John H. Northrop
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Chymotrypsin-Like Peptidases in Insects
Chymotrypsin-like peptidases in insects Dissertation zur Erlangung des akademischen Grades Doctor rerum naturalium (Dr. rer. nat.) Fachbereich Biologie/Chemie der Universität Osnabrück vorgelegt von Gunnar Bröhan Osnabrück, Mai 2010 TABLE OF CONTENTS I Table of contents 1. Introduction 1 1.1. Serine endopeptidases 1 1.2. The structure of S1A chymotrypsin-like peptidases 2 1.3. Catalytic mechanism of chymotrypsin-like peptidases 6 1.4. Insect chymotrypsin-like peptidases 9 1.4.1. Chymotrypsin-like peptidases in insect immunity 9 1.4.2. Role of chymotrypsin-like peptidases in digestion 14 1.4.3. Involvement of chymotrypsin-like peptidases in molt 16 1.5. Objective of the work 18 2. Material and Methods 20 2.1. Material 20 2.1.1. Culture Media 20 2.1.2. Insects 20 2.2. Molecular biological methods 20 2.2.1. Tissue preparations for total RNA isolation 20 2.2.2. Total RNA isolation 21 2.2.3. Reverse transcription 21 2.2.4. Quantification of nucleic acids 21 2.2.5. Chemical competent Escherichia coli 21 2.2.6. Ligation and transformation in E. coli 21 2.2.7. Preparation of plasmid DNA 22 2.2.8. Restriction enzyme digestion of DNA 22 2.2.9. DNA gel-electrophoresis and DNA isolation 22 2.2.10. Polymerase-chain-reaction based methods 23 2.2.10.1. RACE-PCR 23 2.2.10.2. Quantitative Realtime PCR 23 2.2.10.3. Megaprimer PCR 24 2.2.11. Cloning of insect CTLPs 25 2.2.12. Syntheses of Digoxigenin-labeled DNA and RNA probes 26 2.2.13. -
Concentration of Tissue Angiotensin II Increases with Severity of Experimental Pancreatitis
MOLECULAR MEDICINE REPORTS 8: 335-338, 2013 Concentration of tissue angiotensin II increases with severity of experimental pancreatitis HIROYUKI FURUKAWA1, ATSUSHI SHINMURA1, HIDEHIRO TAJIMA1, TOMOYA TSUKADA1, SHIN-ICHI NAKANUMA1, KOICHI OKAMOTO1, SEISHO SAKAI1, ISAMU MAKINO1, KEISHI NAKAMURA1, HIRONORI HAYASHI1, KATSUNOBU OYAMA1, MASAFUMI INOKUCHI1, HISATOSHI NAKAGAWARA1, TOMOHARU MIYASHITA1, HIDETO FUJITA1, HIROYUKI TAKAMURA1, ITASU NINOMIYA1; HIROHISA KITAGAWA1, SACHIO FUSHIDA1, TAKASHI FUJIMURA1, TETSUO OHTA1, TOMOHIKO WAKAYAMA2 and SHOICHI ISEKI2 1Department of Gastroenterological Surgery, Division of Cancer Medicine; 2Department of Histology and Embryology, Graduate School of Medical Science, Kanazawa University, Kanazawa, Ishikawa 920-8641, Japan Received January 23, 2013; Accepted May 30, 2013 DOI: 10.3892/mmr.2013.1509 Abstract. Necrotizing pancreatitis is a serious condition that Introduction is associated with high morbidity and mortality. Although vasospasm is reportedly involved in necrotizing pancreatitis, Acute pancreatitis, particularly the necrotizing type, is the underlying mechanism is not completely clear. In addition, associated with high morbidity and mortality. Necrotizing the local renin-angiotensin system has been hypothesized to pancreatitis is characterized by parenchymal non-enhance- be involved in the progression of pancreatitis and trypsin has ment on contrast-enhanced computed tomography images (1). been shown to generate angiotensin II under weakly acidic Although necrosis is irreversible, certain patients exhibiting conditions. However, to the best of our knowledge, no studies this pancreatic parenchymal non-enhancement recover have reported elevated angiotensin II levels in tissue with with normal pancreatic conditions (2). Vasospasm has been pancreatitis. In the present study, the concentration of pancre- implicated in the development of pancreatic ischemia and atic angiotensin II in rats with experimentally induced acute necrosis (3); however, the precise underlying mechanism is pancreatitis was measured. -
In Escherichia Coli (Synthetic Oligonucleotide/Gene Expression/Industrial Enzyme) J
