George Scheele

Exocytosis occurs at the lateral plasma membrane of the pancreatic acinar cell during supramaximal secretagogue stimulation

In vitro and in vivo studies indicate that the secretory response to both caerulein and carbamylcholine stimulation is biphasic. Over the range of submaximal to maximal concentrations of secretagogues, discharge of exocrine proteins in vitro into the incubation medium and in vivo into the pancreatic duct increased and morphologic analysis indicated that exocytosis of zymogen granules occurred exclusively at the luminal membrane. Under in vivo conditions, supramaximal stimulation with caerulein or carbamylcholine resulted in a dose-dependent decrease in amylase release into the pancreatic duct and increase in the appearance of amylase in the blood circulation. Under in vitro or in vivo conditions, supramaximal secretagogue stimulation resulted in marked inhibition of exocytotic activity at the luminal plasma membrane, the appearance of intergranule contacts and fusions within the cytoplasm, and the appearance of exocytotic activity at the lateral plasma membrane. Lateral exocytotic images were observed with individual and fused zymogen granules and autophagic vacuoles. This redirection in the final step of the secretory pathway provides in part the biological basis for the increased appearance of pancreatic (pro)enzymes in the interstitial fluid and serum during supramaximal secretagogue stimulation.

[7] Methods for the study of protein translocation across the RER membrane using the reticulocyte lysate translation system and canine pancreatic microsomal membranes

Publisher Summary This chapter presents the methods developed to elucidate this mechanism for the transport of pancreatic exocrine proteins across the rough endoplasmic reticulum (RER) membrane. The methods are optimized for the simultaneous study of 17 discrete pancreatic secretory proteins, which include individual members of several families of enzymes and proenzymes. The results show that secretory proteins synthesized by dog pancreas mRNA in the presence of nuclease treated microsomal membranes were similar to those synthesized in vivo . The findings indicate that microsomal membranes isolated from dog pancreas and added to the reticulocyte lysate system are unstable in the presence of 50 μg/ml each of trypsin and chymotrypsin. Using polyacrylamide gel electrophoresis in SDS, the chapter demonstrates that the addition of micrococcal nuclease-treated rough microsomes from dog pancreas results in complete proteolytic processing of pancreatic presecretory proteins to polypeptide chains with apparent molecular weights identical with authentic secretory enzymes and zymogens.

Pancreatic adaptation to dietary lipids is mediated by changes in lipase mRNA

Lipase activity, rates of biosynthesis of lipase (triacylglycerol acylhydrolase, EC 3.1.1.3) and amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1) as well as concentrations of their corresponding mRNAs were measured in the pancreatic tissue of rats fed isocaloric and isoprotein diets with inverse changes in the amounts of lipids and carbohydrates. A control diet (3% sunflower oil--62% starch) and three lipid-rich diets (10% sunflower oil--46.2% starch, 25% sunflower oil--12.5% starch and 30% sunflower oil--1.25% starch) were fed to rats for 10 days. Ingestion of the 10% lipid diet already resulted in a 1.4-fold increase in lipase activity while a 2.4-fold increase was observed with the other 2 high-lipid low-carbohydrate diets. Similarly, 1.3- and 3.1-fold increases in the total rate of protein synthesis were measured in pancreatic lobules of rats fed 10 and 25% or 30% lipid diets, respectively, as compared with control animals. While absolute lipase synthesis showed an important increase during the dietary manipulation (1.7- and 5.9-fold, respectively), amylase synthesis was significantly lower (1.1- and 1.5-fold, respectively). The level of lipase mRNA, as measured by dot-blot hybridization with the corresponding specific cDNA, showed a 2.2-fold increase (10% lipid diet) and a 3.9-fold increase (25% lipid diet), whereas the level of amylase mRNA showed only 1.1- and 1.3-fold increases under the same experimental conditions. These data demonstrated that protein-specific synthesis rates more accurately reflected pancreatic adaptive states than tissue levels of enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of proteins according to biological activity following separation by two-dimensional isoelectric focusing/sodium dodecyl sulfate gel electrophoresis: Analysis of human exocrine pancreatic proteins

