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G Scheele

Publications and source records attributed to G Scheele.

At least 37 records · Page 2Linked to original sources
PubMed1983

Identification of cDNA clones encoding secretory isoenzyme forms: sequence determination of canine pancreatic prechymotrypsinogen 2 mRNA.

A cDNA library has been constructed from canine poly(A)+ mRNA. Clones containing cDNA inserts coding for prechymotrypsinogen 2 (isoelectric point = 7.1; Mr = 27,500), one of three canine pancreatic isoenzyme forms, were selected by colony hybridization using a cDNA probe synthesized from immunoselected prechymotrypsinogen 2 mRNA. To verify that cDNA clones code for prechymotrypsinogen 2 forms that translocate across rough endoplasmic reticulum membranes and fold into stable and identifiable secretory proteins, we conducted in vitro translation of hybrid-selected mRNA in the presence of microsomal membranes and optimal concentrations of glutathione and analyzed nascent translation products in their nonreduced state by two-dimensional isoelectric focusing/NaDodSO4 gel electrophoresis and fluorography. A near full-length chymotrypsinogen 2 cDNA and its primed extension were used to determine the nucleotide sequence for the entire coding region of prechymotrypsinogen 2 mRNA and 87 residues, including a poly(A) addition signal, in the 3' nontranslated region. The deduced amino acid sequence shows a 263-residue presecretory protein containing an 18-residue amino-terminal transport peptide (Met-Ala-Phe-Leu-Trp-Leu-Leu-Ser-Cys-Phe-Ala-Leu-Leu-Gly-Thr-Ala-Phe-Gly ), which we have previously shown to mediate the translocation of chymotrypsinogen 2 across the rough endoplasmic reticulum membrane. Following the transport peptide is a 245-residue proenzyme, which shows 82% and 80% sequence identity with bovine chymotrypsinogens A and B, respectively. Conserved among the three zymogens are 10 Cys residues that form five disulfide bonds in bovine chymotrypsinogens A and B and the residues that are required for zymogen activation, substrate binding, and catalytic activity.

Amino Acid Sequence↗
PubMed1983

[Adaptation to diet of mRNA coding for pancreatic amylase and serine proteases in the rat].

Translation of total pancreatic RNA using the rabbit reticulocyte lysate system allowed us to estimate the relative levels of active mRNA coding for a number of secretory proteins in Rats fed high-carbohydrate or high-protein diets. Quantitative synthesis of secretory proteins in isolated pancreatic lobules was also achieved. Protein synthesis in lobules or as the result of active mRNA translation in a cell-free system was found to give similar results either on a qualitative or quantitative standpoint. We were also able to show that the levels of specific mRNA for pancreatic amylase and serine protease zymogens could directly be related to the amount of carbohydrate or protein in the diet. These findings indicate that nutritional regulation of pancreatic enzyme synthesis occurs through the modulation of levels of specific mRNAs in the acinar cell.

Adaptation, Biological↗
PubMed1983

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.

Animals↗
PubMed1982

Amino acid sequences of transport peptides associated with canine exocrine pancreatic proteins.

Using microsequencing techniques and proteins labeled in vitro with tritiated amino acids we have obtained the following NH2-terminal sequences for six canine pancreatic presecretory proteins: pretrypsinogen 1, pretrypsinogen 2+3, prechymotrypsinogen 2, preproelastase1, preporcarboxypeptidase A1, preamylase. Points of cleavage by the transport peptidase, indicated by the vertical arrows, were located from sequences of authentic products synthesized in the presence of membranes of the rough endoplasmic reticulum. All of the identified residues in the pancreatic transport peptides are hydrophobic. Predictions of secondary structure were calculated for each of the transport peptides. The data indicated neither a common primary of secondary structure which could be interpreted as the signal for functional binding of the nascent presecretory protein to the rough endoplasmic reticulum membrane. These findings suggest that the initial interaction with the membrane or membrane receptor may depend in part, on the hydrophobic nature of the transport peptides. Five of the presecretory proteins showed a region with a high probability of forming a beta-turn immediately following the cleavage point. This feature may give the nascent peptide a region of flexibility that would facilitate both its insertion as a loop structure into the membrane and its cleavage by the transport peptidase. The sequences of authentic secretory products derived from a variety of pancreatic tissues suggest that hydrophilic residues are required immediately following the cleavage point in order to allow translocation of the nascent polypeptide chains across the membrane.