Proc. Nati Acad. Sci. USA Vol. 80, pp. 3671-3675, June 1983 Biochemistry Synthesis of calf prochymosin (prorennin) in Escherichia coli (synthetic oligonucleotide/gene expression/industrial enzyme) J. S. EMTAGE*, S. ANGALt, M. T. DOELt, T. J. R. HARRISt, B. JENKINS*, G. LILLEYt, AND P. A. LOWEt Departments of *Molecular Genetics, tMolecular Biology, and tFermentation Development, Celltech Limited, 250 Bath Road, Slough SL1 4DY, Berkshire, United Kingdom Communicated by Sydney Brenner, March 23, 1983 ABSTRACT A gene for calf prochymosin (prorennin) has been maturation conditions and remained insoluble on neutraliza- reconstructed from chemically synthesized oligodeoxyribonucleo- tion, it was possible that purification as well as activation could tides and cloned DNA copies of preprochymosin mRNA. This gene be achieved. has been inserted into a bacterial expression plasmid containing We describe here the construction of E. coli plasmids de- the Escherichia coli tryptophan promoter and a bacterial ribo- signed to express the prochymosin gene from the trp promoter some binding site. Induction oftranscription from the tryptophan and the isolation and conversion of this prochymosin to en- promoter results in prochymosin synthesis at a level of up to 5% active of total protein. The enzyme has been purified from bacteria by zymatically chymosin. extraction with urea and chromatography on DEAE-celiulose and MATERIALS AND METHODS converted to enzymatically active chymosin by acidification and neutralization. Bacterially produced chymosin is as effective in Materials. DNase I, pepstatin A, and phenylmethylsulfonyl clotting milk as the natural enzyme isolated from calf stomach. fluoride were obtained from Sigma. Calf prochymosin (Mr 40,431) and chymosin (Mr 35,612) were purified from stomachs Chymosin (rennin) is an aspartyl proteinase found in the fourth from 1-day-old calves (1). -
From Renin- Secreting Tumors of Nonrenal Origin
Characterization of inactive renin ("prorenin") from renin- secreting tumors of nonrenal origin. Similarity to inactive renin from kidney and normal plasma. S A Atlas, … , M C Ruddy, M Aurell J Clin Invest. 1984;73(2):437-447. https://doi.org/10.1172/JCI111230. Research Article Inactive renin comprises well over half the total renin in normal human plasma. There is a direct relationship between active and inactive renin levels in normal and hypertensive populations, but the proportion of inactive renin varies inversely with the active renin level; as much as 98% of plasma renin is inactive in patients with low renin, whereas the proportion is consistently lower (usually 20-60%) in high-renin states. Two hypertensive patients with proven renin- secreting carcinomas of non-renal origin (pancreas and ovary) had high plasma active renin (119 and 138 ng/h per ml) and the highest inactive renin levels we have ever observed (5,200 and 14,300 ng/h per ml; normal range 3-50). The proportion of inactive renin (98-99%) far exceeded that found in other patients with high active renin levels. A third hypertensive patient with a probable renin-secreting ovarian carcinoma exhibited a similar pattern. Inactive renins isolated from plasma and tumors of these patients were biochemically similar to semipurified inactive renins from normal plasma or cadaver kidney. All were bound by Cibacron Blue-agarose, were not retained by pepstatin-Sepharose, and had greater apparent molecular weights (Mr) than the corresponding active forms. Plasma and tumor inactive renins from the three patients were similar in size (Mr 52,000-54,000), whereas normal plasma inactive renin had a slightly larger Mr than that from kidney (56,000 vs. -
Serine Proteases with Altered Sensitivity to Activity-Modulating
(19) & (11) EP 2 045 321 A2 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.: 08.04.2009 Bulletin 2009/15 C12N 9/00 (2006.01) C12N 15/00 (2006.01) C12Q 1/37 (2006.01) (21) Application number: 09150549.5 (22) Date of filing: 26.05.2006 (84) Designated Contracting States: • Haupts, Ulrich AT BE BG CH CY CZ DE DK EE ES FI FR GB GR 51519 Odenthal (DE) HU IE IS IT LI LT LU LV MC NL PL PT RO SE SI • Coco, Wayne SK TR 50737 Köln (DE) •Tebbe, Jan (30) Priority: 27.05.2005 EP 05104543 50733 Köln (DE) • Votsmeier, Christian (62) Document number(s) of the earlier application(s) in 50259 Pulheim (DE) accordance with Art. 76 EPC: • Scheidig, Andreas 06763303.2 / 1 883 696 50823 Köln (DE) (71) Applicant: Direvo Biotech AG (74) Representative: von Kreisler Selting Werner 50829 Köln (DE) Patentanwälte P.O. Box 10 22 41 (72) Inventors: 50462 Köln (DE) • Koltermann, André 82057 Icking (DE) Remarks: • Kettling, Ulrich This application was filed on 14-01-2009 as a 81477 München (DE) divisional application to the application mentioned under INID code 62. (54) Serine proteases with altered sensitivity to activity-modulating substances (57) The present invention provides variants of ser- screening of the library in the presence of one or several ine proteases of the S1 class with altered sensitivity to activity-modulating substances, selection of variants with one or more activity-modulating substances. A method altered sensitivity to one or several activity-modulating for the generation of such proteases is disclosed, com- substances and isolation of those polynucleotide se- prising the provision of a protease library encoding poly- quences that encode for the selected variants. -