A novel procedure is described whereby proteins can be identified according to their biological activity after their separation in two dimensions using isoelectric focusing and polyacrylamide gel electrophoresis in sodium dodecyl sulfate (G. Scheele, 1975, J. Biol. Chem., 250, 5375–5385). This procedure includes an optimal staining method for the visualization of two-dimensional gel spots, which avoids the use of chemical fixatives, and a one-step method for elution and renaturation of proteins. Fifteen out of the twenty discrete proteins separated from human pancreatic juice by the two-dimensional gel method were successfully identified by this procedure.

Two-dimensional isoelectric focusing/sodium dodecyl sulfate gel electrophoresis of protein mixtures containing active or potentially active proteases: Analysis of human exocrine pancreatic proteins

Abstract Analysis of human pancreatic juice in two dimensions using isoelectric focusing followed by sodium dodecyl sulfate gel electrophoresis indicated that human pancreatic trypsinogen (IEPn = 6.4) rapidly autoactivated in the absence of the secretory trypsin inhibitor. The addition of 4 to 6 m urea to the protein sample and 8 m urea to the isoelectric focusing gel inhibited this autoactivation process and allowed the analysis of human exocrine pancreatic proteins. Thirteen discrete proteins were separated by the two-dimensional gel procedure including two forms each for trypsinogen, proelastase, and procarboxypeptidase A, and single forms each for amylase, lipase, procarboxypeptidase B, and chymotrypsinogen. The kinetics of inhibition of human trypsin by 8 m urea in the presence of ethylene glycol bis(β-aminoethyl ether)N,N′-tetraacetic acid indicated that samples containing active proteases could also be analyzed by this procedure.

A sensitive and specific enzyme assay for elastase activity using α-[3H]elastin as substrate☆

Abstract A method for the determination of elastase activity is described which uses soluble α-[3H]elastin as substrate. Soluble α-elastin was shown to have the same substrate specificity as natural insoluble elastin. At a substrate concentration of 1 mg/ml, approximately three times half-saturating substrate concentration, the assay is rapid, 1 h, sensitive, 10 ng/ml elastase, and linear up to an enzyme concentration of 250 ng/ml. The addition of 1000 μ/ml Trasylol or 10−4 m N-α-tosyl- l -lysyl chloromethane and 10−4 m tosyl- l -phenylalanyl chloromethane allowed the specific measurement of elastase activity in the presence of trypsin and chymotrypsin activity.

Human pancreatic cancer: analysis of proteins contained in pancreatic juice by two-dimensional isoelectric focusing/sodium dodecyl sulfate gel electrophoresis.

Nineteen discrete proteins contained in normal human pancreatic juice were separated by two‐dimensional isoelectric focusing/SDS gel electrophoresis. Enzyme or proenzyme activity was assigned to 15 of these proteins. Analysis of pancreatic juice obtained from a patient with pancreatic cancer showed a number of additional proteins not observed in pancreatic juice obtained from normals.

Fate of Radioactive Exocrine Pancreatic Proteins Injected Into the Blood Circulation of the Rat:Tissue Uptake and Transepithelial Excretion

