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Poultry Science Dec 1995Day-old male, meat-type chicks raised in brooder batteries were infected by orally administering an inoculum prepared from intestines of broiler chicks infected with...
Stunting syndrome in broilers: effect of age and exogenous amylase and protease on performance, development of the digestive tract, digestive enzyme activity, and apparent digestibility.
Day-old male, meat-type chicks raised in brooder batteries were infected by orally administering an inoculum prepared from intestines of broiler chicks infected with stunting syndrome (SS). Naive controls were kept in a parallel room. The chicks were fed a commercial starter diet supplemented with two levels of enzyme preparations to 14 d of age. The experiment was continued to the age of 6 wk in order to estimate compensatory feed intake and growth. In a parallel study, digestibility of the feed was determined from 1 to 3 wk of age with control or inoculated chicks. The enzymes amylase and proteases were produced by Bacillus subtilis and Penicillium emersonii. Enzyme supplementation had no effect on feed intake, growth, or feed utilization, or on digestibility of fat, starch, protein, or energy. Because enzyme supplementation did not consistently affect performance of chicks and no interactions were observed between enzyme supplementation and infection status, data are presented for effects of infection only. Inoculation of SS-infective material reduced performance to 4 wk. Compensatory growth and feed intake were observed from the age of 4 wk onward. At the age of 6 wk the slight retardation of the inoculated chicks was not significant. On Week 1, retention of fat, starch, protein, and energy was significantly depressed in the inoculated chicks. At the age of 2 wk, retention of starch was not depressed, and at the age of 3 wk, the only consistent depression was that observed for fat. The proventriculus weight and content were consistently higher in inoculated chicks, as were the small intestine and intestinal content. The pH of the gizzard content was higher, and that of the small intestine content was lower, in the inoculated birds than in their control counterparts. Stunting syndrome infection was accompanied by a significant depression of trypsin activity in the pancreas at the age of 1 and 2 wk. At these periods, amylase and chymotrypsin were not affected. At 6 wk of age, the activities of amylase, trypsin, and chymotrypsin in the pancreas were higher in the inoculated than in the control birds. In the intestinal chime, amylase, trypsin, and chymotrypsin activities were lower in the inoculated birds on Week 1 and 2 (NS for amylase on Week 1). On Week 6, the activity of all enzymes assayed was higher in the inoculated birds (NS for amylase). It is suggested that the main factors depressing feed intake and growth in SS-infected birds are most probably beyond those of digestion.
Topics: Aging; Amylases; Animals; Chickens; Chymotrypsin; Dietary Fats; Dietary Proteins; Digestion; Digestive System; Digestive System Physiological Phenomena; Endopeptidases; Energy Metabolism; Food, Fortified; Gizzard, Avian; Intestine, Small; Malabsorption Syndromes; Male; Organ Size; Pancreas; Poultry Diseases; Proventriculus; Starch; Trypsin
PubMed: 8825593
DOI: 10.3382/ps.0742019 -
Proceedings of the National Academy of... Apr 1969The possible mechanisms of action of bovine pancreatic ribonuclease are discussed in the light of the detailed knowledge of the geometry of the active site that has been...
The possible mechanisms of action of bovine pancreatic ribonuclease are discussed in the light of the detailed knowledge of the geometry of the active site that has been derived from studies of inhibitor binding by X-ray diffraction and nuclear magnetic resonance. When combined with a knowledge of the mechanism of phosphate ester hydrolysis, this information imposes severe geometric constraints on possible mechanisms of action of the enzyme. Two types of mechanism can be distinguished, the linear and the pseudorotation. The linear mechanism includes a catalytic role for both histidine residues at the active site and does not involve pseudorotation of the intermediate. In contrast, in the pseudorotation mechanism one histidine residue performs all the catalytic functions, while the other serves only to bind the phosphate anion; this necessarily involves pseudorotation of the intermediate and specific protonation of the leaving group by the enzyme. The mode of binding of the product of the reaction, cytidine-3'-monophosphate, has been elucidated by X-ray diffraction and nuclear magnetic resonance. If the substrate binds in an analogous way, only the linear mechanism is possible. This mechanism is described in detail.