Trypsinogen Isoforms in the Ferret Pancreas Eszter Hegyi & Miklós Sahin-Tóth
www.nature.com/scientificreports OPEN Trypsinogen isoforms in the ferret pancreas Eszter Hegyi & Miklós Sahin-Tóth The domestic ferret (Mustela putorius furo) recently emerged as a novel model for human pancreatic Received: 29 June 2018 diseases. To investigate whether the ferret would be appropriate to study hereditary pancreatitis Accepted: 25 September 2018 associated with increased trypsinogen autoactivation, we purifed and cloned the trypsinogen isoforms Published: xx xx xxxx from the ferret pancreas and studied their functional properties. We found two highly expressed isoforms, anionic and cationic trypsinogen. When compared to human cationic trypsinogen (PRSS1), ferret anionic trypsinogen autoactivated only in the presence of high calcium concentrations but not in millimolar calcium, which prevails in the secretory pathway. Ferret cationic trypsinogen was completely defective in autoactivation under all conditions tested. However, both isoforms were readily activated by enteropeptidase and cathepsin B. We conclude that ferret trypsinogens do not autoactivate as their human paralogs and cannot be used to model the efects of trypsinogen mutations associated with human hereditary pancreatitis. Intra-pancreatic trypsinogen activation by cathepsin B can occur in ferrets, which might trigger pancreatitis even in the absence of trypsinogen autoactivation. Te digestive protease precursor trypsinogen is synthesized and secreted by the pancreas to the duodenum where it becomes activated to trypsin1. Te activation process involves limited proteolysis of the trypsinogen activation peptide by enteropeptidase, a brush-border serine protease specialized for this sole purpose. Te activation peptide is typically an eight amino-acid long N-terminal sequence, which contains a characteristic tetra-aspartate motif preceding the activation site peptide bond, which corresponds to Lys23-Ile24 in human trypsinogens. -
Role of the Amino Terminus in Intracellular Protein Targeting to Secretory Granules Teresa L
In Vitro Mutagenesis of Trypsinogen: Role of the Amino Terminus in Intracellular Protein Targeting to Secretory Granules Teresa L. Burgess,* Charles S. Craik,** Linda Matsuuchi,* and Regis B. Kelly* * Department of Biochemistry and Biophysics, and *Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143 Abstract. The mouse anterior pituitary tumor cell expressed in AtT-20 cells to determine whether intra- line, AtT-20, targets secretory proteins into two distinct cellular targeting could be altered. Replacing the tryp- intracellular pathways. When the DNA that encodes sinogen signal peptide with that of the kappa-immu- trypsinogen is introduced into AtT-20 cells, the protein noglobulin light chain, a constitutively secreted is sorted into the regulated secretory pathway as protein, does not alter targeting to the regulated secre- efficiently as the endogenous peptide hormone ACq'H. tory pathway. In addition, deletion of the NH2-terminal In this study we have used double-label immunoelec- "pro" sequence of trypsinogen has virtually no effect tron microscopy to demonstrate that trypsinogen on protein targeting. However, this deletion does affect colocalizes in the same secretory granules as ACTH. the signal peptidase cleavage site, and as a result the In vitro mutagenesis was used to test whether the in- enzymatic activity of the truncated trypsin protein is formation for targeting trypsinogen m the secretory abolished. We conclude that neither the signal peptide granules resides at the amino (NH2) terminus of the nor the 12 NH2-terminal amino acids of trypsinogen protein. Mutations were made in the DNA that en- are essential for sorting to the regulated secretory codes trypsinogen, and the mutant proteins were pathway of AtT-20 cells. -