[35S]methionine or [35S]methionine-labeled exocrine pancreatic proteins were injected into the bloodstream of conscious rats. Samples of blood, urine, bile, and pancreatic juice were collected at varying intervals through 7 h. Injection of [35S]methionine resulted in the appearance of trichloroacetic acid--soluble radioactivity [( 35S]methionine) in bile and urine within 4 min and trichloroacetic acid-insoluble radioactivity in blood, bile, and pancreatic juice after 20 min. Analysis of these body fluids by two-dimensional isoelectric focusing/sodium dodecyl sulfate gel electrophoresis and fluorography indicated that rat serum, biliary, and pancreatic proteins were labeled, respectively. After the injection of [35S]methionine-labeled pancreatic proteins, half of the trichloroacetic acid-insoluble radioactivity disappeared from the serum in 10-15 min. Radioactive proteins appeared after 5 min in urine and bile, and, over the course of the experiment, accounted for 1%-2% and 0.3%-0.5% of the injected radioactivity, respectively. Analysis of individual radioactive proteins excreted into bile by two-dimensional isoelectric focusing/sodium dodecyl sulfate gel electrophoresis indicated preferential transhepatic transport of negatively charged pancreatic proteins. The majority of pancreatic proteins (approximately 97%) were taken up by a variety of body tissues, particularly kidney, liver, spleen, and lung. Trichloroacetic acid-soluble radioactivity, largely representing [35S]methionine, appeared sequentially in serum, urine, and bile within 2-12 min. At later experimental time points (greater than 60-90 min), radioactive rat serum, biliary, and pancreatic proteins appeared in blood, bile, and pancreatic juice, respectively. After the injection of 35S-labeled guinea pig pancreatic proteins into the blood circulation of the rat, trichloroacetic acid-insoluble radioactivity, observed in pancreatic juice after 60-90 min, exclusively represented rat exocrine pancreatic proteins as judged by the two-dimensional gel procedure. These studies indicate that pancreatic proteins are removed from the blood circulation by at least three separate pathways: (a) uptake and degradation by a variety of tissues in the body (approximately 97% of injected radioactivity), (b) excretion of intact proteins into urine (1%-2%), and (c) transport of intact proteins into bile (0.3%-0.5%). Transport of exocrine pancreatic proteins from the blood circulation to pancreatic juice could not be demonstrated.

Chapter 22 Analysis of the Secretory Process in the Exocrine Pancreas by Two-Dimensional Isoelectric Focusing/Sodium Dodecyl Sulfate Gel Electrophoresis

Publisher Summary This chapter discusses the methodologies used in the analysis of the secretory process in the exocrine pancreas by two-dimensional isoelectric focusing (IEF) electrophoresis. The complete separation of exocrine pancreatic proteins achieved by two-dimensional IEF/sodium dodecyl sulfate (SDS) gel electrophoresis allows the precise measurement of biosynthesis of individual secretory proteins. Around 95% of the radioactivity that is incorporated into soluble pancreatic proteins appears in the two-dimensional spots characterized as secretory proteins. In an experiment reported in the chapter, guinea pig pancreatic lobules were incubated with [ 35 S] methionine for 15 minutes and rapid freezing in liquid nitrogen terminated the incorporation of radioactive methionine. The tissue was then homogenized in 1% Triton X-100 and 25 m M Tris, pH 9.0 and proteins contained in tissue homogenates were separated by two-dimensional IEF/SDS gel electrophoresis. Coomassie Blue stained spots characterized as secretory proteins or unstained regions of the gel representing short-lived precursor forms were then removed from the second-dimension gel and dissolved in hydrogen peroxide, and radioactivity associated with their spots was quantitated by liquid scintillation spectrometry. Thus, the effect of carbamylcholine stimulation on the messenger ribonucleic acid (mRNA)-directed synthesis of the selected guinea pig pancreatic secretory proteins is discussed in the chapter.

Molecular sorting of proteins into the cisternal secretory pathway.

Cotranslational translocation of exportable proteins across the RER membrane prior to their release into the extracellular space has been essentially described by use of canine pancreatic microsomal membranes. Intracisternal segregation of nascent secretory proteins was observed to be irreversible and proteolytic removal of signal sequences resulted in conformationally mature and stable proteins. Structural studies on various translocation peptides from both eukaryotic and prokaryotic preparations showed that many of them have a comparable three-domain organization. A hydrophilic amino-terminal domain is followed by a core region of hydrophobic amino acids and by the region in which the proteolytic cleavage occurs. Membrane components involved in the translocation process namely the signal recognition particle and the SRP receptor as well as the way the vectorial transport mechanism of nascent secretory proteins occurs are also discussed.