Topics: Animals; Binding Sites; Cattle; Chemical Phenomena; Chemistry; Cytosine Nucleotides; Histidine; Magnetic Resonance Spectroscopy; Models, Chemical; Pancreas; Phosphates; Ribonucleases; X-Ray Diffraction
PubMed: 5256413
DOI: 10.1073/pnas.62.4.1151 -
Journal of Biochemistry Mar 2003A survey of DNase I in nine different carp tissues showed that the hepatopancreas has the highest levels of both DNase I enzyme activity and gene expression. Carp...
A survey of DNase I in nine different carp tissues showed that the hepatopancreas has the highest levels of both DNase I enzyme activity and gene expression. Carp hepatopancreatic DNase I was purified 17,000-fold, with a yield of 29%, to electrophoretic homogeneity using three-step column chromatography. The purified enzyme activity was inhibited completely by 20 mM EDTA and a specific anti-carp DNase I antibody and slightly by G-actin. Histochemical analysis using this antibody revealed the strongest immunoreactivity in the cytoplasm of pancreatic tissue, but not in that of hepatic tissue in the carp hepatopancreas. A 995-bp cDNA encoding carp DNase I was constructed from total RNA from carp hepatopancreas. The mature carp DNase I protein comprises 260 amino acids, the same number as the human enzyme, however, the carp enzyme has an insertion of Ser59 and a deletion of Ala225 in comparison with the human enzyme. These alterations have no influence on the enzyme activity and stability. Three amino acid residues, Tyr65, Val67, and Ala114, of human DNase I are involved in actin binding, whereas those of carp DNase I are shifted to Tyr66, Val68, and Phe115, respectively, by the insertion of Ser59: the decrease in affinity to actin is due to one amino acid substitution, Ala114Phe. The results of our phylogenetic and immunological analyses indicate that carp DNase I is not closely related to the mammalian, avian or amphibian enzymes, and forms a relatively tight piscine cluster with the tilapia enzyme.
Topics: Amino Acid Sequence; Animals; Base Sequence; Carps; Deoxyribonuclease I; Fish Proteins; Gene Expression Regulation, Enzymologic; Liver; Molecular Sequence Data; Pancreas; Phylogeny
PubMed: 12761174
DOI: 10.1093/jb/mvg050 -
The Journal of Biological Chemistry Feb 2013Glucokinase is the predominant hexokinase expressed in hepatocytes and pancreatic β-cells, with a pivotal role in regulating glucose-stimulated insulin secretion,...
Glucokinase is the predominant hexokinase expressed in hepatocytes and pancreatic β-cells, with a pivotal role in regulating glucose-stimulated insulin secretion, illustrated by glucokinase gene mutations causing monogenic diabetes and congenital hyperinsulinemic hypoglycemia. A complex tissue-specific network of mechanisms regulates this enzyme, and a major unanswered question in glucokinase biology is how post-translational modifications control the function of the enzyme. Here, we show that the pancreatic isoform of human glucokinase is SUMOylated in vitro, using recombinant enzymes, and in insulin-secreting model cells. Three N-terminal lysines unique for the pancreatic isoform (Lys-12/Lys-13 and/or Lys-15) may represent one SUMOylation site, with an additional site (Lys-346) common for the pancreatic and the liver isoform. SUMO-1 and E2 overexpression stabilized preferentially the wild-type human pancreatic enzyme in MIN6 β-cells, and SUMOylation increased the catalytic activity of recombinant human glucokinase in vitro and also of glucokinase in target cells. Small ubiquitin-like modifier conjugation represents a novel form of post-translational modification of the enzyme, and it may have an important regulatory function in pancreatic β-cells.
Topics: Animals; Carbohydrates; Catalysis; Electrophoresis, Gel, Two-Dimensional; Gene Expression Regulation, Enzymologic; Glucokinase; Insulin-Secreting Cells; Kinetics; Liver; Mass Spectrometry; Mice; Mutation; Pancreas; Protein Isoforms; Protein Processing, Post-Translational; Recombinant Proteins; Sumoylation
PubMed: 23297408
DOI: 10.1074/jbc.M112.393769 -
Bioscience Reports Feb 2018As new nutritional strategies for ruminant are designed to change production efficiency by improving the supply of rumen protect protein, lipid, and even starch, the...