DUAL ROLE of CATHEPSIN D: LIGAND and PROTEASE Martin Fuseka, Václav Větvičkab
Biomed. Papers 149(1), 43–50 (2005) 43 © M. Fusek, V. Větvička DUAL ROLE OF CATHEPSIN D: LIGAND AND PROTEASE Martin Fuseka, Václav Větvičkab* a Institute of Organic Chemistry and Biochemistry, CAS, Prague, Czech Republic, and b University of Louisville, Department of Pathology, Louisville, KY40292, USA, e-mail: [email protected] Received: April 15, 2005; Accepted (with revisions): June 20, 2005 Key words: Cathepsin D/Procathepsin D/Cancer/Activation peptide/Mitogenic activity/Proliferation Cathepsin D is peptidase belonging to the family of aspartic peptidases. Its mostly described function is intracel- lular catabolism in lysosomal compartments, other physiological effect include hormone and antigen processing. For almost two decades, there have been an increasing number of data describing additional roles imparted by cathepsin D and its pro-enzyme, resulting in cathepsin D being a specific biomarker of some diseases. These roles in pathological conditions, namely elevated levels in certain tumor tissues, seem to be connected to another, yet not fully understood functionality. However, despite numerous studies, the mechanisms of cathepsin D and its precursor’s actions are still not completely understood. From results discussed in this article it might be concluded that cathepsin D in its zymogen status has additional function, which is rather dependent on a “ligand-like” function then on proteolytic activity. CATHEPSIN D – MEMBER PRIMARY, SECONDARY AND TERTIARY OF ASPARTIC PEPTIDASES FAMILY STRUCTURES OF ASPARTIC PEPTIDASES Major function of cathepsin D is the digestion of There is a high degree of sequence similarity among proteins and peptides within the acidic compartment eukaryotic members of the family of aspartic peptidases, of lysosome1. -
Hydrolysis of -Lactalbumin by Chymosin and Pepsin. Effect Of
Hydrolysis of α-lactalbumin by chymosin and pepsin. Effect of conformation and pH G. Miranda, G. Hazé, Non Renseigné To cite this version: G. Miranda, G. Hazé, Non Renseigné. Hydrolysis of α-lactalbumin by chymosin and pepsin. Effect of conformation and pH. Le Lait, INRA Editions, 1989, 69 (6), pp.451-459. hal-00929176 HAL Id: hal-00929176 https://hal.archives-ouvertes.fr/hal-00929176 Submitted on 1 Jan 1989 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Lait (1989) 69. 451-459 451 © Elsevier/INRA Original article Hydrolysis of œ-lactalburnin by chymosin and pepsin. Effect of conformation and pH G. Miranda, G. Hazé, P.Scanff and J.P.Pélissier INRA, station de recherches laitières, 78350 Jouy-en-Josas, France (received 21 March 1989. accepted 26 June 1989) Summary - The correlation between change of conformation of a-Iactalbumin and its degradation by gastric enzymes was verified. With citrate buffer (0.1 M), the modification of a-Iactalbumin confor- mation occurred when the pH value was below pH 4.0. This conformational change was influenced by buffer composition and ionic strength. However, the presence of EDTA in the butter did not modi- fy the pH value at which the change of conformation of the protein occurred. -
Structure of the Human Renin Gene
Proc. Nati. Acad. Sci. USA Vol. 81, pp. 5999-6003, October 1984 Biochemistry Structure of the human renin gene (hypertension/aspartyl proteinase/nucleotide sequence/splice junction) HITOSHI MIYAZAKI*, AKIYOSHI FUKAMIZU*, SHIGEHISA HIROSE*, TAKASHI HAYASHI*, HITOSHI HORI*, HIROAKI OHKUBOt, SHIGETADA NAKANISHIt, AND KAZUO MURAKAMI** *Institute of Applied Biochemistry, University of Tsukuba, Ibaraki 305, Japan; and tInstitute for Immunology, Kyoto University Faculty of Medicine, Kyoto 606, Japan Communicated by Leroy Hood, June 27, 1984 ABSTRACT The human renin gene was isolated from a between the intron-exon organization of the gene and the Charon 4A human genomic library and characterized. The tertiary structure of the protein. gene spans about 11.7 kilobases and consists of 10 exons and 9 introns that map at points that could be variable surface loops MATERIALS AND METHODS of the enzyme. The complete coding regions, the 5'- and 3'- Materials. All restriction enzymes were obtained from flanking