Amino Acid Sequence↗
PubMed1981

Characterization of human exocrine pancreatic proteins by two-dimensional isoelectric focusing/sodium dodecyl sulfate gel electrophoresis.

Exocrine proteins contained in human pancreatic juice were separated in two dimensions using isoelectric focusing and sodium dodecyl sulfate gel electrophoresis. Nineteen discrete proteins were found. Fifteen of these were identified by actual or potential enzyme activity and include three forms of trypsinogen, two forms each of procarboxypeptidase A, procarboxypeptidase B, proelastase, and colipase, and one form each for amylase, lipase, chymotrypsinogen, and prophospholipase A2. Lipase and four unidentified proteins were found to contain carbohydrate by the periodic acid Schiff staining method. Each pancreatic protein was characterized by isoelectric point and molecular weight. Proteins were quantitated according to relative mass, as measured by the incorporation of a mixture of 15 3H-amino acids into secretory proteins contained in tissue slices, and according to the distribution of Coomassie blue R stain among proteins contained in pancreatic juice, as determined by two-dimensional gel scanning and computer analysis. The second form of pancreatic procarboxypeptidase B (IEPn6.7) was present in only 4 of 10 subjects tested. Trypsinogens 1 and 3 were covalently labeled with 35SO4. Trypsin derived from trypsinogen 2 showed no inhibition with soybean trypsin inhibitor or Trasylol.

Carboxypeptidase B↗
PubMed1980

Potassium- and ionophore A23187-induced discharge of secretory protein in guinea pig pancreatic lobules. Role of extracellular calcium.

Elevated concentrations of potassium chloride (50 to 120 mM) in the incubation medium stimulated in vitro discharge of secretory protein from guinea pig pancreatic lobules. The effect of potassium was not inhibited by 10(-4) M atropine, sodium substitutes, or 10(-5) M tetrodotoxin. Exposure of lobules to elevated concentrations of potassium chloride did not increase the release of tissue lactic dehydrogenase and resulted in the appearance of exocytotic images detected by electron microscopy. The time course and extent of discharge due to 75 mM KCl were similar to those caused by the ionophore A23187 and the secretory effect of both agents depended on extracellular calcium and intracellular energy reserves. Potassium chloride stimulation of 75 mM increased the influx of extracellular calcium by 49%, as measured by net 45Ca uptake. Optimal carbamylcholine chloride or pancreozymin stimulation consistently showed a greater effect on discharge than optimal KCl or A23187 stimulation and the additional effect depended on the ability of these physiological secretagogues to recruit calcium from intracellular sources. Potassium chloride stimulation did not result in cyclic GMP elevations in the presence of atropine and those elevations due to A23187 stimulation were small (21 to 30%) and dissimilar both in character (calcium dependence) and time course compared to those resulting from the physiological secretagogues. These findings allow us to define two interrelated pathways which couple hormonal stimulation and discharge of secretory protein in the exocrine pancreas.

Anti-Bacterial Agents↗
PubMed1980

Mechanism of compartmentation of secretory proteins: transport of exocrine pancreatic proteins across the microsomal membrane.

