps of intracellular transport, including discharge to the acinar lumen, were independent of protein synthesis. We conclude that: (1) transport and protein synthesis are separable processes; (2) intracellular transport is not the result of a continuous delivery of secretory proteins from attached polysomes to the cisternae of the rough endoplasmic reticulum; and (3) transport is not dependent on the synthesis of "specific" nonsecretory proteins within the time limits tested.

scholarly journals Studies on rat liver plasma membrane. Altered protein and phospholipid metabolism after injection of d-galactosamine

1977 ◽  
Vol 166 (3) ◽  
pp. 455-462 ◽  
Author(s):  
W Bachmann ◽  
E Harms ◽  
B Hassels ◽  
H Henninger ◽  
W Reuitter
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Plasma Membranes ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Composition ◽  
Phospholipid Metabolism ◽  
Morphological Alterations ◽  
Galactosamine Hepatitis

1. The metabolism of protein and phospholipid in rat liver plasma membranes isolated by the method of Neville [(1960) J. Biophys. Biochem. Cytol. 8, 413-422] was investigated 3 and 6 h after the injection of D-galactosamine in vivo. During this time, all the biochemical and morphological alterations associated with hepatitis developed. 2. After the injection of D-galactosamine the concentration of sphingomyelin in the plasma membrane decreased to below 60% of the control values. 3. The activity of 5′-nucleotidase (EC 3.1.3.5), which has been purified as a sphingomyelin-protein complex, decreased in the total homogenate as well as in the plasma-membrane fraction of livers of rats treated with galactosamine, to about 60% of the control values. 4. Protein synthesis, as measured by the incorporation of [14C]leucine into plasma membranes, was decreased to 45% of that of the controls. However, only small differences were observed in the amino acid composition of the plasma membrane after D-galactosamine treatment. 5. The protein composition of the plasma membranes was determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The results showed a change from low- to high-molecular-weight proteins after the injection of galactosamine. 6. These results demonstrate different metabolic processes of the plasma membrane altered during the induction of galactosamine hepatitis.

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