regions, and the exon-intron boundaries were se- either New England Biolabs or Takara Shuzo (Kyoto, Ja- quenced. The active site aspartyl residues Asp-38 and Asp-226 pan). Escherichia coli alkaline phosphatase and T4 DNA li- are encoded by the third and eighth exons, respectively. The gase were from Takara Shuzo. [_y-32P]ATP (>5000 Ci/mmol; extra three amino acids (Asp-165, Ser-166, Glu-167) that are 1 Ci = 37 GBq) and [a-32P]dCTP (=3000 Ci/mmol) were not present in mouse renin are encoded by the separate sixth from Amersham. exon, an exon as small as 9 nucleotides. The positions of the Screening. A human genomic library, prepared from partial introns are in remarkable agreement with those in the human Alu I and Hae III digestion and ligated into the EcoRI arms pepsin gene, supporting the view that the genes coding for of the X vector Charon 4A, was kindly provided by T. -
Caseinolytic and Milk-Clotting Activities from Moringa Oleifera Flowers
View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Food Chemistry 135 (2012) 1848–1854 Contents lists available at SciVerse ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem Caseinolytic and milk-clotting activities from Moringa oleifera flowers Emmanuel V. Pontual 1, Belany E.A. Carvalho 1, Ranilson S. Bezerra, Luana C.B.B. Coelho, Thiago H. Napoleão, ⇑ Patrícia M.G. Paiva Departamento de Bioquímica-CCB, Universidade Federal de Pernambuco, Cidade Universitária, 50670-420 Recife, Pernambuco, Brazil article info abstract Article history: This work reports the detection and characterization of caseinolytic and milk-clotting activities from Received 28 February 2012 Moringa oleifera flowers. Proteins extracted from flowers were precipitated with 60% ammonium sul- Received in revised form 14 May 2012 phate. Caseinolytic activity of the precipitated protein fraction (PP) was assessed using azocasein, as well Accepted 25 June 2012 as a -, b- and j-caseins as substrates. Milk-clotting activity was analysed using skim milk. The effects of Available online 30 June 2012 s heating (30–100 °C) and pH (3.0–11.0) on enzyme activities were determined. Highest caseinolytic activ- ity on azocasein was detected after previous incubation of PP at pH 4.0 and after heating at 50 °C. Milk- Keywords: clotting activity, detected only in the presence of CaCl , was highest at incubation of PP at pH 3.0 and Caseinolytic activity 2 remained stable up to 50 C. The pre-treatment of milk at 70 C resulted in highest clotting activity. Flowers ° ° Milk-clotting Enzyme assays in presence of protease inhibitors indicated the presence of aspartic, cysteine, serine Moringa oleifera and metallo proteases. -
(Enterokinase), Active EK
Catalog # Aliquot Size E535-31H-5 5 ug E535-31H-10 10 µg E535-31H-100 100 µg E535-31H-500 500 µg EK (enterokinase), Active Recombinant swine protein expressed in yeast cells Catalog # E535-31H Lot # L2146-3 Product Description Purity Recombinant swine EK (800-end) was expressed in yeast cells using an N-terminal His tag. The enzyme commission number is EC 3.4.21.9. The purity of EK (enterokinase) Gene Aliases was determined to be >85% by densitometry, approx. MW Enteropeptidase; TMPRSS15; PRSS7; Serine protease 7; 44kDa. ENTK; MGC133046. Formulation Recombinant protein stored in 50mM Tris-HCl, pH 8, 200mM Specific Activity NaCl, 30% glycerol. The specific activity of EK was determined to be Storage and Stability >10,000 units/mg as per the activity assay protocol. Store product at –20oC for up to 1 year. Aliquot enzymes Sample Data: to avoid freeze / thaw cycles. Digestion Conditions Catalytic pH Range: 4.5 ~ 9.5 Catalytic Temperature Range: 4 ~ 45oC Scientific Background Enterokinase (EK) is a member of the trypsin family of serine proteases, which cleaves proteins following the Lys at the HPLC results for digestion of the parathyroid hormone - maltose Asp-Asp-Asp-Asp-Lys recognition sequence. In the binding protein (MBP-PTH) fusion protein by Enterokinase at an stomach, EK converts trypsinogen into trypsin. EK has high enzyme : substrate mass ratio of 1:250 for 8 hr at 25oC. The specificity and high hydrolytic efficiency, making it a digestion products are: PTH and MBP. widely used biochemical tool for cleaving recombinant fusion proteins. EK can tolerate a variety of detergents and some denaturing agents.