As new nutritional strategies for ruminant are designed to change production efficiency by improving the supply of rumen protect protein, lipid, and even starch, the digestive system must fit to utilize these increased nutrient supplies, especially the pancreas. The objective of this study was to investigate the effects of phenylalanine (Phe) on digestive enzymes synthesis or secretion and cellular signaling in pancreatic acinar (PA) cells of dairy calves. The PA cells isolated from fresh pancreas of dairy calves, and cultured in completed RIPA 1640 medium with no fetal serum but 0, 0.15 and 0.45 mM Phe at 37°C in CO incubator for 120 min. The pancreatic tissue segments (PTS) was cut approximately 2 × 2 mm from the fresh pancreas, and incubated in oxygenated Krebs-Ringer bicarbonate (KRB) buffer containing 0 or 0.35 mM Phe at 39°C for 180 min, and the samples were collected every 60 min after incubation. In PA cells, Phe increased ( < 0.05) the α-amylase secretion and mRNA expression, the phosphorylation of ribosomal protein S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E binding protein 1 (4EBP1). In PTS, the Phe increased ( < 0.05) α-amylase and trypsin synthesis, secretion and mRNA expression, as well as the phosphorylation of S6K1 and 4EBP1. Conclusively, these results suggested that Phe regulates the synthesis or secretion of α-amylase, trypsin and lipase through mRNA translation initiation factors - S6K1 and 4EBP1.
Topics: Acinar Cells; Animals; Cattle; Epithelial Cells; Gene Expression Regulation, Enzymologic; Lipase; Pancreas; Phenylalanine; Phosphorylation; Primary Cell Culture; Protein Biosynthesis; Ribosomal Protein S6 Kinases; Signal Transduction; Trypsin; alpha-Amylases
PubMed: 29263147
DOI: 10.1042/BSR20171189 -
The Journal of Physiology May 1956
Topics: Cholecystokinin; Duodenum; Enzymes; Lipid Metabolism; Pancreas; Phospholipids
PubMed: 13320409
DOI: 10.1113/jphysiol.1956.sp005536 -
The Journal of Biological Chemistry Apr 2008The sulfatases constitute a conserved family of enzymes that specifically hydrolyze sulfate esters in a wide variety of substrates such as glycosaminoglycans, steroid...
The sulfatases constitute a conserved family of enzymes that specifically hydrolyze sulfate esters in a wide variety of substrates such as glycosaminoglycans, steroid sulfates, or sulfolipids. By modifying the sulfation state of their substrates, sulfatases play a key role in the control of physiological processes, including cellular degradation, cell signaling, and hormone regulation. The loss of sulfatase activity has been linked with various severe pathophysiological conditions such as lysosomal storage disorders, developmental abnormalities, or cancer. A novel member of this family, arylsulfatase G (ASG), was initially described as an enzyme lacking in vitro arylsulfatase activity and localizing to the endoplasmic reticulum. Contrary to these results, we demonstrate here that ASG does indeed have arylsulfatase activity toward different pseudosubstrates like p-nitrocatechol sulfate and 4-methylumbelliferyl sulfate. The activity of ASG depends on the Cys-84 residue that is predicted to be post-translationally converted to the critical active site C(alpha)-formylglycine. Phosphate acts as a strong, competitive ASG inhibitor. ASG is active as an unprocessed 63-kDa monomer and shows an acidic pH optimum as typically seen for lysosomal sulfatases. In transfected cells, ASG accumulates within lysosomes as indicated by indirect immunofluorescence microscopy. Furthermore, ASG is a glycoprotein that binds specifically to mannose 6-phosphate receptors, corroborating its lysosomal localization. ARSG mRNA expression was found to be tissue-specific with highest expression in liver, kidney, and pancreas, suggesting a metabolic role of ASG that might be associated with a so far non-classified lysosomal storage disorder.
Topics: Arylsulfatases; Cell Line, Tumor; Fluorescent Antibody Technique, Indirect; Gene Expression Regulation, Enzymologic; Humans; Kidney; Liver; Lysosomes; Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; Models, Biological; Pancreas; Protein Binding; Sulfatases; Sulfates
PubMed: 18283100
DOI: 10.1074/jbc.M709917200 -
American Journal of Transplantation :... May 2007A major obstacle to successful human islet isolation has been the variability of the enzymatic digestion phase. The aim of this study was to define optimal enzyme...