The mechanism by which secretory proteins are segregated within the cisternal space of microsomal vesicles was studied using dog pancreas mRNA which directs the synthesis of 14 well-characterized nonglycosylated pancreatic exocrine proteins. In the absence of microsomal membranes, each of the proteins was synthesized as larger polypeptide chains (presecretory proteins). 1,000-2,000 daltons larger than their authentic counterparts as judged by polyacrylamide gel electrophoresis in SDS. Conditions optimal for the study of reconstituted rough microsomes in the reticulocyte lysate system were examined in detail using mRNA and microsomal membranes isolated from dog pancreas. Functional reconstitution of rough microsomes was considerably more efficient in the presence of micrococcal nuclease- treated membranes than in the presence of EDTA-treated membranes. Analysis for segregation of nascent secretory proteins by microsomal vesicles, using post-translational incubation in the presence of trypsin and chymotrypsin, 50 mug/ml each, was shown to be inadequate, because of the disruption of vesicles by protease activity. Addition of 1-3 mM tetracaine or 1 mM dibucaine stabilized microsomal membranes incubated in the presence of trypsin and chymotrypsin at either 0 degrees or 22 degrees C. Each of the pancreatic presecretory proteins studied was correctly processed to authentic secretory proteins by nuclease-treated microsomal membranes, as judged by both one-dimensional and two-dimensional gel electophoresis. Post-translational addition of membranes did not result in either segregation or processing of nascent polypeptide chains. Post- translational proteolysis, carried out in the presence of 3 mM tetracaine, indicated that each of the 14 characterized dog pancreas secretory proteins was quantitatively segregated by nuclease-treated microsomal vesicles. Segregation of nascent secretory proteins was irreversible, since radioactive amylase, as well as the other labeled secretory proteins, remained quantitatively sequestered in microsomal vesicles during a 90-min incubation at 22 degrees C after the cessation of protein synthesis. Studies employing synchronized protein synthesis and delayed addition of membranes indicated that all pancreatic presecretory proteins contain amino terminal peptide extensions. These peptide extensions are shown to mediate the cotranslational binding of presecretory proteins to microsomal membranes and the transport of nascent secretory proteins to the vesicular space. The maximum chain lengths which, during synthesis, allow segregation of nascent polypeptide chains varied between 61 (pretrypsinogen 2 + 3) and 88 (preprocarboxypeptidase A1) amino acid residues among dog pancreas presecretory proteins. Reconstitution studies using homologous and heterologous mixtures of mRNA (dog, guinea pig, and rat pancreas; rat liver) and micrococcal nuclease-treated microsomal membranes (dog, guinea pig, and rat liver; dog pancreas), in the presence of placental ribonuclease inhibitor, suggest that the translocation mechanism described is common to the rough endoplasmic reticulum of all mammalian tissues.

Animals↗
PubMed1979

Role of mammalian RNase inhibitor in cell-free protein synthesis.

Addition of the human placental RNase inhibitor at 10 mu/ml to a mixture of wheat germ extract and translation components, prior to the addition of mRNA from dog pancreas or influenza virus-infected cells, resulted in a significant increase in the yield of proteins synthesized. Analysis of the translation products by sodium dodecyl sulfate/polyacrylamide gel electrophoresis indicated that the inhibitor preferentially increased the yield of the larger proteins. In the presence of the inhibitor, yields of the preprocarboxypeptidases were increased 4.5-fold and yields of preamylase were increased 15-fold. Incubation of the wheat germ extract or individual translation components with dog pancreas mRNA, with or without the placental inhibitor, indicated significant RNase contamination among the fractions. Two other in vitro protein synthesis systems-the reticulocyte lysate system and the Krebs ascites system-were found to contain latent RNase activity (RNase in complex with the inhibitor) and an excess of RNase inhibitor. The addition of placental RNase inhibitor did not increase the yield in these systems, except in those cases in which the RNase contamination approached the amount of endogenous inhibitor. When used during the isolation of rat liver cell fractions, the placental inhibitor increased the yield (as measured by A(260)) of rough microsomes and detached polysomes by 24% and 4.6-fold, respectively. Analysis of translation products indicated that detached polysomes isolated in the presence of the inhibitor were intact; those isolated in the absence of inhibitor were degraded.

Animals↗
PubMed1979

[On the ultrastructure of the lacrimal gland in pigs (Sus scropha L.) (author's transl)].

The lacrimal gland of pigs has been investigated light- and electron microscopically. Among others the results are as follows: 1. The lacrimal gland of pigs is a tubuloacinar or compound acinar gland. Acini often are elongated. Tubuli and acini are enclosed in a basal lamina with some myoepithelial cells. 2. Tubuli and acini consist of mucoserous, mucous and serous cells. Mucoserous cells predominate and show secretory granules with a typical bipartite structure. 3. Like salivary glands, the lacrimal gland of pigs has a prominent duct system. Intercalated ducts have a stratified cuboidal epithelium. The cells of the proximal part contain secretory granules. The intercalated duct continues into the striated duct. Cells here are tall or columnar in shape and show basal striation, which by electron microscopy is resolved as basal invaginations of the plasma membrane with numerous elongated mitochondria in the pockets of cytoplasm so formed. Excretory ducts commence with pseudostratified columnar epithelium. 4. Terminal nerve fibres penetrate the basal lamina and make contakt with the glandular cells, the myoepithelial cells and the duct cells. The terminal axons contain abundantly synaptic vesicles, sporadic dense cored vesicles and mitochondria. Until now, we could not find any specialized presynaptic or postsynaptic membranes.

Animals↗