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 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. 130-149 ◽  
Author(s):  
J. Meldolesi ◽  
J. D. Jamieson ◽  
G. E. Palade
Keyword(s):  
Fatty Acids ◽  
Guinea Pig ◽  
Neutral Lipids ◽  
Liver Microsomes ◽  
Zymogen Granule ◽  
Protein Ratio ◽  
Hepatic Microsomes ◽  
Microsomal Membranes ◽  
E 600 ◽  
Exocrine Cell

The lipid composition of rough and smooth microsomal membranes, zymogen granule membranes, and a plasmalemmal fraction from the guinea pig pancreatic exocrine cell has been determined. As a group, membranes of the smooth variety (i.e., smooth microsomes, zymogen granule membranes, and the plasmalemma) were similar in their content of phospholipids, cholesterol and neutral lipids, and in the ratio of total lipids to membrane proteins. In contrast, rough microsomal membranes contained much less sphingomyelin and cholesterol and possessed a smaller lipid/protein ratio. All membrane fractions were unusually high in their content of lysolecithin (up to ∼20% of the total phospholipids) and of neutral lipids, especially fatty acids. The lysolecithin content was shown to be due to the hydrolysis of membrane lecithin by pancreatic lipase; the fatty acids, liberated by the action of lipase on endogenous triglyceride stores, are apparently scavenged by the membranes from the suspending media. Similar artifactually high levels of lysolecithin and fatty acids were noted in hepatic microsomes incubated with pancreatic postmicrosomal supernatant. E 600, an inhibitor of lipase, largely prevented the appearance of lysolecithin and fatty acids in pancreatic microsomes and in liver microsomes treated with pancreatic supernatant.

scholarly journals PROTEIN SYNTHESIS, STORAGE, AND DISCHARGE IN THE PANCREATIC EXOCRINE CELL

1964 ◽  
Vol 20 (3) ◽  
pp. 473-495 ◽  
Author(s):  
Lucien G. Caro ◽  
George E. Palade
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Complex ◽  
Intracellular Transport ◽  
Secretory Proteins ◽  
Zymogen Granules ◽  
Intravenous Injections ◽  
Pancreatic Exocrine ◽  
Exocrine Cell ◽  
Secretory Products ◽  
Electron Microscopical

The synthesis, intracellular transport, storage, and discharge of secretory proteins in and from the pancreatic exocrine cell of the guinea pig were studied by light- and electron microscopical autoradiography using DL-leucine-4,5-H3 as label. Control experiments were carried out to determine: (a) the length of the label pulse in the blood and tissue after intravenous injections of leucine-H3; (b) the amount and nature of label lost during tissue fixation, dehydration, and embedding. The results indicate that leucine-H3 can be used as a label for newly synthesized secretory proteins and as a tracer for their intracellular movements. The autoradiographic observations show that, at ∼5 minutes after injection, the label is localized mostly in cell regions occupied by rough surfaced elements of the endoplasmic reticulum; at ∼20 minutes, it appears in elements of the Golgi complex; and after 1 hour, in zymogen granules. The evidence conclusively shows that the zymogen granules are formed in the Golgi region by a progressive concentration of secretory products within large condensing vacuoles. The findings are compatible with an early transfer of label from the rough surfaced endoplasmic reticulum to the Golgi complex, and suggest the existence of two distinct steps in the transit of secretory proteins through the latter. The first is connected with small, smooth surfaced vesicles situated at the periphery of the complex, and the second with centrally located condensing vacuoles.

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 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 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.

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

1968 ◽  
Vol 39 (3) ◽  
pp. 589-603 ◽  
Author(s):  
James D. Jamieson ◽  
George E. Palade
Keyword(s):  
Oxidative Phosphorylation ◽  
Golgi Complex ◽  
Intracellular Transport ◽  
Secretory Proteins ◽  
Zymogen Granule ◽  
Antimycin A ◽  
Cell Fractionation ◽  
Pancreatic Exocrine ◽  
Exocrine Cell ◽  
Cell Synthesis

Since in the pancreatic exocrine cell synthesis and intracellular transport of secretory proteins can be uncoupled (1), it is possible to examine separately the metabolic requirements of the latter process. 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 5 x 10-4 M cycloheximide and inhibitors of glycolysis, respiration, or oxidative phosphorylation. In each case, the effect on transport was assessed by measuring the amount of labeled secretory proteins found in zymogen granule fractions isolated from the corresponding slices. This assay is actually a measure of the efficiency of transport of secretory proteins from the cisternae of the rough endoplasmic reticulum (RER) to the condensing vacuoles of the Golgi complex which are recovered in the zymogen granule fraction (16). The results indicate that transport is insensitive to glycolytic inhibitors (fluoride, iodoacetate) but is blocked by respiratory inhibitors (N2, cyanide, Antimycin A) and by inhibitors of oxidative phosphorylation (dinitrophenol, oligomycin). Except for Antimycin A, the effect is reversible. Parallel radioautographic studies and cell fractionation procedures applied to microsomal subfractions have indicated that the energy-dependent step is located between the transitional elements of the RER and the small, smooth-surfaced vesicles at the periphery of the Golgi complex. Radiorespirometric data indicate that the substrates oxidized to support transport are endogenous long-chain fatty acids.

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