A major obstacle to successful human islet isolation has been the variability of the enzymatic digestion phase. The aim of this study was to define optimal enzyme activity ranges normalized by the pancreas weight and to identify valid parameters for the optimal selection of successful lots of collagenase enzyme blends. Our results from 251 islet isolations showed that optimization of thermolysin dosage based on Caseinase unit/g pancreas contributed considerably to islet isolation outcome but that collagenase dosage measured by the manufacturer (Wünsch unit/g pancreas) was not a major determinant of islet isolation outcome. We also found that lot-to-lot inconsistency of enzyme performance was not explained by the activity values provided by the manufacturer, but rather by an in-house assay of class I collagenase (CI) and class II collagenase (CII); using a lot with a lower CII/CI resulted in a higher success rate. The odds of successful isolation was 8.67 times higher when a vial with CII/CI ratio <0.204 was used than when a vial with CII/CI >or=0.204 was used, suggesting that CII/CI ratio may be a strong predictor to distinguish potential lot success. This study provides a framework for improved enzymatic digestion in human islet isolation.
Topics: Adolescent; Adult; Aged; Cell Separation; Child; Collagenases; Diabetes Mellitus, Type 1; Dose-Response Relationship, Drug; Female; Graft Survival; Humans; Islets of Langerhans; Islets of Langerhans Transplantation; Male; Matrix Metalloproteinase 8; Middle Aged; Organ Size; Pancreas; Thermolysin
PubMed: 17359501
DOI: 10.1111/j.1600-6143.2007.01760.x -
The Journal of Biological Chemistry Nov 2018About 20 members of the protein-disulfide isomerase (PDI) family are present in the endoplasmic reticulum of mammalian cells. They are thought to catalyze...
About 20 members of the protein-disulfide isomerase (PDI) family are present in the endoplasmic reticulum of mammalian cells. They are thought to catalyze thiol-disulfide exchange reactions within secretory or membrane proteins to assist in their folding or to regulate their functions. PDIp is a PDI family member highly expressed in the pancreas and known to bind estrogen and However, the physiological functions of PDIp remained unclear. In this study, we set out to identify its physiological substrates. By combining acid quenching and thiol alkylation, we stabilized and purified the complexes formed between endogenous PDIp and its target proteins from the mouse pancreas. MS analysis of these complexes helped identify the disulfide-linked PDIp targets , revealing that PDIp interacts directly with a number of pancreatic digestive enzymes. Interestingly, when pancreatic elastase, one of the identified proteins, was expressed alone in cultured cells, its proenzyme formed disulfide-linked aggregates within cells. However, when pancreatic elastase was co-expressed with PDIp, the latter prevented the formation of these aggregates and enhanced the production and secretion of proelastase in a form that could be converted to an active enzyme upon trypsin treatment. These findings indicate that the main targets of PDIp are digestive enzymes and that PDIp plays an important role in the biosynthesis of a digestive enzyme by assisting with the proper folding of the proenzyme within cells.
Topics: Animals; Disulfides; Enzyme Precursors; Estrogens; HeLa Cells; Humans; Male; Mass Spectrometry; Mice; Mice, Inbred C57BL; Pancreas; Pancreatic Elastase; Protein Binding; Protein Disulfide-Isomerases; Substrate Specificity; alpha-Amylases
PubMed: 30315102
DOI: 10.1074/jbc.RA118.003694 -
Journal of Biochemistry Dec 1977Human pancreatic DNase I was purified extensively from duodenal juice of healthy subjects by a procedure including ammonium sulfate fractionation, ethanol fractionation,...
Human pancreatic DNase I was purified extensively from duodenal juice of healthy subjects by a procedure including ammonium sulfate fractionation, ethanol fractionation, phosphocellulose fractionation, isoelectric focusing, and gel filtration. The final preparation was free of DNase II, pancreatic RNase, alkaline phosphatase, and protease. The enzyme had a molecular weight of approximately 30,000, as determined by gel filtration on Sephadex G-100, and showed maximum activity at pH 7.2-7.6. It required divalent cations for activity, and caused single-strand breaks by endonucleolytic attack on double- as well as single-stranded DNA molecules. The enzyme was inhibited by actin and bovine pancreatic DNase I antibody.
Topics: Deoxyribonucleases; Humans; Immunodiffusion; Molecular Weight; Pancreas
PubMed: 413831
DOI: 10.1093/oxfordjournals.jbchem.a131875