TWO DIMENSIONAL GEL ELECTROPHORESIS
MICROSOMAL MEMBRANE TRANSLOCATION MODELS
PURIFIED RNase INHIBITOR
A method for preparing pancreatic tissue for structural (electron microscopy) and in-vitro functional studies in a manner that minimizes tissue damage and maximizes functional performance. This procedure is used beneficially with pancreatic tissue taken from guinea pig, rat or mouse. The procedure involves using a syringe and a needle to inject Krebs Ringer Bicarbonate (KRB) solution into the interstitial space thereby distending the tissue and revealing the smallest lobular structures perceived by the human eye. Then, using a pair of ophthalmic scissors, these lobular structures may be removed from the duct system and incubated in KRB under in vitro physiological conditions. Normally 5-10 lobules are incubated in a single Erlenmeyer flask at 37 degrees Celsius and in the presence of 95% oxygen and 5% carbon dioxide.
In experiments that conduct pulse-chase studies that rely on rapid transfer of lobules from one flask to another or rapid washing conditions, a piece of nylon mesh cut into a 2 cm circle, may be used to attach the lobules to a single mesh disk in each incubation flask. The connective tissue surrounding the lobules attach spontaneously to the nylon mesh. Following attachment of the lobules to the nylon mesh, which occurs immediately during shaking in a temperature-controlled water-filled incubator, a pair of forceps may be used to pick up the group of lobules for washing or transferring manipulations without introducing injury to the tissue itself.
Slide Presentation - Pancreatic Lobules
Ultilizing enzymatic digestion with collagenase and vigorous shearing forces, pancreatic lobules can be further reduced to pancreatic acini. However, there is an important functional difference between lobules and acini. In pancreatic lobules the acinar lumen is sequestered from the incubation medium allowing use of ductal inhibitors to manipulate the fluid, electrolyte and pH conditions of the acinar lumen. In pancreatic acini the conditions of the acinar lumen can be directly controlled by the conditions of the incubation medium. These functional differences allowed Dr. Freedman and myself to discover the role of bicarbonate secretion from duct cells in regulating the dissolution of secretory enzymes during exocytic release and the cellular uptake of exocytic membranes (secretory granule membranes inserted into the APM) by activated endocytosis at the APM.
Due to the fragile nature of tissue in the mouse pancreas, pancreatic acini obtained after treating with collagenase and shear forces, show relatively poor performance with high background levels of secretion. Thus, it is optimal to use pancreatic lobules when studying the mouse pancreas in vitro. Further, pancreatic lobules taken from mouse pancreas act more like acini in that the conditions of the acinar lumen appear to be directly controlled by the incubation medium. This observation suggests that small pancreatic ductile segments are severed during the tissue distension procedure.
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TWO DIMENSIONAL GEL ELECTROPHORESIS:
Two Dimensional Gel Electrophoresis separating proteins by charge and size - 2D separation of proteins in a slab gel utilizing isoelectric focusing in the first dimension and SDS gel electrophoresis in the second dimension allowed separation of proteins by charge and size, respectively. This procedure, developed independently by Dr. George Scheele and Dr. Patrick O'Farrell, allowed complex mixtures of proteins (up to 2000) to be separated by gel electrophoresis for the first time. Accordingly, it ushered in the era of "proteomics" twenty years before that term was coined. Invention of this technique allowed my laboratory to study the following biological processes for the first time:
- Kinetics of movement of the entire cadre of secretory enzymes through sequential intracellular compartments during the biogenesis and secretion of pancreatic enzymes.
- Kinetic of release of all secretory enzymes from the acinar cell during conditions of rest and secretagogue stimulation.
- Folding of nascent pancreatic enzymes following their sequestration into the RER lumen.
- Kinetics of activation of pancreatic proteases leading to the autoactivation of pancreatic juice, a pathological process believed to play a role in the pathogenesis of acute and chronic pancreatitis in humans.
- Gene and protein expression studies of families of pancreatic enzymes as a function of changes in ratios of nutritional substrates in the diet and as a function of secretagogue stimulation.
Slide Presentation - 2D gel Electrophoresis
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MICROSOMAL MEMBRANE TRANSLOCATION MODELS:
Microsomal membrane models that allowed elucidation of N-terminal signal sequences responsible for the sequestration of secretory enzymes in the RER. The membrane model that proved to show the greatest success was the dog pancreas microsomes for the following reasons:
Compared to other organs, pancreatic microsomal membranes show the highest polypeptide chain translocation efficiencies.
Dog pancreas was superior to pancreas from other species because of low RNase levels (0.5 ug RNase per gm of tissue) and extraordinarily high RNase inhibitor levels (200:1 molar excess over RNase) in the cytosol.
Slide Presentation - Membrane Model for Protein Targeting & RER Sequestration in the Signal Peptide Hypothesis for Translocation of Presecretory Proteins into the RER
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PURIFIED RNase INHIBITOR:
A method by which purified RNase inhibitor is added to in-vitro translation systems to protect against contaminating RNase found in the translation components and the membrane fractions.
Slide Presentation - Purified RNase Inhibitor
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